Current Clinical Pharmacology, 2006, 1, 000-000 1

1574-8847/06 $50.00+.00 ©2006 Bentham Science Publishers Ltd.

The Imaging of Apoptosis with the Radiolabelled Annexin A5: A New Toolin Translational Research

Tarik Z. Belhocine1,*

and Francis G. Blankenberg2

1Department of Diagnostic Radiology and Nuclear Medicine, St. Joseph’s Hospital, London Health Sciences Centre,

University of Western Ontario, Canada, 2Department of Radiology / Division of Pediatric Radiology, Lucile Salter

Packard Children’s Hospital, Stanford, CA, United States

Abstract: Programmed cell death also called apoptosis plays a pivotal role in many physiological and pathological

conditions. In the multi-step process of drug development, a number of medications are being designed to target strategic

checkpoints of the apoptotic cascade either to induce or to inhibit programmed cell death. Conceptually, the assessment of

programmed cell death in response to various therapeutic interventions appears to be critical for evaluating the efficacy of

many drugs that act through apoptotic pathways. In the last decade, nuclear medicine techniques provided proofs of

principle for the imaging of apoptosis both in vitro and in vivo.

The purpose of this article was to review current knowledge on the imaging of apoptosis with radiolabelled annexin A5 in

various pre-clinical and clinical models, and beyond that, to assess the potential integration of such a dedicated technology

into translational research.

Key Words: Apoptosis, radiolabelled rh-annexin A5, drug development, translational research.

INTRODUCTION

Apoptosis is a genetically pre-programmed type of celldeath that plays a key-role in many physiological functionsincluding embryogenesis, homeostasis, and immunity [1, 2].Dysregulation of apoptosis may be the cause for manydisorders, which can have widely different clinical patho-physiologies (see Table 1) [3]. In myocardial infarction,there has been histological evidence of apoptosis around thecentral zone of necrosis. In a similar pattern, apoptotic cellsare observed in the penumbra area surrounding the acutecerebral ischemia. In neurodegenerative diseases, the involve-ment of caspases, key-enzymes in the apoptotic machinery,was evidenced in neuronal loss, particularly in Alzheimer’sdisease and Parkinson’s disease. In inflammatory diseasessuch as Crohn’s disease and rheumatoid arthritis, the over-activation of the immune system inducing increased apop-tosis of target tissues is well demonstrated [4]. Conversely, alack of apoptosis is one of the hallmarks of cancers [5].Improvements in the understanding of the basic mechanismsinvolved in programmed cell death (apoptosis) haveextended the field of pharmacology into the therapeuticmanipulation of a number of newly discovered signalingpathways [6]. For instance, many drugs that are available onthe market have been developed to target one or more of themany apoptotic pathways either by enhancing apoptosis in“hypo-apoptotic” cells or by inhibiting apoptosis in “hyper-apoptotic cells” [7-9]. In the era of health-cost savings, thereis a tremendous need for an appropriate selection of patientswho may benefit from these molecularly designed medi-cations. This requires the development of investigationaltools tailored to the specific mechanism of action of a giventherapy [10, 11].

*Address correspondence to this author at the Department of Diagnostic

Radiology and Nuclear Medicine, London health Sciences Centre, St.Joseph’s Hospital, University of Western Ontario, 268, Grosvenor Street,

London, Ontario, N6A 4V2, Canada; E-mail: tarikbelhocine@yahoo.fr

Table 1. Clinical Pathophysiologies Related to Programmed

Cell Death (Apoptosis)

Increase of apoptosis Decrease of apoptosis

- Myocardial infarction

- Stroke

- Ischemic preconditioning

- Organ transplant rejection

- Atherosclerosis

- Neurodegenerative diseases

(i.e. Alzheimer’s disease;

Parkinson’s disease)

- Chronic inflammatory diseases

(i.e. Rheumatoid arthritis,

Crohn’s disease)

- Infectious diseases

- Autoimmune diseases

(i.e. Hashimoto’s thyroiditis)

- Cancers

- Hyperplasia

- Autoimmune diseases

(i.e. Graves’ disease)

In the last few years, radiolabelled recombinant-humanannexin A5 (rh-annexin A5) has been successfully used forthe imaging of cell death in both animal models and humans[12-15]. This molecular imaging technique was initiallydeveloped to assess non-invasively apoptotic changes invarious experimental and clinical conditions [16]. In trans-lational research, a radiolabelled apoptosis biomarker may bea useful adjunct at each step of the process leading to thepharmacological design of new drugs, and beyond that, tothe pre-clinical and clinical assessment of various pro/anti

apoptotic agents [17-19].

