Therapeutic Drug Monitoring (TDM) Discuss the logic for therapeutic drug monitoring, which refer to as (TDM) List various classes of drugs that require TDM General description of this therapeutic drag TD Discuss the proper sample timing and method for TDM And Discuss analytical methods available for TDM List various drugs that not require TDM Steady state Therapeutic Drug Groups Digoxin, quinidine, procainamide, disopyramide. - Aminoglycosides (amikacin, gentamicin, kanamycin, tobramycin) - vancomycin leucovorin rescue ? First-pass metabolism HPLC methods

1. Al-Balqa Applied University
Department of Medical analysis
Course of Special topics
Supervisor : Dr. Walid Salem Abu Rayyan
By: Majd Ghawanmeh
Therapeutic Drug Monitoring (TDM)

2. OUTLINE
• Objectives
• Definitions
• Indications and requirement
• Drug administration and Metabolism
• Therapeutic drug class & group
• Therapeutic drug demonstration
• Specimens and sampling time
• Analytical methodology for TDM
• References

3. Objectives
• Discuss the logic for therapeutic drug monitoring, which refer to as (TDM)
• List various classes of drugs that require TDM
• General description of this therapeutic drag TD
• Discuss the proper sample timing and method for TDM
• And Discuss analytical methods available for TDM
• List various drugs that not require TDM

4. •Drug Monitoring is process which ensures that a patient is treated
with least expensive, most effective therapeutic agent, in a manner that
will maximize efficiency and minimize side effects".
•Therapeutic drug monitoring is a process in clinical pharmacology
which specializes in measuring the concentration of certain drugs in the
body fluids and clinically interpreting it to obtain useful and often
lifesaving information.
• The purpose of these actions is to ensure that a given drug dosage
produces maximal therapeutic benefit and minimal toxic adverse
effects.
 Definitions

5.  Why do I need TDM?
• You may need testing when you first start taking a medicine. This
helps your provider figure out the most effective dose for you. Once
that dose is determined, you may be tested regularly to make sure the
medicine is still effective without being harmful. You may also need
testing if you have symptoms of a serious side effect. Side effects
vary depending on the medicine.

6. Routes of administration
For a drug to express a therapeutic benefit, it must be at the appropriate
concentration at its site of action. Measuring drug concentration at the site
of action would be ideal. Unfortunately, for most drugs, this cannot be done.
Drugs can be :
• Intravenous [IV].
• Intramuscular [IM].
• Subcutaneous [SC].
• Absorbed through the skin (transcutaneous).
 The focus of the current discussion is on oral and IV administration.

7. Drug Metabolism
• Convert drugs into more hydrophilic metabolites to enhance the elimination.
• The consequences of drug metabolism is:
• The termination of pharmacological activity of the drug.
• Activated pharmacological activity for prodrugs, such as codeine.
• Decreases the bioavailability of the drug.
The biotransformation of drugs may produce metabolites that are
pharmacologically active. In such instances the metabolite should also be
measured because it is contributing to the effect of the drug on the patient
The role of TDM is important for drugs with variability in the rate of
metabolism in patients that may have a single nucleotide mutation in the
gene for the drug metabolism enzymes

9. • Figure 1 the upper is an idealized plot of
elimination after an IV does. it assumes
there is no distribution of this drug.
• A drug that does distribute outside
of vascular space would produce an
elimination graph such as in Figure 2.
• The rapid rate of change seen
immediately after the initial IV is a
result of distribution and elimination.[1]

10. • FIGURE 3: After oral administration at time 0, serum concentration increases, and
peak reached. The rate of absorption exceeds distribution and elimination.
*elimination (k) (dotted line) occurs when absorption and distribution are
complete.[1](in general)

11.  Steady state concentration is achieved when the amount of drug administered
at a constant rate, is equal to the amount of drug eliminated from the body.
 It achieved after 5-7 half-lives of the drug after it has been administered.
 E.g.) drug has a half-live of 24h and it is administered once a day, then it will
take 5 to 7 days of daily dosing to reach steady state concentration.
 The goal of a multiple dosage regimen is to achieve a trough that is in the
therapeutic range and a peak that is not in the toxic range.
Steady state

12. • The therapeutic range
is the concentration range
of drug in plasma where
the drug has been shown to
be efficacious without
causing toxic effects in
most people. FIGURE 4[1]
 By the end of the seventh dose, a single dose administered is equal to the
amount eliminated during the dosage period. At this point, steady state is
established, and peak and trough concentrations can be evaluated.

