Figure S1B . The same DAG as in figure S1A, but with depicted
outcome/exposure ancestors controlled for by different means depicted as
open, black-outlined circles - minimal sufficient adjustment set. Black
circle is the instrument, gray/black-outlined circles depict unmeasured
exposure defined by the instrument (ABCG2 activity) and a hypothetical
mediator (MPAG levels) on a causal path - direct (thick full black
arrow) and/or indirect (dashed black arrow) – between the instrument
(unmeasured exposure) and the outcome. Completely gray circles are
unmeasured variables – enzyme/transporter activities that are
(presumable) mediators on some of the biasing paths (descendants of
outcome/exposure ancestors). They did not need to be adjusted for, since
their ancestors were included in the adjustment set. One suggested
confounder and one ancestor of the outcome (based on some of the
literature data) remained unmeasured – the SLCO1B3 c.334 SNP andUGT1A9 c.98 SNP. Two green circles depict ancestors of exposure
that did not need to be in the adjustment set, since their descendant –
CNI concentration – is adjusted for (see text for details).
xiii) Additionally – estimated creatinine clearance – by matching and
statistical adjustment; xiv) Hypoalbuminemia – by inclusion criteria:
patients had to have serum albumin >31 g/L; xv) Medical
conditions that may interfere with bioavailability of both MPA and CNIs
– by inclusion criteria: patients had to be generally “well-doing”
(free of surgical complications, infections, relevant metabolic,
gastrointestional, hepatic or cardiovascular conditions); xvi) Drugs
interfering with MPA pharmacokinetics – by inclusion criteria: patients
had to be free of treatment known to affect MPA during the pre-study
days and on the study day (antacids, phosphate binders, proton pump
inhibitors, antibiotics, rifampicin, oral iron, calcium or magnesium;
also, co-medication was restricted by exclusion based on comorbidities);
xvii) Drugs interfering with CNIs – by inclusion/exclusion criteria: no
specific list was formed, but co-medication was restricted already
regarding MPA, and comorbidities; also, CNI concentrations were
accounted for in matching; xviii) Drugs interfering with ABCG2 activity
(apart from CNIs) – by inclusion/exclusion criteria: no specific list
was formed, but considering the known ABCG2 substrates and inhibitors
[11], relevant co-medication was practically completely restricted
already regarding MPA and comorbidities.
Variables that did not need to be included in the adjustment set
(depicted as green circles in Figure S1B): i) CNI dose – as it is
represented by its descendant, CNI concentrations; ii) The same applies
to the two genotyped CYP3A4/5 SNPs. CYPs are of marginal relevance for
MPA clearance, hence the two genotyped CYPs and other possible SNPs
affecting CYP3A4/5 or other CYPs and/or transporters are of relevance
primarily for their effect on CNI concentrations [1, 10] – and are
thus controlled for by adjustment for CNI concentrations. By the same
logic, drugs affecting CNI concentrations (by any means) did not
necessarily need to be included in the adjustment set, but by
comorbidity and co-medication restrictions pertinent to MPA, they were,
to a great extent. As reviewed [3], in vitro data and a few
studies in humans (vs. several “negative” studies) suggest thatSLCO1B3 c.344 SNP and UGT1A9 c.98 SNP may be relevant for
exposure to MPA – in the present study, these SNP was not genotyped,
i.e., remained “unobserved” (indicated as gray circles [for
unobserved/unmeasured variables] in Figure S1B.