C O M M U N I C A T I O N S
AGE or anti-MSA response was present. All initial immunizations
were performed using an alternative injection regimen that used
frequent doses in small amounts to more closely simulate the “binge
and crash” pattern of methamphetamine abuse. Indeed, in these
binging periods, chronic users are known to participate in “runs”
in which as much as a gram of the drug is injected every 2 to 3 h
to maintain a suitable response.9
is administered, a user could develop an immune response against
a 1-AGE-modified protein, and the produced antibodies could bind
some proportion of the serum methamphetamine, thereby reducing
the available concentration of the drug and ensuing high. Further-
more, autoantibodies against methamphetamine-modified proteins
could have undesirable consequences such as the misregulated
activation of inflammatory pathways, leading to extensive tissue
damage.
After the preliminary series of injections, an appreciable titer10
was discovered against 1-AGE (1:1000-1:2000), while no signifi-
cant titer was observed against the MSA carrier protein or in control
injections of MSA alone. This finding was particularly surprising
as aberrant covalent protein modification has been shown to yield
a detectable titer against the carrier protein.11 Presumably, autore-
active B cells were selected against through various mechanisms
(e.g., anergy, clonal deletion) prior to affinity maturation and
subsequent secretion of IgG. Furthermore, it is noteworthy that no
adjuvant was required to achieve significant titers against 1-modified
MSA, suggesting the immune system needs little priming to
recognize foreign glycation motifs. The obtained titer against the
1-AGE-MSA was long-lasting and could be increased to 1:4000-
1:32 000 by standard booster injections. Interestingly, these titers
against 1-AGE-MSA are 1 order of magnitude larger than those
obtained against a methamphetamine-KLH conjugate used in the
development of an active vaccine for methamphetamine addiction.12
The presence of antibodies against proteins that have been
covalently modified by a drug of abuse has been described in the
context of chronic cocaine addicts.13 As such, we assessed if 1-based
glycation gives rise to antibodies capable of binding methamphet-
amine, leading to an unrecognized mechanism of tolerance.
Competition studies were performed by ELISA using the obtained
anti-1-AGE pAb serum. Gratifyingly, negative control competitions
with MSA, AGE-MSA prepared from glucose and MSA, a small
molecule AGE-MSA analogue prepared from NR-acetyllysine (Ac-
Lys-OH) and glucose, and synthetically prepared 1-derived Amadori
rearrangement product14 all showed no appreciable competition with
1-glycated RNase A for antibody binding.
Although competition with free 1 was poor (Kd,app ≈ 10 mM), a
small molecule analogue of the 1-AGE-MSA prepared from
Ac-Lys-OH did effectively compete (Kd,app ≈ 10 µM) with 1-AGE-
RNase A. This compound was prepared such that the lysine side
chain that becomes glycated in MSA is mimicked, thereby providing
a similar reaction environment to 1-AGE formation reactions.
Glycation inherently generates a complex heterogeneous mixture
of products, and as such, exact quantitation of the extent of binding
was not possible. However, our results do provide clear evidence
that the primary epitope of the anti-1-AGE serum incorporates the
methamphetamine moiety as well as the glucose-derived cross-
linking region and, furthermore, that detectable methamphetamine
binding does occur.
In total, our results provide an intriguing possibility for an
unrecognized mechanism underlying methamphetamine addiction
and the associated health consequences. Further studies into the
importance of this process in vivo are underway and will be reported
in due course.
Acknowledgment. This work was supported by The Skaggs
Institute for Chemical Biology and the National Institute on Drug
Abuse (DA 015700 to K.D.J. and DA 015973 to T.J.D.). The Eisai
Co. Ltd. is gratefully acknowledged for sabbatical support to N.Y.
We thank Mr. Gunnar F. Kaufmann for helpful discussions.
Supporting Information Available: Experimental procedures for
immunogen preparation, immunization conditions, and ELISA experi-
ments (PDF). This material is available free of charge via the Internet
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