13. o Terms in Therapeutic Drug Monitoring (TDM)

14. When TMD is required ?
 Low therapeutic index and range of drugs e.g. lithium, digoxin
• Prevent consequences of overdosing and underdosing.
 When drug interaction may be occur (a reaction between two drugs or drug
and food).
 Appropriate analytical techniques are readily and easily present.
 There is a poor relationship between the dose of drug and circulating
concentrations but a good correlation between circulating concentrations
and therapeutic or toxic effects.

15. GROUP REPRESENTATIVE DRUGS
Cardioactive Digoxin, quinidine, procainamide, disopyramide.
Antibiotics
- Aminoglycosides (amikacin, gentamicin, kanamycin, tobramycin)
- vancomycin
Antiepileptics
Antineoplastic
Phenobarbital, phenytoin, valproic acid, carbamazepine.
gabapentin, Methotrexate.
Psychoactives Lithium, Tricyclic antidepressants
Analgesics Salicylates, acetaminophen
Immunosuppressants Cyclosporine, tacrolimus (FK-506)
 Therapeutic Drug Groups

16.  Drug demonstration
Cardioactive drug
1)Digoxin : is a glycoside used in the treatment of congestive heart failure. It
causes a decrease in intracellular potassium (by inhibiting Na, K-ATPase) that
led to increased intracellular calcium in cardiac myocytes to improves cardiac
contractility. It have a low therapeutic range of 0.8–2 ng/mL.
Higher serum concentrations (> 2 ng/mL) :
1. Decrease the rate of ventricular depolarization.
2. Toxic adverse effects (nausea, vomiting, and visual disturbances).
3. Premature ventricular contractions (PVCs).
4. Atrioventricular node blockage, are also common.

17. Evaluation of peak digoxin levels
• The timing is crucial. for adult, serum levels peak between 2 and 3 hours
after an oral dose. However, uptake into tissue is a relatively slow
process.
• As a result, peak serum concentrations do not correlate with tissue
concentrations.
• It has been established that the serum concentration 8–10 hours after an
orally administered dose correlates with tissue concentration. Peak
levels collected before this time are misleading and not valid.

18. 2)Quinidine: used to treat various cardiac arrhythmic situation. By block the
sodium channel, decreasing of rapid depolarization of the action potential.
The two most common formulations are:
I. Quinidine sulfate : Gastrointestinal absorption is rapid.
Peak serum concentrations are reached about 2 hours .
II. Quinidine gluconate: is a slow-release formulation.
Peak serum concentration is reached 4–5 hours after an oral dose.
 Cardiovascular toxicity, such as Premature ventricular contractions
PVCs,it seen at twice the upper limit of the therapeutic range is 2-6µg/ml

19. 3)Procainamide: Like quinidine, it used to treat cardiac arrhythmia
Gastrointestinal absorption is rapid and Peak plasma concentrations occur at
about 1 hour and therapeutic range 4-10µg/ml.
4) Disopyramide: It commonly used as a quinidine substitute when
quinidine adverse effects are excessive.
• Absorption is rapid, with serum concentrations peaking at about 1–2 hours.
It have a therapeutic range of 3–7.5 µg/mL

20. Antibiotic drug
1) Aminoglycosides: effective against resistant gram-negative bacteria.
It impair of proximal tubules of the kidney led to electrolyte imbalance and
proteinuria. High-level exposure led to necrosis of these cells and renal failure.
 TDM is necessary in compromised renal function patient. figure 5
2) Vancomycin: effective against gram-positive cocci and bacilli.
 Major toxicities of vancomycin are:
• Red-man syndrome: allergic reaction to vancomycin that typically presents
with a rash on the face, neck, and upper torso.
• Ototoxicity: When peak serum concentrations exceed 40 µg/mL
• Nephrotoxicity: When trough greater than 10 µg/mL

21.  Vancomycin prevents the cell wall from forming the cross-linking
necessary to keep it strong.[3]

22. Antiepileptics
1) Phenobarbital: slow-acting barbiturate that effectively controls of seizures.
• Peak serum concentration is reached about 10 hours after an oral dose.
• The half-life is 70–100 hours
• Toxic adverse effects include drowsiness, fatigue, depression.
2) Phenytoin(Dilantin): used in treatment for seizure disorders, And as a short-
term prophylactic agent in brain injury to prevent loss of functional tissue.
• The therapeutic range for free serum phenytoin is 1–2 µg/mL.
The major toxicity of it the initiation of seizures. Thus, seizures may be a result
of subtherapeutic or toxic concentrations.

