Download \chapter{Erythrocyte MTX and 6MP metabolite levels} During the

\chapter{Erythrocyte MTX and 6MP metabolite levels}
During the maintenance therapy of children with Acute Lymphoblastic Leukaemia the
children receive weekly oral doses of methotrexate (MTX) and daily oral doses of 6mercaptopurine (6MP).
The metabolites of MTX and 6MP are accumulated in the erythrocytes and the levels
of metabolites in the erythrocytes serve as a surrogate measure of the levels of
the metabolites in the diseased cells. The inter individual variation of the
metabolisation of 6MP and MTX is substantial and previous studies have demonstrated
that these differences are important for the rate of relapse such that patients
with high metabolite levels show a decreased risk of relapse (\cite{lilleyman1994}
and \cite{schmiegelow1995}).
To gain insight into the processes generating the metabolite levels I will here
formulate statistical models for the erythrocyte metabolite levels as a function of
the doses and the characteristics related to the patients.
When formulating appropriate models for the erythrocyte metabolite levels it is
important to focus on the biology. In Section \ref{biology} I therefore give a
short introduction to the biology and how the metabolites are accumulated in the
erythrocytes. Next the available data are described in Section \ref{Data} and the
class of statistical models I will use for analyses is described in \ref{Methods}.
In Section \ref{emtxana} and \ref{e6tgnana} I apply the methods described in
Section \ref{Methods} for each drug separately. Finally, I discuss the results and
comment on the obtained models in Section \ref{discussion} .
\section{Biology} \label{biology}
Blood consists of leukocytes (white blood cells), erythrocytes (red blood cells)
and platelets. The bone marrow supports stem cells which are large primitive
undifferentiated cells. The stem cells differentiate to become one of these kinds
of cells and only the mature cells are released into the blood stream. The
leukocytes consist of five different types of white blood cells. In patients with
ALL, precursors of the lymphocytes (lymphoblasts) continuously replicate
themselves. The resulting leukemic clone does not function normally. %do not fight
infections
When the precursors of the lymphocytes form a clone, the DNA-sequences are
replicated. When a patient is given 6MP and MTX, the replication of the DNA is
disrupted, thereby controlling the proliferation of the lymphoblasts.
The most important cytotoxic metabolite of 6MP is 6-thioguanin nucleotide
(6TGN) which is incorporated in the replicated DNA sequences. Doer cellen saa?
%isf. guanin
The level of active metabolites in the lymphocytes within the bone marrow cannot
easily be measured but 6TGN is also accumulated in the erythrocytes for which the
level is relatively easily determined in a blood sample.
The level of erthrocyte 6TGN is used as a surrogate measure of the level of 6TGN in
the cells containing a nucleus and thereby as a surrogate measure of the influence
of 6TGN on the DNA synthesis in the lymphocytes.
MTX and methylated 6MP metabolites (MeMP) inhibit the purine de novo synthesis
(i.e. the synthesis of the building blocks for the DNA synthesis). When the cells
contain less purines, more 6TGN is incorporated in the replicated DNA sequences.
MTX is also accumulated in the erythrocytes and the level of MTX in these cells
(eMTX) similarly serves as a measure of the level of MTX in the cells containing a
kernel and thereby as a measure of the influence of MTX on the DNA synthesis in the
lymphoblasts.
\\
The erythrocytes do not constitute a homogenous population. They have a life span
of approximately 120 days and during that time a progressive ageing process is
seen.
The kinetics and metabolism of MTX and 6MP in the erythrocytes are qualitatively
well described in the literature. For 6MP there is an extensive literature from
several authors (e.g. \cite{rostami1995}, \cite{lennard1992}, \cite{lennard2001})
whereas for MTX most of the literature is due to Schr\o der (e.g.
\cite{schroder1986}, \cite{schroder1989}). Below I shortly describe how the
metabolites accumulate in the erythrocytes for each drug separately.
\\
\subsection{Accumulation of MTX in erythrocytes}\label{sec:MTXbio}
Due to accumulation, MTX is detectable in the erythrocytes for
2-3 months after the administration has been discontinued. The mature erythrocytes
are not able to form MTX metabolites %polyglutamates but instead the metabolites
are thought to be incorporated in the erythroid precursors of the bone
marrow:
\\
After a single oral dose of MTX, the serum MTX concentration reaches a peak after
1-2 hours. After 24 hours the serum MTX concentration will be almost 0. Initially
the MTX concentration in the erythrocytes is kinetically similar but has a lower
peak value (\cite{schroder1989}).
\\
Three to five days after administration of a MTX dose, the concentration of MTX in
the erythrocytes starts to rise and continues to increase for 10-14 days. This time
delay in the MTX concentration in the ery\-thro\-cytes corresponds to the ery\thro\-cyte maturation time.
\\
The erythroid precursors metabolise MTX to polyglutamates which are retained in the
mature erythrocytes due to the size and loading
(=ladning??) of the polyglutamates. However, during erythrocyte ageing, part of the
MTX disappears from the cells through efflux. The long-chain MTX polyglutamates are
retained in the erythrocytes until their age dependent destruction whereas the
short-chain polyglutamates disappear and are thought to serve as a slow release MTX
store. After 6-7 weeks no loss of MTX polyglutamates from the erythrocytes can be
detected.
During unaltered weekly oral MTX therapy a steady state MTX concentration in the
erythrocytes is achieved.
\cite{schroder1986} found that the steady state erythrocyte MTX concentration is
obtained after at least 8 weeks of constant MTX dose for children on maintenance
therapy.
The level of MTX metabolites in the erythrocytes is measured in \ehx.
\subsection{Accumulation of 6MP in the erythrocytes} \label{sec:6mpbio}
After an oral dose of 6MP many potentially active metabolites are produced, the
most important cytotoxic metabolite being 6TGN. The metabolites accumulate slowly
within the erythrocytes and the e6TGN levels reflect the cumulative dose of 6MP and
not the dose taken e.g. the day before the blood sample was taken. Whilst the e6TGN
levels increase slowly during treatment, the plasma concentrations are low and fall
to the lower limit of detection by 5 hours post-dose.
%on a time scale of days and weeks .
\cite{rostami1995} found that the 6TGN metabolites are found at the same level in
all cell age groups indicating that the metabolites do not originate in the bone
marrow as is the case for the MTX metabolites. The erythrocytes metabolise 6MP
while in circulation.
The inter individual variability is high due to differences in the activity of the
enzyme thiopurine methyltransferase (TPMT).
This enzyme methylates 6MP and some of the metabolites of 6MP. This enzyme is thus
important for the intensity of the treatment with 6MP as well as the risk of
toxicity.
\\
The distribution of TPMT activity in the Caucasian population is
trimodal: Approximately 89\% of the population have a high TPMT activity
(homozygous, TPMT activity $>$ 14 U/ml erythrocytes), 11\% have intermediate levels
of the activity (heterozygous) whereas 1 in 300 has no functional activity (TPMT
activity $<$ 2 U/ml erythrocytes). Hvad er U? Several studies have demonstrated an
inverse relation between TPMT activity and e6TGN (e.g. \cite{lennard1990} Lennard
et al 1990 lancet)
During unaltered daily oral 6MP therapy, steady state is obtained. The time until
steady state depends on the TPMT activity such that the heterozygous patients
quickly reach high erythrocyte levels whereas the homozygous patients have a much
slower increase, reaching steady state levels after 3 weeks (Lennard 1992 siger 4
uger. Jeg kan desvaerre ikke faa adgang til KS ref).
The level of 6TGN in the erythrocytes is measured in \ehmp.