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Blood, 1 July 2002, Vol. 100, No. 1, pp. 367-369
CORRESPONDENCE
To the editor:
Binding of imatinib by  1-acid
glycoprotein
In their recent report Jørgensen et al1 raised
doubts on the ability of  1-acid glycoprotein (AGP) to
bind and inhibit imatinib (STI571), as shown in our previous
report.2 We would like to comment on this paper,
on some methodological inaccuracies of their paper, and on additional
in vivo data that in our opinion strongly indicate an important role
for AGP in modulating imatinib bioavailability and
pharmacokinetics (PK). First, it is well known that chromatographically isolated AGP, the one
used by Jørgensen et al, show less-efficient binding of drugs
in general than chemically isolated AGP, the one used in our
paper.3 It is surprising in this respect that
Jørgensen et al never used as a control our preparation of AGP. Second, in their paper the authors state that our AGP
preparation, supplied by Sigma, "risks desialylation of the
protein." 1(p714) But the authors fail to acknowledge
that such a phenomenon has been associated with a decrease
(or with no change at all) in drug binding,3-5 and
not with an increase in binding, as their data apparently suggest. Third, the drug-binding assay shown is misleading. Quenching of AGP
fluorescence requires detailed information on a given drug's binding
site to AGP, since several binding sites for drugs on AGP are
known3; this information was not provided for imatinib. In
addition, quenching should be shown using progressively increasing concentrations of the drugs being studied and not, as done by Jørgensen et al, by comparing 2 different drugs, used at a single concentration, which differed in the 2 drugs studied (imatinib at 1 µM, chlorpromazine at 2.5 µM). Fourth, in vitro experiments using unmanipulated AGP (in the form
of sera containing different concentrations of AGP) performed by 2 independent groups2,6 show that the inhibition on imatinib activity is proportional to the content of AGP and can be blocked by the coincubation with erythromycin, a known binder of AGP. Fifth, additional ex vivo experiments were performed using unseparated
blood samples from patients on treatment with imatinib and
clinically resistant to it. Although plasma levels of imatinib exceeded
3 µM levels in these patients, Bcr/Abl was highly phosphorylated; short-term incubation (1 h) with erythromycin resulted in almost total
(> 85%) phosphorylation inhibition.7 Sixth, in vivo studies in patients treated with imatinib show
that there is a significant correlation between AGP levels and some PK
data such as Cmax.8 In addition, the
coadministration of imatinib and clindamycin, another antibiotic known
to bind AGP, resulted in significantly reduced Cmax and AUC and in
increased free fraction of imatinib; in particular, clindamycin induced within 5 minutes a fall in plasma imatinib concentrations ranging from
2-fold to 5-fold (Gambacorti-Passerini et al9 and
Gambacorti-Passerini, April 9, 2002, manuscript submitted for publication). For the above-mentioned reasons, the data from Jørgensen et al are
difficult to evaluate and their in vivo relevance is questionable.
Carlo Gambacorti-Passerini, Philipp le Coutre, Massimo Zucchetti, and Maurizio D'Incalci
Correspondence: Carlo Gambacorti-Passerini, Oncogenic Fusion
Protein Unit, Istituto Nazionale Tumori, Via venezian, 1-20133 Milan,
Italy; e-mail: gambacorti{at}istitutotumori.mi.it
References
1.
Jørgensen HG, Elliott MA, Allan EK, Carr CE, Holyoake TL, Smith KD.
1-Acid glycoprotein expressed in the plasma of chronic myeloid leukemia patients does not mediate significant in vitro resistance to STI571.
Blood.
2002;99:713-715[Abstract/Free Full Text].
2.
Gambacorti-Passerini C, Barni R, le Coutre P, et al.
Role of alpha1 acid glycoprotein in the in vivo resistance of human BCR-ABL(+) leukemic cells to the abl inhibitor STI571.
J Natl Cancer Inst.
2000;92:1641-1650[Abstract/Free Full Text].
3.
Kremer JM, Wilting J, Janssen LH.
Drug binding to human alpha-1-acid glycoprotein in health and disease.
Pharmacol Rev.
1988;40:1-47[Medline]
[Order article via Infotrieve].
4.
Friedman ML, Wermeling JR, Halsall HB.
The influence of N-acetylneuraminic acid on the properties of human orosomucoid.
Biochem J.
1986;236:149-153[Medline]
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5.
Wong AK, Hsia JC.
In vitro binding of propranolol and progesterone to native and desialylated human orosomucoid.
