Background

[PubMed]

Deep vein thrombosis (DVT), which is the formation of blood clots in veins and is also known as venous thromboembolism (VT), is preventable, yet it occurs in the lower extremities of approximately two million Americans each year (1). A majority of DVT patients will experience pulmonary embolism (PE; ~30% are symptomatic, and another 40% are asymptomatic and at high risk) because the blood clot is unstable and can travel to, and lodges in, the lungs. An estimated 100,000 individuals are reported to die every year as a result of PE, and an equal number of people may die from PE-associated conditions such as heart disease and cancer (1). Contrast-enhanced venography is the gold standard for the diagnosis of DVT, but compression ultrasonography is usually the most common technique used to detect VT in the lower extremities. The primary limitation of these procedures is that neither technique can distinguish between chronic and unstable thrombi (2).

Formation of a blood clot is a complex process that requires recruitment of platelets (thrombocytes are cytoplasmic bodies derived from megakaryocytes and have many other functions besides facilitating blood clot formation) for the formation of a thrombus, and the process is described in detail elsewhere (3). One of the important components of the blood clotting process is platelet activation, which leads to the expression of a glycoprotein IIb/IIIa receptor (GPIIb/IIIa receptor) that binds fibrinogen and promotes platelet–platelet interaction, resulting in platelet aggregation and the formation of a secure plug. Several investigators have used radiolabeled monoclonal antibodies targeting fibrin and platelets to identify acute thrombi in humans, but these molecules have limited use because they are cumbersome to manufacture, are large in size, have a long blood circulation time, and present a danger of developing human antibodies directed against mouse antibodies (HAMA) (4). These limitations can be alleviated by the use of synthetic peptides that are much smaller than the monoclonal antibodies, are cleared quickly from circulation, and do not have a potential to produce the HAMA response. In this regard, synthetic peptides containing or mimicking the arginine-glycine-aspartate (RGD) sequence were shown to specifically bind the GPIIb/IIIa receptor, and a radioactive technetium (^99mTc)-labeled RGD mimetic peptide, ^99mTc-apcitide (^99mTc-P280), was shown to selectively accumulate in fresh venous thrombi in dogs (4-6). The study with dogs showed that the thrombus could be detected with scintigraphy 1 h after administration of the radiochemical, and at 4 h the thrombus/muscle ratio was 11 (6). The investigators also studied the biodistribution of ^99mTc-apcitide in rats and rabbits. In another study, Muto et al. conducted a preliminary study using ^99mTc-apcitide to detect DVT in humans (4). Apcitide is approved by the United States Food and Drug Administration for the detection and localization of acute VT in humans and is commercially available as a nonradioactive freeze-dried kit that can be labeled with ^99mTc for clinical use (2, 7-9).

Synthesis

[PubMed]

The synthesis of ^99mTc-apcitide was described by Lister-James et al. (6). Briefly, a trifluoroacetate salt of P280 was synthesized on a peptide synthesizer using FastMoc™ chemistry on a Rink amide resin. The peptide was purified with preparative reverse-phase (RP) high-performance liquid chromatography (HPLC) on a C₁₈ Delta-Pak column. Identification of the P280 peptide was established with amino acid analysis and electrospray mass spectrochemistry. Purity of the peptide was determined to be ≥90% with RP-HPLC as described above.

Labeling of the P280 peptide with ^99mTc was also described by Lister-James et al. (6). The P280 peptide was labeled with the use of ^99mTc-glucoheptonate, a ligand exchange reagent. The peptide was dissolved in normal saline and mixed with commercially available glucoheptonate previously mixed with ^99mTc-sodium pertechnetate. The reaction was allowed to proceed for 15 min at 100°C, and after cooling the solution was sterile-filtered through a low-protein binding 0.22-micron filter. The radiochemical yield and purity were both determined to be ≥90% with instant thin-layer chromatography and HPLC analysis. The specific activity of the labeled product was reported to be ~2,230 GBq/mmol (~60 Ci/mmol) P280 peptide.

The commercially available freeze-dried kit has been described in the draft package insert of nonradioactive apcitide. Each vial of the kit contains bibapcitide (a dimer of apcitide), sodium glucoheptonate dihydride, stannous chloride dihydride, and sufficient sodium hydroxide or hydrochloric acid to adjust the pH to 7.4 before freeze-drying. The freeze-dried powder, containing no preservative, is then sealed with a rubber stopper under a nitrogen atmosphere. The peptide was labeled with ^99mTc as described above, and the specific activity of the labeled product was calculated to be 11.2–33.6 MBq/μM (303–909 μCi/μM) depending on final volume (1–3 mL) of the labeled product (7). ^99mTc-Apcitide was used within 6 h of labeling.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Lister-James et al. (6) determined the 50% inhibition concentration (IC₅₀) of P280 for human platelet aggregation using inhibition curves as described by Zucker (10). A fixed number of the platelets were respectively exposed to varying concentrations of the peptide to obtain curves representing percent platelet aggregation versus peptide concentration. The IC₅₀ of P280 for platelet aggregation was determined from the curves to be 0.079 ± 0.017 μM. The investigators also compared the inhibition platelet aggregation by P280 in dogs and humans (6). The IC₅₀ for dog platelets was reported to be 0.20 ± 0.11 μM compared with 0.056 ± 0.011 μM for the human platelets. This showed that the peptide was 2.5–3.5 times more potent in inhibiting human platelet aggregation than dog platelet aggregation.