2 Current Clinical Pharmacology, 2006, Vol. 1, No. 2 Belhocine and Blankenberg

A NEW MOLECULAR IMAGING PROBE

In recent years, a new imaging agent, radiolabelled rh-annexin (human, MW = 36 kDa) was designed for theassessment of programmed cell death (apoptosis and necrosis)in a variety of pre-clinical and clinical studies [13-18, 20-22]. During the early phase of the apoptotic cascade (i.e. 60to 90 min after the caspases activation), rh-annexin A5reversibly binds with a nanomolar affinity (10

-10 M) to the

externalized phosphatidylserine (PS) residues selectivelyexpressed on the surface of apoptotic cells in presence ofcalcium ions [23-25]. This phenomenon has been shown toplay a key-role in the phagocytosis of dying cells by

activated macrophages and neighboring cells [26, 27].

Many commonly used isotopes and linkers have beenused for the radiolabelling of rh-annexin A5 [16, 28]. So far,the most used chemical form in basic and clinical researchhas been

99mTc-HYNIC-annexin A5 [29]. This form of

radiolabelled rh-annexin A5 permits the imaging of apop-tosis using conventional nuclear medicine modalities such assingle photon emission computed tomography (SPECT) andwhole-body planar gamma-camera imaging [16, 17, 21, 22].Other forms of radiolabelled rh-annexin A5 are currentlybeing refined in order to improve the technical and clinicalproperties of the radiotracer [16, 17, 28-49].

Radiolabelled rh-annexin A5 imaging provides a non-invasive technique easily applicable in animal models andclinical trials involving one or even several intravenousinjections of tracer without significant side effects [16-18].Within 20 to 40 minutes a SPECT exam can be performed ofthe head, neck, chest, abdomen, or pelvis, while the entirebody can be imaged with planar imaging. Dynamic, early,and late images may be obtained depending on the acquisi-tion protocol [21, 22]. As a rule, the

99mTc-HYNIC-rh-

annexin A5, as well as the initial form using the N2S2 linkermethodology has been well tolerated by patients in multiplecontrolled trials [15, 17, 21, 22, 50, 51].

The localization of radiolabelled rh-annexin A5 at sites ofapoptosis has also been confirmed by a number of goldstandards including TUNEL staining of apoptotic nuclei andimmunohistochemical assays of caspase-3, the terminalenzyme in the apoptotic cascade just prior to or coincidentwith the expression of PS on the cell surface [13, 16, 17, 28,52-57].

PRE-CLINICAL STUDIES

Radiolabelled-rh-annexin A5 has been widely used inanimal models of apoptosis [17,18]. Various kinds of diseaseshave been assessed including malignancies, cardio vascularpathology, neurological disorders, rheumatoid arthritis, organtransplant rejection, autoimmune and infection/inflammatorydiseases [12-14, 30, 35, 49, 58-83].

99mTc-labeled rh-annexin

A5 uptake can be qualitatively (visually) and quantitatively(using relative measures of activity) assessed with radio-nuclide imaging [84, 85]. Comparative analyses with patho-logical studies showed a close correlation between increaseduptake of

99mTc-labeled rh-annexin A5 and detection of

apoptosis by TUNEL method and/or anti-caspase 3 immuno-staining [52-56, 64]. Moreover, autoradiography studies

provided strong evidence for the localization of 99m

Tc-labeled rh-annexin A5 at sites of apoptosis [78-81]. Table 2reports the literature data in pre-clinical studies aimed atevaluating the potential uses of rh-annexin A5 imaging.