23. 3) Valproic Acid : used as a monotherapy for the treatment of petit mal or
absence seizures (person suddenly stops all activity) and Migraine Headaches.
• The therapeutic range is relatively wide (50–120 µg/mL).
 The most common adverse effects (>120g/mL):
• Nausea, lethargy, and weight gain.
• Pancreatitis and hallucinations associated with high serum levels (200 g/mL).
• Hepatic dysfunction occasionally occurs in some patients even at therapeutic
serum concentrations; therefore, hepatic indicators should be checked
frequently for the first 6 months after initiation of therapy.

24. 4) Carbamazepine(Tegretol): effective in treatment for seizure disorders
and bipolar mania, it have a narrow therapeutic range is 4–12 µg/mL.
 Have a several idiosyncratic toxic effects including:
• Rashes, leukopenia, nausea and vertigo.
• Liver dysfunction may result so Liver function testing and Leukocyte counts is
also done during this period.
• It metabolized through oxidative pathways in the liver, leading to toxic
aromatic metabolites, ( carbamazepine 10.11-oxide) These will induce direct
damage to the bone marrow's erythroid progenitors, leading to aplastic anemia.
 Because of its serious toxic adverse effects, it is less frequently used, except
when patients do not respond to other drugs.

25. Psychoactives drug
• Lithium is an orally administered drug used to treat bipolar disorder. is a
cationic metal that does not bind to proteins. It is eliminated predominately
by renal filtration and is subject to reabsorption.
TDM to maintain serum concentrations in the range of 0.5–1.2 mmol/L.
 If it greater than 2 mmol/L are possible to muscle rigidity, seizures and coma.
•Tricyclic antidepressants (TCA): used to treat depression, insomnia,
extreme apathy, and loss of libido.
• peak serum concentrations are reached in the range of 2–12 hours.
• Toxicity in Higher levels may cause seizure, cardiac arrhythmia.

26. Immunosuppressive
 Drugs used to prevent rejection in Transplantation medicine program.
1) Cyclosporine: use in suppression of host-versus-graft rejection of
heterotopic transplanted organs.
• It is a Calcineurin inhibitors that plays a central role in signal transduction
upon TCR-ligand binding.[2]
• The toxic effects (350–400ng/mL) of cyclosporine are primarily renal
tubular and glomerular dysfunction, which may result in hypertension.
2) Tacrolimus: it is 100 times more potent than cyclosporine.
• Narrow therapeutic rang (5 to 15µg/L )
• Due to the high potency of it; circulating therapeutic concentrations are low.
• At concentrations above therapeutic, it associated with thrombus formation.

27. Antineoplastic (Chemotherapy)
• Methotrexate: Methotrexate is one of the few antineoplastic drugs in which
TDM offers benefits to a therapeutic regimen; due it followed by leucovorin.
• The therapy depends on the relative rate of mitosis of normal vs neoplastic cells.
• Neoplastic cells have a rapid rate of division, so it have a higher requirement for
DNA and are susceptible to depravation of this essential constituent before
normal cells.
• Methotrexate inhibits DNA synthesis in all cells.
• The efficacy of the therapy is dependent on a controlled period of inhibition, one
that is selectively detrimental to neoplastic cells.

28.  leucovorin rescue ?
 Administration of leucovorin, which reverses
the actions of methotrexate at a specific time
after methotrexate infusion.
Figure 6: Mechanism of action of Methotrexate (FH2 – Dihydrofolate, FH4–
Tetrahydrofolate, dTMP- deoxythymidine monophosphate, dUMP-
deoxyuridine monophosphate)[3]
 Failure to stop methotrexate actions results
in cytotoxic effects to most cells.

29. sampling time
An appropriate specimen is necessity.
This requires that the patient is at steady state, except when suspected toxicity
is being investigated.
 Blood specimens for drug monitoring can be taken at two different times:
• during the drug's highest therapeutic concentration ('peak' level)
• or at lowest ('trough' level).
Trough values are the least variable concentrations and are most often used to
establish therapeutic ranges. Drugs with short half-lives require trough
concentration monitoring. Drugs with a long half-life can be monitored at any
point in the dosage interval.

