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British Journal of Radiology (2003) 76, 553-560
© 2003 British Institute of Radiology
doi: 10.1259/bjr/30385847
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Full Paper

Comparison between human pharmacokinetics and imaging properties of two conjugation methods for 99mTc-Annexin A5

H H Boersma, PharmD 1,2 I H Liem, MD 1 G J Kemerink, PhD 1 P W L Thimister, MD, PhD 1 L Hofstra, MD, PhD 3 L M L Stolk, PharmD, PhD 2 W L van Heerde, PhD 4 M-T W Pakbiers, BSc 2 D Janssen, BSc 1 A J Beysens, PharmD 2 C P M Reutelingsperger, PhD 5 and G A K Heidendal, MD, PhD 1

Departments of 1 Nuclear Medicine, 2 Clinical Pharmacy and Toxicology and 3 Cardiology, University Hospital Maastricht, P.O. Box 5800, NL-6202 AZ Maastricht, 4 Central Hematology Laboratory, University Medical Center St. Radboud, P.O. Box 9201, 6500 HB Nijmegen and 5 Department of Biochemistry, Cardiovascular Research Institute, University of Maastricht, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands

Correspondence: Hendrikus H Boersma, PharmD, Department of Clinical Pharmacy and Toxicology, University Hospital Maastricht, P.O. Box 5800, NL-6202 AZ Maastricht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Annexin A5 (AnxA5) is a protein with high affinity for phosphatidyl serine, a phospholipid exposed on the cell surface during apoptosis. This phenomenon has been used for determination of cell death after myocardial infarction. To evaluate the potential of 99mTc-AnxA5 for in vivo scintigraphy of apoptotic cells, the pharmacokinetics and imaging properties of two radiopharmaceuticals, 99mTc-(n-1-imino-4-mercaptobutyl)-AnxA5 (I-AnxA5) and 99mTc-(4,5-bis(thioacetamido)pentanoyl)-AnxA5 (B-AnxA5), were studied. I-AnxA5 was administered intravenously to seven patients and one healthy volunteer, and B-AnxA5 was administered to 12 patients. All patients in the pharmacokinetic study had myocardial disease. Additionally, imaging was performed in a patient with acute myocardial infarction, as well as in three patients with different malignancies. The plasma concentration, excretion and biodistribution of 99mTc-AnxA5 were measured, as well as levels of AnxA5 antigen. The kinetic data of both radiopharmaceuticals in plasma fitted a two-compartment model. Both preparations had similar half-lives, but a different distribution over the two compartments. Plasma levels of AnxA5 antigen showed a broad variation. Both radiopharmaceuticals accumulated in the kidney, liver and gut. B-AnxA5 was excreted significantly faster than I-AnxA5. Both compounds can be used for imaging of the head/neck region, the thorax and the extremities. B-AnxA5 has a faster clearance and a lower radiation dose. Imaging of apoptosis in the abdomen will be difficult with both radiopharmaceuticals, and especially with B-AnxA5 because of its faster appearance in the gut.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Apoptosis or programmed cell death plays a pivotal role not only in normal physiology, but also in pathological processes. Many drugs, such as cytostatics, affect apoptosis [1]. Thus, the detection and quantification of apoptosis in vivo might provide insight into the disease status and/or treatment efficacy of a patient.

The externalization of phosphatidyl serine (PS) at the cell membrane is one of the first events of apoptosis and persists until the cell is destroyed [2]. A naturally occurring protein, annexin A5 (AnxA5, 36 kDa), binds with high affinity to PS on apoptotic cells. For this reason, radioactively or fluorescently labelled AnxA5 has been investigated in vitro as well as in vivo in animal models as a tool to detect apoptosis [3–6]. Recently, our group and others demonstrated that 99mTc-AnxA5 can be used successfully to image apoptosis in patients [7–9]. In this study, two preparations of 99mTc-AnxA5 have been used: I-AnxA5 and B-AnxA5.