In pre-clinical studies, 99m

Tc-labeled rh-annexin A5 hasbeen able to detect an apoptotic signal in response to pro-apoptotic drugs such as Cyclophosphamide, Cyclopentenyl-cytosine, Paclitaxel, Gemcitabine, Doxorubicin, Taxol,Cycloheximide, and Methylprednisolone [12, 20, 30, 35, 49,64-67, 80-84]. This holds true with anti-apoptotic agentssuch as caspase inhibitors [70, 71, 73]. The effects ofradiation therapy and ischemic-reperfusion injury of thebrain and heart have also been successfully imaged withradiolabelled rh-annexin A5 [30, 31, 62, 63, 69-71, 74, 77,79]. Interestingly, rh-annexin A5 imaging can directlymeasure the pre-to-post-therapeutic changes induced bytherapeutic interventions, thereby, providing an in vivoapoptotic index as shown in Fig. (1 ). Importantly, noincrease of radiolabelled rh-annexin A5 uptake has beenobserved with non-apoptotic drugs including anti-proliferative chemotherapeutic agents [35].

To date, pre-clinical studies with 99m

Tc-rh-annexin A5were performed with conventional SPECT devices usinggamma emitting isotopes including

99mTc-(technetium) and

111In-(indium) [13, 35]. Accordingly, a significant uptake of

radiolabelled-rh-annexin A5 at sites of increased apoptosishas been observed in virtually all organs and tissues of thebody including the brain, the lungs, the myocardium, thespleen, the liver, the bone marrow, and the vessels [58-85].Owing to the non-specific uptake and retention of HYNIC-rh-annexin A5 within the urinary tract (kidneys and bladder),the imaging of renal apoptosis has been suboptimal [50, 51,86]. In general, rh-annexin A5 imaging has a detection limitapproaching 10% of apoptotic cells within a 2-3 gramsvolume of tissue, which appears to be the minimum requiredfor the imaging of cell death on SPECT devices [87].

Alternative methods to radiolabel rh-annexin A5 havebeen recently developed including the use of self-chelatingannexin A5 mutants that have an endogenous Tc chelationsite (Ala-Gly-Gly-Cys-Gly-His) added to the N-terminus ofannexin A5 [25, 33]. These mutants under reduced condi-tions can be easily radiolabelled with Tc99m using tin/glucoheptonate as an exchange reagent. Two mutants, A5-117 and the newer A5-128 annexin A5, also have 50% to75% less uptake in the kidneys in mice and rats as comparedwith HYNIC-annexin A5, thereby, permitting the imaging ofrenal apoptosis [28, 32, 86, 89]. In another approach, rh-annexin A5 has been successfully labeled with fluorine 18(

18F) using N-succinimidyl 4-fluorobenzoate [43-49].

18F-rh-

annexin A5 designed specifically for positron emissiontomography imaging (PET), eventually combined withcomputed tomography (PET/CT), offers a number ofadvantages over SPECT agents, especially in terms of easierpreparation and better spatial resolution. Moreover,

18F-

annexin A5 has an advantageous biodistribution with muchlower uptake in the liver, spleen, and kidney compared toHYNIC-annexin A5. Additionally, the use of PET imagingwith annexin A5 should allow the absolute (not relative)quantification of uptake (i.e. % injected dose or % injected

The Imaging of Apoptosis with the Radiolabelled Annexin A5 Current Clinical Pharmacology, 2006, Vol. 1, No. 2 3

dose per gram of tissue) in the tumor before and after theinitial dose of therapy.

CLINICAL STUDIES

In recent years, a number of clinical trials have estab-lished the value of radiolabelled-rh-annexin A5 as in vivobiomarker of cell death. These studies have included phasesI/II and phase II/III controlled trials. Single site andmulticenter studies are still on going to gain more experiencein this emerging field of clinical research [15, 21, 22, 28, 52-57, 88-94, 97-99]. Available data from literature have

confirmed the ability of the radiolabelled-rh-annexin A5to localize at sites of apoptosis in humans. Additionally,intra-observer and inter-observer analyses highlighted thereproducibility as well as the reliability of annexin A5imaging for detecting an apoptotic signal in vivo [52-57, 88].

