Study on Biodistribution and Radioimmunoimaging of 131 Iodine-Labeled Monoclonal Antibody D-D3 Against Progastrin-Releasing Peptide (31

Abstract

This study was aimed at investigating the biodistribution and radioimmunoimaging of ¹³¹I-D-D3 in nude mice bearing different types of tumor xenografts. Radioiodination of the D-D3 antibody was performed with the chloramine-T method. The radiochemical purity was determined through thin-layer chromotography. ¹³¹I-D-D3 was injected into healthy Kunming mice via a tail vein, and the %ID/g for various organs was obtained. Similarly, the %ID/g and tumor/nontumor tissue ratio of ¹³¹I-D-D3 in nude mice bearing small cell lung cancer (SCLC) xenografts were obtained. Planar images of ¹³¹I-D-D3 in tumor-bearing nude mice were acquired at different times after injection. The ¹³¹I-D-D3 labeling rate was 86.56% ± 3.8%. The radiochemical purity of ¹³¹I-D-D3 was 99.27% ± 0.6%. After 12 hours of incubation in 37°C water bath, the radiochemical purity was 97.64% ± 0.5% and remained at 88.38% ± 0.4% after 48 hours. After being mixed with healthy human serum for 24 hours, the radiochemical purity was more than 64%. The metabolism of ¹³¹I-D-D3 in healthy Kunming mice was consistent with a two-compartment model with first-order absorption; T _1/2α and T _1/2β were 0.25 and 37.89 hours, respectively. The %ID/g of ¹³¹I-D-D3 in SCLC xenografts was much higher than those of other tissues at 48 hours after injection, and the tumor/nontumor tissue ratio also gradually increased with time. After 24 hours of injection, planar imaging was obtained, which clearly showed a contrasting tumor on the right armpit of nude mice bearing SCLC with high concentrations of radioactivity. Also, nude mice bearing gastric cancer showed similar results as that of the SCLC with a lower radioactivity level. No observable accumulation was observed in nude mice bearing pancreatic cancer or lung adenocarcinoma. The labeling rate and radiochemical purity of ¹³¹I-D-D3 were high and stable. ¹³¹I-D-D3 selectively accumulated at tumors that highly expressed progastrin-releasing peptide; therefore, it is a promising radioimmunoimaging reagent for SCLC.

Introduction

Recently, the incidence and mortality rates of lung cancer have greatly increased. Lung cancer has become the first killer of Chinese patients. Approximately 20%–25% of lung cancers are categorized into small cell lung cancer (SCLC), which is fast growing and very aggressive, with early metastasis. SCLC is the most malignant lung cancer, and it is usually at the late stage when a definitive clinical diagnosis is examined. The treatment for SCLC is very different from that of non-SCLC (NSCLC). Therefore, making a definitive diagnosis of pathological subtypes of lung cancer is an absolute requirement. Radioimmunoimaging and radioimmune-targeted therapy has the advantages of high specificity, good targeting capability, and low toxicity. Developing a new generation of antibodies with optimal biodistribution in vivo will benefit the early diagnosis and effective treatment of SCLC.

In 1978, a peptide resembling bombesin at the carboxy-terminus was isolated from the porcine stomach. The peptide resembling bombesin comprises of 27 amino acids and functions as a stimulant of gastrin; therefore, it was named gastrin-releasing peptide (GRP).^1,2 However, it has been found to perform many other functions including stimulation of the secretion of a variety of gastrointestinal hormones and pancreatic enzymes as well as the control of intestinal transit, metabolism, and behavior.^3,4 GRP has also been found to be expressed in the lungs of fetus and newborns and in primary lung cancer, especially SCLC.⁵ It has been discovered that SCLC can stimulate the secretion of GRP, and it also has the GRP receptors, indicating the presence of a self-regulatory circuit that can promote cell proliferation and the unlimited growth of tumors.⁶ GRP was originally demonstrated to be an important product of SCLC and an important tumor marker by Maruno et al.⁷ in 1989. In addition, 72% of SCLC patients have increased serum GRP. Recent research demonstrated that GRP was an auto-stimulatory growth factor of SCLC cells and could promote rapid growth of tumor cells.⁸ However, clinical application of GRP is limited because of the instability of serum GRP, and it is difficult to extract GRP from the serum because of its tremendously short half-life.⁹