We hypothesise that both compounds can be used for imaging of the head-neck region, the thorax and extremities. Until now, published pharmacokinetic and imaging data for I-AnxA5 and B-AnxA5 are incomplete, because only their biodistribution has been studied [10, 11]. Biodistribution profiles alone are, however, not sufficient to determine target organs for optimal 99mTc-AnxA5 imaging. Moreover, it is useful to compare the initially used I-AnxA5 with the second, improved preparation of 99mTc-AnxA5, B-AnxA5. The purpose of the present study therefore was to characterize and compare the pharmacokinetics and imaging properties of I-AnxA5 and B-AnxA5. The study was performed in a group of patients with myocardial disease. A patient with acute myocardial infarction and patients with different malignancies were studied in order to evaluate the imaging properties of the agents.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Conclusion
 References
 
Patients
I-AnxA5 was administered to seven patients (mean age 56 years; range 47–66 years) and one healthy volunteer (35 years), and B-AnxA5 was administered to 12 patients (mean age 58 years; range 35–85 years). Each radiopharmaceutical was administered within 4 h of its preparation. All 19 patients had myocardial disease—12 patients had acute myocardial infarction, 4 patients suffered from heart failure, and another three patients had other myocardial diseases (angina pectoris, an intracardial tumour and myocardial contusion, respectively). For imaging purposes four patients suffering from acute myocardial infarction, leg sarcoma, non-Hodgkin's lymphoma and breast cancer were studied. All patients had a normal kidney and liver function. The study was approved by the Medical Ethics Committee of the University Hospital Maastricht.

Radiolabelling
Both radiolabelled compounds are experimental. An application as an investigational new drug (IND) has been filed for B-AnxA5.

I-AnxA5 was prepared by incubating approximately 1000 MBq 99mTc-pertechnetate with 1 mg freeze-dried, (n-1-imino-mercaptobutyl)-AnxA5 (Mallinckrodt, Petten, The Netherlands) in the presence of 10.8 µg Sn2+ as reducing agent for 2 h at ambient temperature. Radiochemical purity was assessed by column chromatography using a Sephadex PD-10 column (Pharmacia, Uppsala, Sweden) and 0.9% NaCl (B. Braun, Uden, The Netherlands) containing 1% bovine albumin (Sigma, St Louis, MO) as eluent [10]. I-AnxA5 was eluted, whereas free pertechnetate and TcO2 remained on the column.

B-AnxA5 (Apomate®, Theseus Imaging Corporation, Cambridge, MA) was prepared by synthesis of a phentioate ester ligand, followed by coupling to 1 mg AnxA5 and subsequent purification of the obtained B-AnxA5 as previously described [10]. The product was filter sterilized (0.2 µm pores) after preparation. Purification yield of the AnxA5-protein was about 80% ({approx}800 µg). The integrity of the filter was checked by a bubble point test. The pH was measured (limit pH 6–8) and radiochemical purity was assessed by instant thin layer chromatography (ITLC) (Gelman Sciences, Ann Arbor, MI) using 12% trichloroacetic acid (Merck, Darmstadt, Germany) in water as eluent. B-AnxA5 remains at the position of application (Rf=0), whereas most technetium-containing impurities are at the solvent front (Rf=1). Minor impurities, e.g. colloidal 99mTc, were found in the middle section of the ITLC glass fibre sheets (Rf{approx}0.5).

Administration of the radiopharmaceuticals
I-AnxA5 (about 2.5 ml) and B-AnxA5 (about 7 ml) were given by intravenous bolus injection. Patients received 555±89 (mean±standard deviation (SD)) MBq I-AnxA5 (1 mg) or 575±114 MBq B-AnxA5 (about 800 µg).

Plasma, urine and faeces sampling
Blood samples were obtained before administration of the radiopharmaceutical, and at 5, 10, 15, 20, 40, 60, 90, 120, 150, 180, 210, and 240 min afterwards. From eight patients (B-AnxA5 group) one to six additional blood samples were obtained to monitor the clearance of B-AnxA5 over 24 h. The radioactivity in plasma was measured using a universal gamma counter (1282 Compugamma CS, LKB-Wallack, Turku, Finland) and corrected for background radiation, individual patient dose, and decay.