Can J Biochem Cell Biol.
1983;61:1114-1116[Medline]
[Order article via Infotrieve].
6. Larghero J, Mahon FX, Madeleine-Chambrin I, et al. Elevated levels of
the plasma protein alpha 1 acid glycoprotein in chronic myelogenous
leukemia in blast crisis mediate pharmacological resistance to Gleevac
(STI571, imatinib) in vitro and are associated with primary resistance
in vivo. Presented at the 43rd annual meeting of the American Society
of Hematology, Orlando, FL, Dec 10, 2001.
7. Gambacorti-Passerini C, Rossi F, Verga M, et al. Differences between in
vivo and in vitro sensitivity to imatinib of Bcr/Abl+ cells obtained
from leukemic patients. Blood Cells Mol Dis. 2002. In press.
8. Gambacorti-Passerini C, Zucchetti M, Russo D, et al. Alpha 1 Acid
Glycoprotein (AGP) binds to STI571 and substantially alters its
pharmacokinetics in chronic myeloid leukemia patients. Presented at the
43th annual meeting of the American Society of Hematology, Orlando, FL,
Dec 9, 2001.
9.
Gambacorti-Passerini C, Corneo G, D'Incalci M.
Roots of clinical resistance to STI-571 cancer therapy.
Science.
2001;293:2163[CrossRef][Medline]
[Order article via Infotrieve].
Response:
Further observations on the debated ability of AGP to bind
imatinib
We thank Gambacorti-Passerini et al for their comments on our
paper, which we note with interest. We are indeed encouraged by the
debate provoked by the publication of our brief report1 and welcome objective discussion from scientific colleagues. But we
feel that some of the points made by Gambacorti-Passerini et al
potentially arise from a lack of appreciation of the methods utilized
for the purification and characterization of glycoproteins, the
importance of glycosylation as a secondary modification of proteins,
and its implication for drug binding. Our responses to the specific
points raised are as follows: First, historically the majority of techniques for the isolation
of human 1-acid glycoprotein (AGP) were chromatographic procedures with strongly acidic buffer. Indeed, the commercial AGP
product assayed by Gambacorti-Passerini et al2 was
isolated according to the process of Hao and
Wickerhauser,3 which is a combination diethyl-amino ethyl
(DEAE)-Sephadex/carboxymethyl (CM)-cellulose chromatographic
method at pH 4.7, not a chemical method as stated by
Gambacorti-Passerini et al. Thus, both the commercial product and our
AGP are chromatographically isolated. Review of the literature,
including Kremer et al,4 indicates that acidic isolation
methods will damage AGP oligosaccharide (principally by desialylation)
and polypeptide components. Thus we are satisfied that, by
avoiding harsh acidic conditions, our published purification method
yields AGP without any structural degradation5 and thus
gives a valid representation of the actual in vivo presentation of the
glycoprotein. Furthermore, the fluorescence data (see the third point
below) presented indicates that chlorpromazine, a known AGP binder,
effectively binds to our isolated AGP, which is not in keeping with
"less-efficient binding." As clearly presented in our paper, the main aim was to examine AGP in
the CML setting, as it is well documented that the glycoprotein alters
both quantitatively and qualitatively in disease. Thus, commercial AGP
isolated from normal plasma would not satisfy this requirement.
Nonetheless, the most logical approach was to isolate AGP from normal
plasma as a control by the same method as for CML-derived AGP. Second, glycosylation, in the form of oligosaccharide chains covalently
bound to protein, is a significant presence on the surface of
glycoproteins, such as AGP, and functions to affect the conformation of
the underlying polypeptide largely owing to the huge hydrodynamic
volume occupied relative to amino acids. Thus, the presence of a
particular glycosylation pattern may influence the protein conformation
and thus the degree of access to the drug-binding site. Any change in
glycosylation, including the removal of sialic acid, may result in a
conformational rearrangement that could conceivably increase, decrease,
or leave unaltered the access to the drug-binding site. Our isolation
method does not involve denaturing steps, such as preliminary acidic
preparation and/or exposure to strongly acidic buffers during
chromatography, and has been proven not to result in structural
degradation. The quoted phrase was included to emphasize
that the latter could not be used to explain our results. In other
words, our observed lack of binding of imatinib to CML-derived AGP is
not an artifact of processing, but rather reflects more truly the
nature of the in vivo interaction. We do not read pretense of
increased binding into our data. Third, the fluorescence-quenching experiment, a method utilized to
directly study AGP drug binding,6 was simply employed to
demonstrate the retention of drug-binding potential by our purified
glycoprotein, which was amply shown with the known AGP-binder, chlorpromazine. An inability to bind the control substance,
chlorpromazine, would have been indicative of loss of AGP structural
integrity, which clearly had not been induced by our
purification processing. As this assay revealed no interaction between
CML-derived AGP and imatinib in the face of proven chlorpromazine
binding, we feel we cannot comment further on the nature of the
purported imatinib-binding site on AGP. It is not accepted that
chlorpromazine and imatinib should be tested at identical
concentrations providing clinically relevant concentrations are chosen.