In oncology trials, 99m

Tc-labeled rh-annexin A5 has beenused to image patients with breast cancer, non-small celllung cancer, small-cell lung cancer, Hodgkin’s lymphoma,non-Hodgkin’s lymphoma, head and neck cancer, leukemia,and soft tissue sarcomas [21, 22, 53-55, 88, 97-99]. Thesepatients were subjected to conventional chemotherapy

Table 2. Imaging of Apoptosis with the Radiolabelled Annexin A5 – Pre-Clinical Data

Authors Ref. Animal models

Blankenberg et al. [61] Lung transplant rejection

Ogura et al. [60] Liver transplant rejection

Vriens et al.

Kown et al.

[58]

[59]

Heart transplant rejection

Heart transplant treated by Zinc chloride

Blankenberg et al. [13, 14] Fulminant hepatitis

Blankenberg et al. [80] Subacute and chronic infection

Peker et al.

Tokita et al.

[68]

[76]

Subacute Myocarditis

Autoimmune Myocarditis

Taki et al.

Taki et al.

Doue et al.

[79]

[71]

[70]

Myocardial ischemia

Myocardial ischemia treated by ischemic preconditioning and caspase inhibitor

Myocardial ischemia treated by anti-apoptotic agents

Bennink et al.

Schimmel et al

[66]

[67]

Cardiotoxicity induced by Doxorubicin

Cardiotoxicity induced by Cyclopentenyl cytosine

Kolodgie et al.

Johnson et al.

[77]

[69]

Atherosclerosis

Atherosclerosis

Sarda-Mantel et al.

Zhao et al.

Sarai et al.

[72]

[75]

[73]

Mural thrombus in aortic aneurysm

Atherosclerosis in ApoE-/- mice

Repression of apoptosis in atherosclerotic lesions

D'Arceuil et al.

Mari et al.

Blankenberg et al.

[62]

[63]

[74]

Neonatal brain ischemia

Cerebral ischemia

Brain ischemia treated by anti-FasL antibody

Post et al. [81] Rheumatoid arthritis treated by Corticosteroids

Blankenberg et al. [64] Intra-medullary and splenic apoptosis

induced by Cyclophosphamide

Blankenberg et al.

Mochizuki et al.

[13]

[65]

Lymphoma cell-lines treated

by Cyclophosphamide

Lee et al.

Yang et al.

[31]

[30]

Lung cancer cell-lines treated by gamma-irradiation

Breast cancer cell-lines treated by radiation and Paclitaxel

Ke et al.

Erba et al.

[35]

[82]

Breast cancer cell-lines treated by Taxol

Breast cancer cell-lines treated by Taxol

Kuge et al.

Takei et al.

Yagle et al.

Wang et al.

Kumar et al.

[20]

[78]

[49]

[83]

[84]

Hepatoma cell-lines treated by Cyclophosphamide

Hepatoma cell-lines treated by Gemcitabine

Liver apoptosis treated by Cycloheximide

Sarcoma cell-lines treated by Cyclophosphamide

Thymoma cell-lines treated by Cyclophosphamide and radiation

4 Current Clinical Pharmacology, 2006, Vol. 1, No. 2 Belhocine and Blankenberg

regimens and/or radiation therapy; chemotherapy protocolsincluded various cytotoxic drugs such as Mitomycin,Ifosfamide, Cis platinum, Vepeside, Cyclophosphamide,Doxorubicin, Vincristine, Prednisone, Melphalan, Cycloidal,Bleomycin, Carboplatinum, and Taxane. Radiolabelled rh-annexin A5 uptake was compared either with responseevaluation criteria in solid tumors (RECIST) or withcytological and pathological gold standards. An intriguingnumber of different temporal patterns of

99mTc-rh-annexin

A5 uptake have been described in these oncology trials. Forinstance, increased annexin A5 uptake could be detected insome tumors prior to any therapy suggesting spontaneousapoptosis that later responded well to chemotherapy. In othertumors, increases in annexin A5 uptake were found as earlyas 24 to 72 hours after completion of the first courseof chemotherapy and/or radiation suggesting intervention-induced apoptosis, which all responded to anti-tumortherapy. Surprisingly, some patients had decreased uptake of99m

Tc-rh-annexin A5 within the first 24 to 48 hours after thestart of treatment followed by a significant tumor response[21, 53-55, 88, 97-99].

