Introduction

Sucralfate, the aluminum hydroxide complex of sulfated sucrose (Fig. 1), which have remarkable adherence ability to the ulcers forming a barrier against acid, pepsin, and bile acid penetration and promotes the healing of peptic and duodenal ulcers [1]. This unlabeled drug has been studied by many authors showed its various actions of mechanisms in animals and humans [2–5]. Many investigators employed 99mTc–sucralfate complex to diagnose few types of ulcers, such as esophageal, peptic, and duodenal ulcers [6–17]. Among them was Pera et al. [10], who reported a direct in vivo technique for labeling sucralfate used for the diagnosing gastric ulcers. The technique involve oral administration of a sucralfate/stannous ion suspension followed by oral administration of radioactive pertechnetate after 2 h. Vasquez et al. [12], successfully imaged gastric ulcers with sucralfate complex with 99mTc-human serum albumin (99mTc-HSA–sucralfate). Billinghurst et al. [13], studied extensively two formulations, the first one was 99mTc–sucralfate (by direct in vitro method), and the other one was 99mTc-HSA–sucralfate (by using modified version of Vasquez method) provided studying the various factors affecting its stability. Naumovski et al. [15], prepared 99mTc-DTPA–Suc (by modification of the method presented by Scopinaro et al. [18]) and suggested that scintigraphy with 99mTc-DTPA–sucralfate can be considered as additional method, complementary to routine investigations in detecting mucosal lesions.

Fig. 1
figure 1

Structure of sucralfate

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The present work aims to test and improve the validity of the intended freeze-dried kit production process of the formulation introduced by Billinghurst et al. [13] for the benefit of Iraqi nuclear medicine centers. The work is to be supported by radioanalytical studies with the in vivo biological behavior in laboratory animal to demonstrate the stability extent of the in vitro labeled formulation and its usefulness in medical applications for the detection of gastrointestinal ulcers.

Experimental

Preparation of new formulation of tin–sucralfate freeze-dried kit (F.D.K.)

The preparation of developed formulation of tin–sucralfate (F. D. K.) was carried out by using chemicals of commercial sources without further purification (sucralfate was imported by The State Company for Marketing Drugs & Medical Appliances/Iraq, and dihydrated stannous chloride was obtained from Riedel-De-Haen, AG-Germany). This developed formulation was prepared by mixing acid solution of a constant concentration of hydrated stannous chloride (11.3 mg/5 mL) with different amounts of sucralfate powder at various pH values. The suspension mixture was incubated at 37 °C for 1 h then centrifuged to separate the precipitate from the supernatant. The precipitate was resuspended in normal saline (0.9% NaCl solution) after each vigorous shaking a homogenized sample of 2.0 mL suspension dispensed in each vial (10 mL vial), then lyophilized in freeze dryer for 48 h.

The labeling method proceeded by resuspension of the F. D. K. with a suitable volume (2.0–5.0 mL) of technetium eluate (Na99mTcO4) taken from 99Mo–99mTc generator (CIS–biointernational, France) of certain radioactivity (0.5–10 mCi). For the determination of the radiochemical purity of labeled preparations the labeled sucralfate suspension were rotated for homogenization then different sample volumes (0.1–0.2 mL) were withdrawn at various incubation time (5, 15, 30, 60 and 120 min) and filtered through 3 mm filter paper (25 mm in diameter). Each precipitate was then washed twice with 1 mL normal saline. The filter and the filtrate were counted separately in well-type scintillation counter (sodium iodide crystal). The labeling efficiency was calculated according to the published formula by Billinghurst et al. [13].

Parameters studied related to the preparation of the new formulation

Effect of pH

The pH of the preparation suspension was adjusted by using either 0.1 N HCI or 0.1 N NaOH to get different pH values (2, 4, 6, 7, 8, 9, 10, and 11). Samples were taken for assessment at various incubation time intervals (5, 15, 30, 60 and 120 min) after 99mTc-eluate addition. These samples were incubated and analyzed at ambient temperature.

Effect of sucralfate amount

Various quantities of sucralfate amounts (50, 100, 150, 200, 250 and 500 mg) were selected with fixed amount of hydrated stannous chloride (11.3 mg) at optimal pH value (7.0). These preparations were directly labeled with 99mTc-eluate and analyzed by filtration for the detection of the radiochemical purity.

Effect of specific activity on complex stability

A series of experiments were conducted to determine at what specific activity the 24 h stability of 99mTc–sucralfate complex would be compromised. 99mTc sucralfate preparations using one tenth of our developed formulation (1.13 mg hydrated stannous chloride and 10 mg sucralfate) were incubated with various values of 99mTc-radioactivity concentrations (0.5, 1.0, 3.0, 5.0, 8.0 and 10.0 mCi). Analyses were carried out after 3 incubation time intervals (1, 4 and 24 h) at ambient temperature.