Urine excreted over 20 h was collected for B-AnxA5 in two fractions (0–4 h and 4–20 h; five patients). Patients were asked to empty their bladders before B-AnxA5 was injected. Faeces were collected for B-AnxA5 over the first 20 h.

Plasma AnxA5 concentrations were measured using a validated ELISA (Hyphen Biomed, Andresy, France) according to the manufacturer's protocol. Also, levels of endogenous AnxA5 were measured in a control group of patients with acute myocardial infarction, to detect whether endogenous AnxA5 levels interfere with the measurement of administered I-AnxA5 or B-AnxA5. These levels were compared with measurements of endogenous concentrations of AnxA5 in healthy volunteers.

Determination of the in vivo stability of B-AnxA5
The in vivo stability of B-AnxA5 was assessed between 1 h and 20 h after administration of the radiopharmaceutical. 1 ml to 2 ml of patient plasma (pre-treated with 1% Triton X-100; Sigma, St. Louis, MO, USA) was added to a Sephadex PD-10 column (Pharmacia, Uppsala, Sweden), pre-eluted with 15 ml eluent (saline containing 1% bovine albumin; Sigma, St. Louis, MO, USA). The sample was eluted with 7 ml of eluent. The percentage of intact radiolabel was determined by measuring B-AnxA5 in the eluent and unbound pertechnetate retained on the Sephadex PD-10 column.

Biodistribution and pharmacokinetics
Six (I-AnxA5) and five (B-AnxA5) patients underwent imaging with a dual-head gamma camera (Multispect2, Siemens Gammasonics, Hoffman Estate, IL). The gamma camera was equipped with high-resolution collimators, and a 15% energy window around the photo peak of 99mTc was used. Upon injection of the radiopharmaceutical, a dynamic study of the thorax region was started, lasting 30–60 min. Subsequently, whole-body scans were performed with a scan speed of 10 cm min-1, two to four scans for the I-AnxA5 group and three scans for the B-AnxA5 group. The whole-body acquisitions were typically performed at 2 h, 4 h, and 20 h after radiopharmaceutical administration.

The pharmacokinetic parameters were derived using the KINFIT module of the MW/PHARM computer program package (Version 3.30, MediWare, Groningen, The Netherlands) [12]. The data, consisting of the plasma concentration of 99mTc radioactivity or AnxA5 versus time, were analysed by non-linear regression analysis using a least-squares weighted simplex algorithm, with data weighted with the reciprocal of the observed value. The following parameters were obtained: t1/2;,{alpha}, distribution half-life; t1/2;,ß, elimination half-life; f{alpha}, fß, fractions of the total radioactivity concentration in the plasma, making up the corresponding component; V{alpha}, volume of distribution of the central ({alpha}) compartment; Vtot, total distribution volume; CL, total clearance. A similar analysis was performed for endogenous AnxA5 in plasma.

Total body radioactivity and the uptake of radioactivity by organs were calculated as previously described [10, 11].

SPECT and/or planar imaging of patients
Patient with acute myocardial infarction
The diagnosis of acute myocardial infarction was made by electrocardiographic criteria and confirmed by biochemical detection of cardiac enzyme release. I-AnxA5 (638 MBq) was administered intravenously 2 h after reperfusion, which was achieved by performing percutaneous transluminal coronary angioplasty (PTCA), resulting in thrombolysis in myocardial infarction (TIMI) III flow. Images were obtained 22 h after injection. A 64 x 64 matrix was used and 60 angle views, counting 45 s at each angle. Studies were reconstructed using a filtered back-projection method, using a Butterworth filter with a cut-off frequency of 0.55 and an order of 5. All SPECT studies were converted to transverse slices to compare the reperfusion defect with the uptake of I-AnxA5. After 7 weeks, sestamibi imaging was performed to evaluate the reperfusion defect [7].