In summary, rh-annexin A5 imaging revealed thatsignificant changes involving the annexin A5 uptake pre-to-post therapy could be predictive of treatment response. In thefield of nuclear medicine, PET imaging using

18FDG (

18F-

fluorodeoxyglucose) is considered as the standard of care forthe assessment of oncology patients [100]. Such a metabolicimaging relies upon the increased glucose metabolismof malignancies. Another PET tracer,

18FLT (

18F-fluorothy-

midine), has been recently proposed to assess the proli-feration activity in the course of cancer therapy [101].Within the tumor microenvironment, however, these radio-pharmaceuticals cannot specifically image the cell death asmain mechanism of action for most cytotoxic agents [102].In addition, confounding imaging patterns may occur in18

FDG PET imaging within the two first weeks following thetreatment (i.e. reactive inflammation versus residual tumor)

[102]. The imaging of apoptosis by means of radiolabelledannexin A5, immediately before and after therapy, maytimely complement the PET monitoring of malignancies with18

FDG or 18

FLT [78, 102, 103]. Further investigations willhelp clarify the biological and clinical significance of multi-

tracer imaging patterns.

Cardiology trials have included patients with acutemyocardial infarction and heart transplant rejection [15, 52,56, 57]. In clinical studies of myocardial infarction,increased rh-annexin A5 uptake matched areas of myocardialhypoperfusion. In heart tranplants, histological evidence ofrejection was also found in patients with increased uptake ofthe apoptosis imaging agent. New strategies againstischemia-reperfusion injury of myocardium include ischemicpreconditioning, which consists in short episodes of non-lethal ischemia followed by reperfusion [104]. This thera-peutic approach has been shown to protect the myocardiumfrom apoptosis, thereby, potentially limiting the extent ofischemic damage. Recently, the annexin A5 imaging waselegantly used to assess the efficacy of physiological andpharmacological interventions in a new model of ischemicpreconditioning [90, 91]. This human model used ischemicpreconditioning of forearm skeletal muscle to test variousdrugs such as dipyridamole, adenosine, and phentolamine.This model as it deals with peripheral muscle avoids many ofthe biases related to classical human experiments with

cardiac ischemia.

Neurology trials have included patients with acutestrokes who were examined by rh-annexin A5 imaging inconcert with MR imaging [92, 93]. Patterns of vascularlesions on MRI coincided with increased rh-annexin A5uptake. However, the clinical symptoms correlated better

with the extent of uptake of rh-annexin A5 uptake.

Preliminary data in patients with Crohn’s disease (CD)also demonstrated the feasibility of radiolabelled annexin A5

Fig. (1). Planar whole body imaging of the different temporal patterns of doxorubicin uptake in murine flank melanoma. K1735 M2 murine

melanoma tumor bearing C3H/HeN adult mice received 1-2 mCi of 99m

Tc-HYNIC-rh-annexin A5 injected via tail vein at baseline (pre), 24,

and 48 hours after treatment with one dose of doxorubicin (10 mg/kg i.p.). Serial planar whole body images are shown of two representative

treated right flank tumor bearing mice one hour after injection of tracer. Note that in animal #1 the initial decrease at 24 hours is followed by

a marked increase in tracer uptake at 48 hours after treatment. In mouse #2 there is an initial increase in tracer uptake with a subsequent

decrease at 48 hours.

The Imaging of Apoptosis with the Radiolabelled Annexin A5 Current Clinical Pharmacology, 2006, Vol. 1, No. 2 5

for monitoring treatment-induced apoptosis. In a small seriesof five patients with proven CD who were submitted to anti-TNF alpha antibodies (Infliximab), increased uptake ofannexin A5 as early as 24 hours post-therapy matched areasof colitis localized by colonoscopy [94].