ProGRP is the precursor of neuropeptide GRP and has shown good clinical performance in lung carcinoma patients for discriminating between NSCLC and SCLC as well as for classification of tumor subtypes.¹⁰ There are three types of ProGRP that share a common region (amino acids 31–98, ProGRP_(31–98)). The serum ProGRP level is stable and parallels the expression of GRP at gene and protein levels. As a new SCLC tumor marker, studies on ProGRP were very active.^{11

–20}

More recently, the recombinant ProGRP_(31–98) was produced by the Chinese Institute for Radiation Protection. The recombinant ProGRP_(31–98) was used as an antigen to immunize Balb/c mouse, and a monoclonal antibody D-D3 subtyping as IgG₁ subtype was successfully prepared. In the present study, the D-D3 antibody was labeled using ¹³¹Iodine and an SCLC nude mouse model was established by xenografting NCI-H446, an SCLC cell line. The biodistribution of ¹³¹I-D-D3 in vivo and the specificity of ¹³¹I-D-D3 in detecting SCLC were investigated via radioimmunoimaging study, to provide a specific and effective way to diagnose and treat SCLC.

Materials and Methods

All procedures involving animals were performed in accordance with an institutional guideline (Guide for the Care and Use of Laboratory Animals in Experimental Animal Center of Soochow University).

Labeling, isolation, purification, and stability of ¹³¹I-D-D3

D-D3 (obtained from Chinese Institute for Radiation Protection, Taiyuan, China) was labeled using ¹³¹Iodine via chloramine-T method. The labeling rate was determined by thin-layer chromotography using Xinhua #1 filter paper as support and saline as the solute. The R _f of ¹³¹I-D-D3 equaled to 1/10 and that of ¹³¹I equaled to 1.

The labeled D-D3 antibody was separated from unbound reactants by chromatography on a Sephadex G-50 column (Amersham Phamacia Biotech) and eluted with phosphate-buffered saline (0.05 M, pH 7.5). Column fractions were collected at an elution rate of 0.2 mL/minute (2.5 minutes/fraction). The radiochemical purity was determined by thin-layer chromotography.

The labeled D-D3 was incubated in 37°C water bath for 2, 4, 6, 10, 12, and 24 hours in either the presence or absence of healthy human serum separately. Then, the radiochemical purity was further determined.

Flow cytometric analysis for ProGRP expression in tumor cells

After being washed with the flow cytometry staining medium (phosphate-buffered saline, 2% BSA, and 0.2% NaN₃) at room temperature, SCLC NCI-H446 cells, gastric cancer SGC-7901 cells, pancreatic cancer Patu8988 cells, and lung adenocarcinoma A549 cells were collected and permeabilized with 0.01% Triton X-100 for 1 hour at room temperature, respectively. The cells were then incubated with 2 μL (1 μg/μL) D-D3 antibody overnight. Then, the cells were incubated with FITC-mouse IgG₁ isotype antibody for 1 hour at room temperature. Finally, the relevant data were collected by flow cytometry (FACSCalibur; Becton Dickinson) and analyzed with Cell Quest Analysis Software.

Pharmacokinetic study of ¹³¹I-D-D3 in normal Kunming mice

Fifty (50) healthy Kunming mice (Experimental Animal Center, Soochow University, Suzhou, China), including half males and half females, with a body weight of about 30 g, were divided into 10 groups randomly; 0.1 mL (1.48 × 10^–1 MBq) of ¹³¹I-D-D3 was injected into each mouse via a tail vein, and all mice were sacrificed at 5, 15, and 30 minutes and at 1, 2, 4, 8, 12, 24, and 48 hours after injection, respectively. The blood, heart, spleen, lung, kidney, intestine, bone, muscle, and brain samples were collected and weighed. Then, the radioactivity counts (cpm) were determined. Finally, the %ID/g for various organs was calculated.