In another in vitro assay described elsewhere (11, 12), P280 was shown to inhibit the binding of fibrinogen to the GPIIb/IIIa receptor with an IC₅₀ of 6.8 nM (6). Under the same conditions, the IC₅₀ of P280 to inhibit the binding of vitronectin to the vitronectin receptor was 12,900 nM, indicating that the P280 peptide was three logs more selective for the GPIIb/IIIa than the vitronectin receptor.

Animal Studies

Human Studies

[PubMed]

Muto et al. reported the use of ^99mTc-apcitide imaging to detect DVT in nine patients (4). No adverse effects on the patients were reported, and the thrombi were visible in eight of the nine patients 1 h after treatment with the radiochemical. All the positive cases had an onset of DVT in <3 weeks, and the only negative case was diagnosed ~6 weeks earlier. PE was detected in two patients, and a third patient showed an accumulation of ^99mTc-apcitide in a hemagioblastoma. From these results the investigators concluded that the use of ^99mTc-apcitide scintigraphy is a safe and sensitive procedure for the diagnosis of DVT and PE.

Two preliminary reports detailing the use of ^99mTc-apcitide for DVT imaging in clinical trials of humans are available (9, 15). A detailed account of two clinical trials comparing the use of ^99mTc-apcitide with contrast venography for the imaging of DVT was later published by Taillefer et al. (16). Two hundred eighty patients either showing signs and symptoms of acute DVT or having a high risk of DVT after surgery were reported to be enrolled in the two clinical trials. Imaging with ^99mTc-apcitide and contrast venography was performed on the patients within 36 h of enrollment. Only 243 patients participating in the trials were evaluable. Compared with contrast venography, ^99mTc-apcitide scintigraphy had a sensitivity, specificity, and agreement of 73.4%, 67.5%, and 69.1%, respectively. In one of the institutions involved in the clinical trial, data obtained from a subset of patients (n = 63), who had no history of acute DVT or PE and presented at the institute within 3 days of the onset of DVT symptoms, showed that ^99mTc-apcitide scintigraphy compared with contrast venography had a sensitivity, specificity, and agreement of 90.6%, 83.9%, and 87.3%, respectively. On the basis of this information the investigators concluded that ^99mTc-apcitide scintigraphy is a new, sensitive modality that could be used for imaging acute DVT.

Bates et al. (7) evaluated the use of ^99mTc-apcitide for the detection of recurrent DVT in 78 patients. Of these, 38 patients were newly diagnosed with DVT (group 1), and 40 patients were previously diagnosed (group 2) with DVT and showed postthrombotic symptoms. The investigators expected that scintigraphy with ^99mTc-apcitide would be positive only in individuals with the acute (recent) condition because GPIIb/IIIa is expressed only on activated platelets that participate in the formation of the thrombus. Patients with old, inactive thrombi were expected to be negative for DVT with this technique. The images were interpreted by two expert nuclear medicine physicians in a blinded fashion. The sensitivity and specificity of ^99mTc-apcitide were respectively based on the proportion of group 1 patients who were positive for acute DVT and the proportion of group 2 individuals who were negative for the condition. On the basis of the results of the two experts, who had excellent agreement on interpretation of the images, it was concluded that ^99mTc-apcitide has a sensitivity of 92% and a specificity of 82%. The investigators concluded that ^99mTc-apcitide scintigraphy could be used to detect acute DVT and has few false positive results in patients having a previous history of DVT. However, accuracy of the interpretation was dependent on the training and experience of the interpreting physicians.

Dunzinger et al. investigated the use of ^99mTc-apcitide for the detection of DVT and PE in 19 patients (2). The patients received the radiochemical through an intravenous injection within 24 h of presentation at the hospital. Scintigraphic images were compared with results obtained from ultrasonography and/or contrast venography, and patients suspected to have PE underwent computed tomography or lung perfusion scans. Scintigraphy was reported to detect acute DVT in 14 of 16 patients diagnosed with the condition using the other modalities. In the 14 DVT-positive patients, the thrombi were visible with scintigraphy up to 17 days after the clinical symptoms were apparent. Three patients without DVT, as determined with other modalities, were reported to be negative even with scintigraphy. Only 1 of 6 patients with PE was positive on the basis of scintigraphy. From these results the investigators concluded that ^99mTc-apcitide scintigraphy had a sensitivity and specificity of 87% and 100%, respectively, for patients with DVT. However, ^99mTc-apcitide scintigraphy did not detect PE in 83% of the patients.

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