Optimizing the clinical use of rh-annexin A5 imagingremains a challenge owing to the molecular complexity ofprogrammed cell death and the heterogeneity of theapoptotic response among identical cells in various tissularenvironments. The optimal timing for the imaging of

apoptosis will likely have to be defined for each type ofdisease as well as for each pro/anti apoptotic therapy [16, 21,95, 96]. To this end, more data are still needed on the utilityof annexin A5 imaging for the assessment of molecularlydesigned therapies. Additionally, the rh-annexin A5 imagingrequires further technical improvements to best capture theapoptotic signal [105, 106]. Multidisciplinary research groupsincluding pharmacologists, clinicians, imaging professionalsand pathologists may prompt further development ofradiolabelled rh-annexin A5 for the in vivo non-invasiveserial imaging of apoptosis in humans. Table 3 summarizes

Table 3. Imaging of Apoptosis with the Radiolabelled Annexin A5 – Clinical Data

Authors Ref. Clinical models Study type End-points

Belhocine et al. [21] NSCLC, SCLC, BC, NHL, HL

treated by chemotherapy

Phase I/II (n = 15) Toxicity

Spontaneous apoptosis

Drugs-induced apoptosis

Steinmetz et al. [97, 98] NSCLC, SCLC treated by chemotherapy Phase II/III

(n = 24)

Toxicity

Spontaneous apoptosis

Drugs-induced apoptosis

Van de Wiele et al. [54] Primary staging of HNC

treated by surgery

Phase I/II

(n = 20)

Toxicity

Spontaneous apoptosis

Vermeersch et al. [88] Primary staging of HNC

treated by surgery

Phase I/II

(n = 18)

Toxicity

Spontaneous apoptosis

Hass et al. [55] Follicular lymphoma treated

by radiation therapy

Phase I/II

(n = 11)

Toxicity

Spontaneous apoptosis

Radiation-induced apoptosis

Kartachova et al. [53] NHL, HL, Leukemia, NSCLC, HNC treated

by radiation therapy and/or chemotherapy

Phase I/II

(n = 33)

Toxicity

Spontaneous apoptosis

Drugs-induced apoptosis

Kartachova et al. [99] Malignant lymphoma, Leukemia treated by

radiation

Phase I/II

(n = 32)

Toxicity

Spontaneous apoptosis

Radiation-induced apoptosis

Kalinyak et al. [92] Non-hemorrhagic ischemic stroke Phase I

(n=2)

Toxicity

Spontaneous apoptosis

Loberboym et al. [93] Acute non-hemorrhagic stroke Phase I (n=12) Toxicity

Spontaneous apoptosis

Hofstra et al. [15] Acute myocardial infarction Phase I/II

(n = 7)

Toxicity

Spontaneous apoptosis

Narula et al. [56] Cardiac transplant rejection Phase I/II

(n = 18)

Toxicity

Allograft rejection-induced apoptosis

Known et al.

Thimister et al.

[57]

[52]

Cardiac transplant rejection

Acute myocardial ischemia

Phase I/II

(n = 10)

Phase I

(n = 9)

Toxicity

Acute allograft rejection-induced apoptosis

Toxicity

Spontaneous apoptosis

Rongen et al. [90] Ischemic preconditioning Phase I/II

(n = 43)

Toxicity

Intervention-reduced apoptosis

Riksen et al.

Bennink et al.

[91]

[94]

Ischemia-reperfusion injury treated

by dipyridamole

Crohn’s disease treated by

anti-TNF alpha antibodies

Phase I/II (n=9)

Phase I

(n=5)

Toxicity

Intervention-reduced apoptosis

Toxicity

Intervention-induced apoptosis

6 Current Clinical Pharmacology, 2006, Vol. 1, No. 2 Belhocine and Blankenberg

the literature data in clinical studies aimed at evaluating therh-annexin A5 imaging.

