Monitoring Adherence to Antihypertensive Drug Treatment by using a Dried Blood Spot test-test validation

Resistant hypertension, defined as uncontrolled blood pressure despite
concurrent use of three antihypertensive drugs including a diuretic is a common
health issue leading to costs and suboptimal cardiovascular prevention. Around
12% of patients with hypertension fulfill this definition.(1) A major part of
these patients (estimated 40-60%) considered to be *therapy-resistant* have a
problem in adherence to their diet and intake of their medication explaining
their assumed resistance to therapy.(2) To distinguish these individuals from
those with secondary causes of hypertension or *true* therapy-resistant
patients can be difficult. Current practice when non-adherence is suspected to
be the cause of resistant hypertension is hospitalization for at least half a
day for supervised intake of prescribed medication.(3) This is costly,
time-consuming and even in supervised intake it is not certain that the patient
did swallow the given drug.
Measurement of serum or urine levels of antihypertensive drugs by liquid
chromatography-mass spectrometry (LC-MS) has been performed for research
purposes but is not yet endorsed for use in clinical practice.(4, 5) Measuring
drug levels has become available in dried blood spots (DBS) which can be easily
obtained by a finger prick.(6-8) Due to this convenient and patient-friendly
sampling method, this technique is becoming more and more popular in
therapeutic drug monitoring (TDM) and is most commonly used in organ
transplantation and psychiatry. In hypertension, using DBS enables immediate
sampling in the office when suspecting non-adherence or during a sitting
automatic (*datascope*) or 24 hours ambulatory blood pressure measurement (24h
ABPM) without an additional visit to the laboratory.(6)

In short, blood is obtained by a finger prick and applied to a piece of filter
paper on a *DBS card* where it can dry. In the laboratory, this dried blood
spot is punched out, extracted and further analyzed using liquid
chromatography-mass spectrometry (LC-MS). We are currently developing this test
for the most commonly used antihypertensive drugs of four different classes.
Also some active metabolites are measured, they are mentioned between brackets:
-angiotensin converting enzyme (ACE) inhibitors: enalapril (enalapril-d5
maleate) and perindopril
-angiotensin II receptor blockers (ARB): losartan (losartan carboxylic acid)
and valsartan
-diuretics: hydrochlorothiazide and spironolactone (canrenone)
-calcium channel blockers: amlodipine and nifedipine

The method will be validated according to the guideline on bioanalytical
methods of the European Medicine Agency.(9) Different concentrations of the
different drugs and metabolites will be added to drug-free blood from blood
donors. These samples will be used to define selectivity, calibration curves,
the lower limit of quantification, accuracy, precision, dilution integrity,
recovery, stability, carry-over and the matrix effect including effects of
different hematocrit levels.
The current study is necessary to validate the developed method in patients
before further use in research and clinic. To ascertain measurable drug levels,
we will select patients expected to be adherent based on blood pressure levels
below target. The next step will be a trial in patients with resistant
hypertension to assess (non-)adherence and study the effect of feedback on drug
levels on adherence and as a consequence reaching blood pressure targets. For
this study, we recently received a ZonMW Grant (Rational Pharmacotherapy)

Primary Objective:
to validate the measurement of drug levels of eight antihypertensive drugs in
DBS against their corresponding plasma concentrations
Secondary Objective:
to study the trough levels to assess expected range of drug levels at a random
time point

Cross-sectional study needed for validation of a newly developed test.

Patients will be asked to undergo a finger prick to obtain a dried blood spot
(DBS) at the day they undergo automatic 45-minutes blood pressure measurement.
This is usually a couple of weeks before their visit to the physician. Usually,
also blood is taken on this day. We will ask for an extra tube. Half of the
patients will be asked to not take the measured drug in the morning (trough
level) while the other half wil be measured after drug intake in the morning
(peak level)

The risk and burden is limited to the risk and burden of venipuncture and a
finger prick . This is the same risk they will also have at regular blood
sampling or undergoing 24h ABPM. The risk of venipuncture is very low and
mainly exists of syncope, bleeding complications or formation a pseudoaneurysm.
At the day of the trough level, the studied medication will be taken slightly
later than normally. We will only select patients visiting their physician in
the morning. This will have no direct negative consequences when the drugs will
be taken right after the venipuncture.