Sarcoma of the leg
A 58-year-old woman was admitted for resection of a large tumour in the upper right thigh (approx. 9 x 9 x 18 cm). 10 months earlier surgical biopsy diagnosed the tumour as a soft tissue sarcoma, of the epithelioid type. Further evaluation revealed lung metastases. Chemotherapy (adriamycin 3 times 75 mg m-2; 6 months before operation), regional perfusion with melphalan and tumour necrosis factor alpha (3 months before operation) and radiotherapy (3 times 6.0 Gy; 1 to 21/2; weeks before operation) were without effect on tumour size and lung metastases (MRI). Owing to local tumour progression with ulceration, a hip-exarticulation of the right leg was planned as a palliative procedure. One day prior to the operation 400 MBq of B-AnxA5 was administered intravenously followed by static images after 4 h (including SPECT) and 16 h.

Breast carcinoma
A 37-year-old female was diagnosed with carcinomatic mastitis. Furthermore, left axilliary palpable lymph nodes were found. Further metastatic disease was not detectable. Prior to chemotherapy, 603 MBq B-AnxA5 was injected intravenously to determine the apoptotic status of the disease. SPECT scintigraphy was performed 5 h and 15 h after administration of the radiopharmaceutical.

Non-Hodgkin's lymphoma
A 63-year-old male was diagnosed with a grade 1 small cell follicular Non-Hodgkin's lymphoma. A CT scan of the thorax revealed a large mass in the neck region. Prior to chemotherapy, 391 MBq of B-AnxA5 was injected intravenously in order to determine the apoptotic status of the disease in this patient. SPECT scintigraphy was performed 4 h and 8 h after administration of B-AnxA5.

Data were statistically evaluated using Student's t-test. A probability value <0.05 was considered significant. Results are generally presented as means±SD.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Conclusion
 References
 
Radiolabelling of AnxA5 yielded I-AnxA5 and B-AnxA5 with a radiochemical purity of 79±3% and 96±2%, respectively. The radiolabelling procedures did not affect the PS binding ability of AnxA5 as evaluated by ellipsometry [13]. Both radiopharmaceuticals were stable for up to 4 h after their preparation. No side effects were observed after the administration of the radiopharmaceuticals to the patients. After evaluation of the data for patients receiving I-AnxA5, it was decided to collect additional information (urine and faeces samples, stability in plasma) for B-AnxA5.

Figure 1Go (left) shows the time courses of the radioactivity in plasma for I-AnxA5 and B-AnxA5. The AnxA5 antigen concentration versus time curves are shown in the right panel. The kinetics of both radiolabelled AnxA5 and AnxA5 antigen in plasma could satisfactorily be described by a two-compartment model. Curve fitting of the pharmacokinetic data with a three-compartment model did not improve the fit as can be judged from the r2 for both the 2-compartment and 3-compartment model in Table 1Go. This table gives the kinetic parameters for I-AnxA5 and B-AnxA5 in plasma, assuming a two-compartment model. The calculated clearance of B-AnxA5 (2.8±1.2 l h-1) was significantly higher than that of I-AnxA5 (1.2±0.4 l h-1; p<0.01). The fast alpha-component of I-AnxA5 radioactivity in plasma (52±13%) was significantly smaller than the corresponding component of B-AnxA5 (81±13%; p<0.001).



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  		Figure 1. Concentration-time curves of I-AnxA5 ({blacklozenge}) and B-AnxA5 ({blacksquare}) of (a) 99mTc-radioactivity in plasma and (b) AnxA5 concentration in plasma. For I-AnxA5, data for 8 individuals are presented, whereas for B-AnxA5 values for 10 (radioactivity) and 12 (plasma concentration AnxA5) patients are given. Radioactivity data were normalized with the administered activity by dividing the obtained plasma radioactivity levels by the administered dose in GBq. All values are depicted as means±standard deviation.

 

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  		Table 1. Pharmacokinetic parameters of 99mTc radioactivity and annexin A5 concentration in plasma

 
Plasma levels of AnxA5 antigen ranged from 4 µg l-1 to 104 µg l-1 (>1 h after administration) in patients injected with B-AnxA5. Owing to the great data spread, median and ranges are given for the half-life and volume of distribution of the radiopharmaceuticals in the elimination phase. In the control group of patients who did not receive the radiopharmaceutical, plasma levels of AnxA5 ranged from 3 µg l-1 to 68 µg l-1. In contrast, AnxA5 levels were much lower in healthy volunteers, 0–9 µg l-1 (WL van Heerde, private communication, 2001) (Table 2Go).