30. • The percentage of a drug that enters the circulation in an unchanged(non-
metabolized form).
• Oral drugs undergo first-pass metabolism and lead to a decrease in bioavailability.
• Drugs administered intravenously will bypass first-pass metabolism in the liver
and have 100% bioavailability in systemic circulation.
• Drug bioavailability can be impacted by serum protein binding of drugs, which
affect drug distribution into the tissues and elimination(Propranolol 90–95%).
• The amount of drug available for transport across a membrane depends on the
concentration of free, non-bound, drug.
Bioavailability:

31. FIGURE:- Factors that influence the circulating concentration of
an orally administered drug. (GI gastrointestinal).

32.  First-pass metabolism
• All substances, including drugs, absorbed from the intestine then enter the
hepatic portal system. In this system, all the blood from the gastrointestinal
tract is routed through the liver before it enters general circulation.
• Certain drugs are subject to significant hepatic uptake and metabolism during
this passage through the liver, inconsequence decrease in bioavailability
• In these instances, predicting the final circulating concentration from a
standard oral dose can be difficult.
• By TDM, an effective oral dosage regimens can be determined.

33. Drugs with significant First Pass effect
or low Bioavailability :
• Propranolol ~26% Bioavailability because 75-85 % is metabolized
by the liver before it can reach the circulation when taken orally.

34.  For oral administered the peak levels usually occur 1-2 hours after the dose
and the trough serum concentration shortly after the dose is administered.
• Digoxin is one exception. The serum level to determine peak should be drawn
after the serum digoxin equilibrate with the tissue; 6-10 hours after the oral dose.
 Intravenous medications should also be given time to equilibrate before the
peak level is drawn. They should be sampled ½-1 hour after administration.
• For example, the peak serum level of Gentamicin should be drawn thirty
minutes after infusion of the drug ends.
 Intramuscularly administered medications if injected in an area with good
blood flow will achieve peak levels within a similar time frame as
intravenous.

35. • Plasma or serum :- the most commonly used.
• Whole blood:- for Cyclosporine, tacrolimus, sirolimus, as there are large shifts
of drug between red cells and plasma.
• Saliva:- gives a measure of the unbound drug concentration, it useful when
blood samples are difficult to collect. Ex: Phenytoin, Lithium.
 Heparinized plasma is suitable for most drug analysis.
 For some drug avoid serum-separator tubes due it affect drug concentration by
adsorption of it into the gel after prolonged contact.(Quick separation(
 Storage of samples: Plastic cryovial type tubes are acceptable.
 CSA: Whole blood to be collected in an EDTA tube.
Specimens

37. Analytical methodology for TDM
1) Spectrophotometric methods:
• Prior to advent of GLC and HPLC, drug samples were analyzed by
spectrophotometric methods.
• It can provide a much-derived simplicity in assay procedure when the level
of sensitivity required is not too low (in the µg/ml range).
2) Immunoassay methods:
1) Enzyme immunoassay (EMIT).
2) Enzyme-linked immunosorbent assay (ELISA).
• These assays are specific; however, TCAs use polyclonal antibodies, which
cross-react among the different TCAs and their metabolites, so it used for
TCA screening rather than TDM.

38. 1. HPLC methods used in most of the commonly measured drugs, including
theophylline, antiarrhythmics, antibiotics and tricyclic antidepressants,
pentobarbital (PTB), phenobarbital (PHB), phenytoin (PHT).
The major advantage is that it may be quantitate several drugs in one separation.
The main disadvantage the need to employ specialist expertise.
3)Chromatographic methods
Physical method in which separation of components takes place between
two phases-a stationary phase and a mobile phase; it provide separation and
quantitation of the parent drug from metabolites.

39. HPLC methods
• It mainly consists of a column that holds packing material (stationary phase),
a pump that moves the mobile phase through the column, and a detector that
shows the retention times of the molecules.
• Stationary phase: substance which adsorption of the analyte (substance
that separated) takes place; It can be a solid, gel or solid liquid
combination.
• Mobile phase: solvent which carries the analyte (a liquid or a gas).
• The time at which a specific analyte elutes (comes out of the end of the
column) is called the retention time.

42. • Solvent Reservoir: mobile phase contained in a glass contents.
• It usually a mixture of polar and non-polar liquid components.
• Pump: aspirates the mobile phase from the solvent reservoir and forces it
through the system's column and detector.
• Depend on several factors including column dimensions, particle size of the
stationary phase, the flow rate and composition of the mobile phase,
operating pressures of up to 42000 kPa can be generated.
• Sample Injector: can be a single or an automated injection system.
• An injector for an HPLC system should provide injection of the liquid
sample under high pressure (40000 kPa).