(A correlation was observed between quenching and chlorpromazine
concentration up to 250 µM; data not shown). Fourth, we would consider the evidence presented suggesting a
stoichiometric interaction specifically between AGP and erythromycin in
whole sera to be circumstantial and difficult to evaluate. For this
reason, we deliberately isolated AGP from contaminating, nonspecific
plasma-protein drug binders. Additionally, can 2 groups be described as
independent when they have shared membership? Regarding the fifth and sixth points, we feel unable to comment on
unpublished manuscripts but look forward to scrutinizing the data once
in print. We do, however, wish to reiterate the findings of
Gorre et al,7 exponents of the "cell intrinsic" theory
of resistance, which our data supports: (a) cells taken from relapsing
patients exhibited reduced sensitivity to imatinib compared with
pretreatment cells; (b) relapsing patients did not have significantly
reduced imatinib plasma concentrations,8 despite a
presumed concomitant increase in plasma AGP concentration with disease
progression mirroring the findings in our cohort of
patients1(Fig1); and (c) dose escalation has not proven
successful in inducing remissions in relapsing, resistant patients (our
observations, and Gorre et al9). We trust that our responses will facilitate further evaluation of our data.
Heather Jørgensen, Moira Elliott, Sarah Paterson, Tessa Holyoake, and Kevin Smith
Correspondence: Heather Jørgensen, Academic Transfusion
Medicine Unit, University Department of Medicine, Queen Elizabeth
Building, Glasgow Royal Infirmary, 10 Alexandra Parade, Glasgow G31
2ER, Scotland; e-mail: hgj1b{at}clinmed.gla.ac.uk
References
1.
Jørgensen HG, Elliott MA, Allan EK, Carr CE, Holyoake TL, Smith KD.
1-Acid glycoprotein expressed in the plasma of chronic myeloid leukaemia patients does not mediate significant in vitro resistance to STI571.
Blood.
2002;99:713-715[Abstract/Free Full Text].
2.
Gambacorti-Passerini C, Barni R, le Coutre P, et al.
Role of alpha-1-acid glycoprotein in the in vivo resistance of human BCR-ABL(+) leukemic cells to the abl inhibitor STI571.
J Nat Cancer Inst.
2000;92:1641-1650[Abstract/Free Full Text].
3.
Hao Y-H, Wickerhauser M.
A simple method for the large scale preparation of AGP.
Biochim Biophys Acta.
1973;322:99-108[Medline]
[Order article via Infotrieve].
4.
Kremer JMH, Wilting J, Janssen LHM.
Drug binding to human alpha-1-acid glycoprotein in health and disease.
Pharmacol Rev.
1988;40:1-47[Medline]
[Order article via Infotrieve].
5.
Smith KD, Elliott MA, Elliott HG, McLaughlin CM, Wightman P, Wood GC.
The heterogeneity of alpha-1-acid glycoprotein in rheumatoid arthritis.
J Chrom Biomed Appl.
1994;661:7-14[CrossRef].
6.
Parikh HH, McElwain K, Balasubramanian V, et al.
A rapid spectrofluorimetric technique for determining drug-serum protein binding suitable for high-throughput screening.
Pharm Res.
2000;17:632-637[CrossRef][Medline]
[Order article via Infotrieve].
7.
Gorre ME, Mohammed M, Ellwood K, et al.
Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification.
Science.
2001;293:876-880[Abstract/Free Full Text].
8.
Druker BJ, Sawyers CL, Kantarjian H, et al.
Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.
N Engl J Med.
2001;344:1038-1042[Abstract/Free Full Text].
9.
Gorre M, Shah N, Ellwood K, Nicoll J, Sawyers CL.
Roots of clinical resistance to STI-571 cancer therapy.
Science.
2001;293:2163a[Free Full Text].
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