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  		Table 2. Annexin A5 (AnxA5) levels in patients with myocardial disease and in healthy volunteers

 
More than 75% of B-AnxA5 present in plasma remained chemically stable for at least 20 h after administration of the radiopharmaceutical (Table 3Go).


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  		Table 3. Percentage intact radiolabel for B-AnxA5 (99mTc-(4,5-bis(thioacetamido)pentanoyl)-annexin A5) in plasma of patients

 
Representative whole-body scintigrams, obtained after administration of I-AnxA5 and B-AnxA5, are given in Figure 2Go to illustrate the biodistribution of the two radiopharmaceuticals. There was a high uptake of radioactivity in the kidneys and the liver. The faster excretion of B-AnxA5 radioactivity is shown by the earlier presence of radioactivity in the gut in the images to the right in Figure 2Go. Cardiac uptake of radiolabelled AnxA5 was seen in patients with acute myocardial infarction (Figure 4Go, see next section) and in a patient with an intracardial tumour [8]. Cardiac tracer uptake was assessed visually, because it was not possible to quantify the cardiac accumulation of radiolabelled AnxA5 due to its low radiation intensity compared with the radiation in adjacent organs such as liver and kidneys.



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  		Figure 2. Views of whole-body scan of a male volunteer (35 years, left) and a male patient (53 years, right), taken between 4 h and 5 h after administration of 440 MBq I-AnxA5 and 566 MBq B-AnxA5, respectively. A, anterior view; P, posterior view.

 


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  		Figure 4. Transverse tomographic image obtained with I-AnxA5 in a patient suffering from acute anteroseptal myocardial infarction. The arrow indicates increased I-AnxA5 uptake in the anteroseptal region 22 h after reperfusion.

 
Figure 3Go depicts the clearance of I-AnxA5 (left panel, n=6) and B-AnxA5 (right panel, n=5) from the body. B-AnxA5 was excreted faster than I-AnxA5: the biological half-life of B-AnxA5 was 17±8 h and that of I-AnxA5 was 62±13 h (p<0.001). Data from five patients showed that 26.0±9.3% of injected B-AnxA5 was excreted in urine up to 4 h and 57±11% up to 20 h. The amount of radioactivity excreted in faeces in 20 h was estimated to be about 25% for both radiopharmaceuticals.



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  		Figure 3. Decay corrected 99mTc-uptake in the total body after (a) injection of I-AnxA5 and (b) B-AnxA5, obtained with the gamma-camera. Curves with mean and (mean±1 standard deviation) are shown. ID, injected dose.

 
In vivo apoptosis detection
Acute myocardial infarction
Figure 4Go shows an example of I-AnxA5 uptake in the anteroseptal area of the patient with an acute anterior wall myocardial infarction at 22 h after injection [7].

Sarcoma of the leg
Planar images at 16 h after injection of B-AnxA5 clearly show increased activity in the right upper leg of the patient with sarcoma, indicating PS expression in this region. This is best seen in the anterior image (Figure 5Go). The central region without activity correspond to an area of necrosis on a CT scan (posterior image, Figure 5Go).



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  		Figure 5. Planar images of a patient with leg sarcoma, 16 h after the injection of B-AnxA5. There is increased uptake of B-AnxA5 in a major part of the upper right leg, with a central zone of decreased activity. Almost no uptake is seen in the left leg.

 
Breast carcinoma
Figure 6Go shows several SPECT images, obtained 5 h after injection, of B-AnxA5 from the patient with mastitis carcinomatosa of the breast. All generated SPECT views show clearly uptake of B-AnxA5 in the anterior part of the left breast, indicative for PS-expression in this tumour (indicated by the arrows).