43. • Columns: usually made of stainless steel, and it have an internal diameter
between 2 and 5 mm. They are commonly filled with a stationary phase with
a particle size of 3-10 μm.
• Detector: located at the end of the column detect the analytes as they elute
from the column; Commonly used detectors are:
• UV-spectroscopy, fluorescence, Mass spectroscopy.
• Data Collection Devices: collected Signals from the detector then the
computer integrates the response of the detector to each component and
places it into a chromatograph that is easy to read and interpret; store it.

45. Type of HPLC
I. Normal phase chromatography: separates analytes based on polarity.
• uses a polar stationary phase and a non-polar mobile phase. The polar analyte
interacted and retained by the polar stationary phase.
• Adsorption strengths increase with increased analyte polarity, and the
interaction between the polar analyte and the polar stationary phase increases the
elution time.
II. Reversed phase chromatography:
• The stationary phase is nonpolar (hydrophobic), while the mobile phase
is a polar liquid, such as mixtures of water and methanol.
• It works on the principle of hydrophobic interactions hence the more
nonpolar material is, the longer it will be retained.

46. III.Size exclusion chromatography: separates particles based on size.
• The column is filled with material having precisely controlled pore sizes.
• Larger molecules are rapidly washed; smaller molecules penetrate inside the
porous of the packing particles and elute later.
IV.Ion exchange chromatography:
• Retention is based on the attraction between solute ions and charged sites at
the stationary phase. Ions of the same charge are excluded.
• The mobile phase is an aqueous buffer, where both pH and ionic strength
are used to control elution time.

47. 2. Gas chromatography: technique which separates and analyzes volatile
compounds in the gas phase using very high temperatures to cause sample
vaporization. The separation is unique for each drug and establishes a
"fingerprint" for identification.
• GC is the gold standard method for the identification of drugs of abuse.
(GSC) Gas solid
chromatography
(GLC) Gas liquid
chromatography
Stationary phase solid liquid
Technique adsorption partition
Retention Time long Short compared to GSC.
Mobile phase Gas Gas

49. 4) Radio immune assay (RIA)
• It is sensitive, precise but requires the use of radioisotope.
• The hazard of using radioactive material is a considerable limitation of this
method. e.g., barbiturates and amphetamine.
5)Ion-selective electrode(ISE):
• Determination of serum lithium is done by ion-selective electrode.
6)Fluorescence Polarization Immunoassay (FPIA):
• It is a competitive biochemical assay used to detect antigens or Ab.
• This method uses a fluorescent molecule as the label instead of an enzyme,
making it more sensitive. Chlorophor polarized light ab ag.

50.  Drug measured in other body fluid:
Urine: Benzodiazepines
Sweat: Cocaine & Heroin
Saliva: Marijuana, Cocaine, Alcohol
Breath: Alcohol

51.  When TDM is unnecessary ?
1. Drugs with large therapeutic index e.g. Cephalosporin’s. and wide
therapeutic range e.g., Beta blockers.
2. Dosage need not to be individualized.
3. The pharmacological effects and clinical response can be clinically
quantified e.g., blood pressure, blood glucose.
4. The therapeutic range for many of these drugs includes
concentrations associated with toxic effects e.g., Methotrexate

52. Drug not suitable for TDM
1. Drugs having wide therapeutic index (Most antibiotics).
2. Toxicity is not a realistic concern (Penicillin).
3. Effects can be measured using functional laboratory tests (Anticoagulants)
4. Plasma concentration not predictably related to effects.
5. Effect of the relationship remains undefined (Antidepressants).
6. The ‘hit and run’ drugs e.g., Omeprazole, the half-life is less than 1 hour.

54. Reference
1. Clinical Chemistry: Techniques, Principles, Correlations (Bishop, Clinical
Chemistry) North American Edition 6th (sixth) edition Hardcover –
January 1, 2009
2. Demkes, E.J., Rijken, S., Szymanski, M.K. et al. Requirements for Proper
Immunosuppressive Regimens to Limit Translational Failure of Cardiac
Cell Therapy in Preclinical Large Animal Models. J. of Cardiovasc. Trans.
Res. 14, 88–99 (2021). https://doi.org/10.1007/s12265-020-10035-2
3. https://biopharmanotes.com/pharmacology-of-methotrexate/.
4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY: A SHORT
REVIEW Malviya R, Bansal V*, Pal O.P. and Sharma P.K.