Biological studies

White Swiss albino mice weighing 15–25 gram were applied for biological comparative studies. The animals were grouped into three groups (according to the presence of ulcer and nature of the radioactivity to be orally administered) as follows:

  1. 1.

    First group (control group) The animals of this group were none fastend nor ulcerated and orally administered with 99mTc-eluate (Na99mTcO4).

  2. 2.

    Second group (ulcerated group) The animals received nothing orally 15 h and orally administered with 99mTc-eluate (Na99mTcO4).

  3. 3.

    Third group (ulcerated group) The animals received nothing orally for 15 h and orally administered with 99mTc–tin–sucralfate.

Ulceration could be made by successive multidoses of glacial acetic acid to the fasted animals. The animals of all groups were administered orally 0.1 mL sample of radioactivity (50–100 μCi 99mTc-eluate or 99mTc–sucralfate preparation). The animals were let to drink water then killed 90 min post-administration. The organs of interest and blood samples were taken from the dissected animals and counted in a well-type scintillation counter. The total blood activity was calculated assuming that blood constitutes 7% of the total body weight.

Results and discussion

Labeling kinetics and labeling yield of the new formulation of tin–sucralfate

Our good method of preparation of the developed formulation based on the mixing of an acid solution of hydrated stannous chloride (11.3 mg/5 mL) with suitable amount of sucralfate (100 mg) at optimal pH value (7.0).The best kit contents leads to obtaining a very high value of radiochemical purity (>95%) as illustrated in Fig. 2. The high labeling yield obtained at early time as indicated in Fig. 2 was very fast which is may be due to rapid process of binding of 99mTc with sucralfate and optimal kit contents. The labeled complex is consistent and stable as shown in Table 2. The labeling kinetics of the labeled complex was achieved by direct labeling without any successive steps. These results were comparable with one of the author’s results [13] and better than other investigators followed in vivo and transchelating methods [9, 10, 14, 15].

Fig. 2
figure 2

Represents the formation rate of the labeled complex (99mTc–sucralfate) as a function of pH

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Parameter studied related to the preparation of the new formulation

Effect of pH

Figure 2 shows the results of the formation rate of the 99mTc–tin–sucralfate complex as a function of pH (from 2.0 to 11.0). The results showed high labeling yield (>95%) in the range studied except pH 2.0 the labeling yield showed significant lower percentage (70–80%). Better labeling yield was obtained in pH range upper than 9.0 when compared with results reported by Bilinghurst et al. [13].

Effect of sucralfate amount

The results shown in Table 1 indicate that the amounts of the chelating agent (sucralfate) were not affected the radiochemical purity of the labeled complex. High labeling yield (>94%) obtained in a wide range of chelate amounts (50–500 mg) at a fixed amount of dihydrated stannous chloride (11.3 mg) at optimal pH value 7.0.

Table 1 Represents the data of radiochemical purity of 99mTc–sucralfate as a function of sucralfate amount at optimal pH value (pH = 7.0)
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Effect of specific activity on complex stability

Data of stability studies conducted on samples of the labeled preparations (99mTc–tin–sucralfate) containing various concentrations of radioactivity were shown in Table 2. No breakdown due to the radioactivity was seen in the range studied [up to 10 mCi/10.0 mg sucralfate (1.0 mCi/1.0 mg)]. Since this new upper level is above the need for clinical use (it corresponds to 1,000 mCi per gram of sucralfate). This value is higher than that published earlier (700 mCi/gram sucralfate) [13].

Table 2 Represents the stability of 99mTc–sucralfate versus total radioactivity
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Biological studies

Organ distribution data of free pertechnetate (Na99mTcO4) in none fasting nor ulcerated mice (control group-first group) are summarized in Table 3. The results have shown that high radioactivity uptake was observed in the stomach (41.30%) and intestine (33.73%). Lower radioactivity uptake was seen in the other organs at 90 min post-administration. These high percentages of radioactivity accumulation were attributed to the adherence of the radioactivity with food present (non fastened animals).

Table 3 Represents the biodistribution data of labeled complex (99mTc–sucralfate) 90 min post-oral administration in the fasted and ulcerated mice
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The results of ulcerating and fasting (second group) effects on the biological behavior of free pertechnetate are presented in Table 3. Data clear that the stomach radioactivity uptake (40.06%) was the same as in the control group (41.30%) except that intestine showed lower radioactivity uptake (15.38%).

To demonstrate the variation in the biological behavior in a comparison with the previous groups (first and second groups), the results of organ distribution of the labeled preparation (99mTc–tin–sucralfate) in the ulcerated and fasted animals (third group) were given in Table 3. The data show that very high radioactivity uptake were accumulated in both stomach and intestine (87.92 and 11.43%) respectively. This mean that about more than 99% of administered activity was accumulated in the gastrointestinal tract with trace amounts in the other listed organs.

The radioanalytical results together with the biological findings of our developed formulation of 99mTc–tin–sucralfate, demonstrate its stability, efficacy and usefulness in clinical applications specially in the detection of gastro duodenal ulcers.