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  		Figure 6. SPECT (5 h post injection of B-AnxA5) images of a patient with mastitis carcinomatosa. The increased uptake, of B-AnxA5, indicated by arrows, can be seen in the anterior part of the left mamma.

 
Non-Hodgkin's lymphoma
As an example a localization in the neck of a non-Hodgkin's lymphoma in the described patient is shown in Figure 7Go. The picture indicates that B-AnxA5 is taken up by the lymphoma, 8 h after intravenous injection of the radiopharmaceutical.



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  		Figure 7. Coronal SPECT image of a patient with non-Hodgkin's lymphoma. Increased B-AnxA5 accumulation is seen in the neck region, corresponding to a large mass on a CT image.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Conclusion
 References
 
This paper describes characteristics of two different preparations of radiolabelled AnxA5. The procedure yielding I-AnxA5 is much easier to perform than the procedure giving B-AnxA5. Both radiopharmaceuticals showed different radiochemical and pharmacokinetic characteristics. First, I-AnxA5 was less pure than B-AnxA5. The rather low radiochemical purity of I-AnxA5, which decreased from 83% to 76% over 6 months, is probably due to the decrease in the amount of Sn2+ in the kit with time. The radiochemical purity of B-AnxA5 remained stable for the prescribed shelf-life of 12 weeks. The low radiochemical purity of I-AnxA5 might affect, in principle, the radiation dose administered, pharmacokinetics and imaging results. Assuming that the free 99mTc in I-AnxA5 is present in the form of pertechnetate, the effect on the patient effective dose is small (<10% increase) [10]. Also, the effect of radiochemical impurities on the pharmacokinetics of I-AnxA5 is small. After correction of the plasma data for free 99mTc pertechnetate, by subtraction of the weighted pharmacokinetic curve for pertechnetate [14], the difference in amplitude of the alpha- and beta-components between the two radiopharmaceuticals was still present. This suggests that the radiochemical instability of I-AnxA5 did not affect its biodistribution and imaging properties.

Owing to the radiochemical problems of I-AnxA5, the stability of B-AnxA5 was investigated more extensively. It was stable in vivo, for at least 20 h after injection (Table 3Go).

The clearance of the two radiopharmaceuticals from plasma was also different. Although similar half-lives were found for the kinetics in both cases for the corresponding components, the relative magnitude of the components differed significantly between I-AnxA5 and B-AnxA5 (p<0.001). The large amplitude of the alpha-component for B-AnxA5 corresponds to the large initial decline in Figure 1Go (a). The greater amplitude of the beta-component for I-AnxA5 is reflected in Figure 2Go, where the large blood vessels are still visible at 4–5 h after injection of I-AnxA5, but not after injection of B-AnxA5. Kemerink et al reported a somewhat larger t1/2;,{alpha} (26±5 min) and t1/2;,ß (6.9±1.4 h) for B-AnxA5 in the blood of five patients [11]. This difference is mainly due to the use of different measurement times in the two studies. While Kemerink et al measured radioactivity in the blood up to 20 h, we measured samples up to 4 h after administration. This finding suggests that the pharmacokinetic model must be more complicated than a simple two-compartment model. Nevertheless, a two-component model gave a satisfactory fit to the data for all time points, whereas a three-component model did not provide a better fit, as judged from r2 (Table 1Go). The pharmacokinetics of 123I-AnxA5 in pigs were also described best by a two-compartment model (t1/2;,{alpha}=6.4 min, t1/2;,ß=71 min) [15]. In our study, both radiopharmaceuticals showed similar pharmacokinetics for AnxA5-antigen (Table 2Go). This is probably because most of the administered AnxA5 (>99%) was not labelled with 99mTc, and thus the unlabelled AnxA5 should have the same biochemical properties for both preparations. Although the labelling with 99mTc clearly affected the kinetics of the antigen, this could not be observed for the whole pool of AnxA5 because 99% of AnxA5 was not labelled.