TRANSLATIONAL RESEARCH

Tremendous advances in molecular biology allowed thedevelopment of more specific therapies aimed at targetingstrategic checkpoints in the physiopathology of manydiseases [6-9]. Conventional clinical and radiologicalevaluation criteria, however, cannot assess the biologicaleffect of new therapies at the molecular level [10, 107]. Thegoal of translational research is to provide new tools forbridging basic sciences into clinical setting [11, 19, 108,109]. Accordingly, the National Institutes of Health (NIH)and the Food and Drug Administration (FDA) emphasizedthe use of biotechnologies for a better assessment of cancerfrom genotype to phenotype [110, 111]. Similarly, theEuropean Organization for Research and Treatment ofCancer (EORTC) clinical trial cooperative group advocatedthe introduction of biological criteria into the multi-phaseassessment of cancer [112]. This holds true for non-cancerrelated diseases. From this perspective, specific biomarkersused as surrogate endpoints on dedicated technologies(genomics, proteomics, and molecular imaging) should beintegrated in timely fashion, with new pre-clinical andclinical trials [10, 86, 113].

A recent report on current trends in the drugs marketrevealed that more than 40 therapeutic companies areinterested in developing pharmacological agents aimed attargeting the cell death pathway, especially p53, Bcl-2,TRAIL, IAP, and caspases [114]. Therefore, the assessmentof apoptotic signaling pathways provides with exquisitetargets in the course of pharmacological design, and more soin patient management [6, 7, 17, 18]. In both animal andhuman models, radiolabelled rh-annexin A5 imaging may beused to serially assess the in vivo effect(s) of a certain drug(pharmacokinetic) as well as the dose/response relationshipsof a given molecule (pharmacodynamic) [115-117]. More-over, in all preclinical and clinical phases, pharmacokinetic-pharmacodynamic modeling may be repeatedly performedwith non-invasive rh-annexin A5 imaging. As an example,the incorporation of biological criteria in phase I and IIstudies may help to better assess the classical toxicity andresponse end-points. To this end, radiolabelled rh-annexinA5 may be used to: 1) document the actual pro/anti apoptoticeffects of a newly designed drug; 2) quantify the apoptoticchanges pre-to-post therapy; 3) optimize the dose-effect ofthe combination of different drugs; 4) document theapoptosis-mediated toxicity of a newly designed drug; 5)select different categories of patients on the basis of theirapoptotic competence.

Similarly, in phases II and III studies, there is a clinicalneed for diagnostic and prognostic biomarkers aimed atevaluating the specific effect of a molecular therapy. A non-invasive reliable and reproducible molecular imaging maycomplement conventional response and survival end-points:1) to provide molecular insights onto the apoptotic capacityof disease (spontaneous apoptosis and intervention-inducedapoptosis); 2) to predict the ability of disease to respondapoptotically after the first course of treatment; 3) to assessthe apoptotic changes following several courses of treatment.

Even after the release of new drugs on the market,complementary investigations are still needed to define themost appropriate treatment protocol. In such phase IVstudies, radiolabelled rh-annexin A5 may help optimize thedose/schedule for an already approved therapy.

CONCLUSION

The development of molecularly targeted therapies forbasic and clinical research requires a well-designed, sensitiveand specific molecular assay to assess their efficacy.Radiolabelled rh-annexin A5 can contribute in bridging thegap between molecular biology and clinical medicine.Controlled studies will help gather more data to better definethe optimal imaging use of radiolabelled rh-annexin A5imaging, especially in terms of timing and signal-to-background ratio.

ACKNOWLEDGEMENTS

Special thanks to Dr. Jean-Luc Urbain (Department ofDiagnostic Radiology and Nuclear Medicine, University ofWestern Ontario, Canada) for reviewing this paper. Manythanks also to Pr. Chun Li (Division of Diagnostic Imaging,MD Anderson Cancer Center, Houston, Texas, USA) forsharing his unique experience in the imaging of apoptosiswith 111In-DTPA-PEG annexin A5.

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Received: 11 August, 2005 Revised: 25 January, 2006 Accepted: 01 January, 2006