The kinetics of AnxA5-antigen were difficult to describe >1 h after radiopharmaceutical administration. Because this could be caused by fluctuations in endogenous AnxA5 levels, we decided to compare plasma AnxA5 concentrations in patients receiving B-AnxA5 and in patients with a myocardial infarction not receiving the radiopharmaceutical (Table 3Go). In both groups AnxA5 levels showed a broad variation. Corresponding values were much lower in a group of healthy volunteers in whom AnxA5 antigen levels had been determined before (Table 3Go). Thus measurement of the concentration of radiolabelled AnxA5 antigen seems to be hampered by fluctuations in endogenous AnxA5 levels. This fluctuation in the concentration of AnxA5 in patients with myocardial infarction has been described before [16]. At present, it is still not clear what causes these fluctuations.

The biological half-life in the body was 62±13 h for I-AnxA5 and 16±7 h for B-AnxA5, which is not optimal for imaging purposes. The excretory pathway for both radiopharmaceuticals was identical. Extrapolation of data obtained in the first 20 h showed that about 25% of the radiopharmaceutical is excreted in faeces and the remainder in urine. Recently, the biodistribution of AnxA5 derived mutants containing endogenous chelation sites labelled with 99mTc was described in mice. Compared with I-AnxA5 and B-AnxA5, these mutants show considerably lower uptake in the liver and a similar to lower uptake in the kidney measured in the same time frame after administration. No pharmacokinetic data were shown [17].

The difference in pharmacokinetics between I-AnxA5 and B-AnxA5 must be due to the different ligands used to attach 99mTc to the AnxA5 molecule. In I-AnxA5 an n-1-imino-4-mercaptobutyl group is used, which is chemically unrelated to the 4,5-bis(thioacetamido)pentanoyl group used for incorporating 99mTc in B-AnxA5. Changes in biodistribution and pharmacokinetics caused by differences in conjugation methods have been described before for radioimmunoconjugates [18].

Furthermore, the high uptake of the two AnxA5 radiopharmaceuticals in the liver and kidneys, and in the case of B-AnxA5 the relatively fast appearance of radioactivity in the gut, hampers imaging of the abdomen. The study of the thorax and the head/neck region appears feasible [11]. Here, B-AnxA5 has the advantage of its faster clearance, whereas I-AnxA5 would be more favourable for imaging of the abdomen.

We were able to show that 99mTc-AnxA5 is suitable for imaging several targets: acute myocardial infarction and malignant tumours. The imaged tumours were confirmed with CT imaging and histology, indicating that the observed PS-expression is related to the tumour cell death programme. As can be judged from the additional information present in this applied scintigraphy, the optimal imaging time generally depends on the target to be imaged. A SPECT study revealed the best time for imaging of myocardial infarction to be between 15 h and 20 h after administration of the radiopharmaceutical [7]. The imaging studies of tumours in the extremities and in the female breast suggest that this kind of scintigraphy can be performed best 5–15 h after administration of B-AnxA5, dependent on the tumour type. For imaging of cell death, an agent with no apparently non-specific uptake in organs like liver, kidney and gut would be favourable. This goal seems hard to achieve but remains an essential target for further research.


   Conclusion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
References
 
This article describes the pharmacokinetic and imaging properties of I-AnxA5 and B-AnxA5, two radiopharmaceuticals suitable for the detection of apoptosis by in vivo imaging of the heart, extremities, breast and head-neck region. Firstly, it should be stressed that the behaviour of the 99mTc labelled Annexin A5 protein in the human body is affected by the labelling method used. Secondly, B-AnxA5 is the most stable radiopharmaceutical with good and reproducible labelling characteristics. Labelling of pertechnetate with I-AnxA5, however, was less reliable and the obtained radiochemical purity lower. B-AnxA5 has the advantage of a somewhat faster clearance from the body. Imaging of apoptosis in the abdomen will be difficult with both radiopharmaceuticals, and especially with B-AnxA5 because of its faster appearance in the gut.


   Acknowledgments
 
We thank Petra Lux for technical support.


   Footnotes
 
This study was supported in part by grants from the Dutch Heart Foundation (NHS 98.125 and NHS D96.025). Back

Received for publication October 31, 2002. Revision received March 24, 2003. Accepted for publication May 20, 2003.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 

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