Population pharmacokinetic analyses were performed using nonlinear mixed effect model program, NONMEM (version V, level 1.1). The concentration time course of ¹³¹I-D-D3 was well described by a two-compartment model with first-order absorption.

Establishment of tumor-bearing nude mice

SCLC NCI-H446 cells (laboratory stock) were maintained regularly. Cells at log phase were collected and prepared in single-cell suspension at a concentration of 2 × 10⁷ per mL in DMEM. The single-cell suspension (0.2 mL) was injected subcutaneously into the right armpits of each BALB/c-neu nude mice (Shanghai Experimental Animal Center, Shanghai, China), which were 3–4 weeks old and half male and half female, with an average body weight of 20 g. The recipient mice were kept in a sterile incubator and were observed regularly for general condition, food intake, and tumor growth. The mice were used for further studies when the xenograft grew to 1.5 × 1.5 × 1.0 cm³.

Nude mice bearing SGC-7901 gastric cancer, Patu8988 pancreatic cancer, or A549 lung adenocarcinoma xenografts were established accordingly.

In vivo biodistribution study of ¹³¹I-D-D3 in nude mice bearing SCLC xenografts

Thirty (30) nude mice bearing SCLC xenografts were randomly divided into six groups and each mouse was injected with 0.1 mL of ¹³¹I-D-D3 (0.148 MBq) via a tail vein. All nude mice were sacrificed at 1, 24, 48, 72, and 96 hours after injection. The blood, heart, spleen, lung, kidney, small intestine, bone, muscle, brain, and tumor samples were collected and weighed. Then, their radioactivity counts (cpm) were determined. Finally, the %ID/g was calculated for each time point. The radioactivity count ratio of tumor/nontumor tissue (T/NT) at different times was also determined.

Radioimmunoimaging study

Five (5) nude mice of each group of SCLC, gastric cancer, Patu8988 pancreatic cancer, and lung adenocarcinoma models were fed with 0.1% KI in 5% glucose solution at 3 days before the imaging study to saturate the thyroid; 0.2 mL of ¹³¹I-D-D3 (7.4 MBq) was injected into each mouse via a tail vein, and the static planar imaging was performed at 1, 4, 8, 12, 24, 48, 72, and 96 hours, using the Axis Dual Head SPECT (Philips Healthcare). The static planar images were collected at a preset 1000 kcts, a magnification of 1.6, and a matrix of 64 × 64. The ratio of tumor to the contralateral corresponding normal tissue (T/B ratio) was then calculated by ROI technique.

Statistical analysis

Differences in the mean values (the %ID/g of ¹³¹I-D-D3 in nude mice bearing SCLC xenografts, T/NT ratio, and T/B ratio) were evaluated statistically by the SPSS 13 program (SPSS, Inc.). Probability values <0.05 were considered significant (Student's t-test).

Results

Labeling, isolation, purification, and stability of ¹³¹I-D-D3

The labeling rate of ¹³¹I-D-D3 was 86.56% ± 3.8%; the radiochemical purity was 99.27% ± 0.6%; the specific radioactivity was 2.25 ± 0.05 MBq/μg. The radiochemical purity of ¹³¹I-D-D3 was 97.64% ± 0.5% after being incubated in 37°C water bath for 12 hours, and it remained at 88.38% ± 0.4% after 48 hours. The radiochemical purity was more than 64% at 24 hours after being mixed with healthy human serum.

ProGRP expression ratio in tumor cells

Intracellular flow cytometric analysis indicated that NCI-H446 (SCLC cell line) and SGC-7901 (gastric cancer cell line) cells expressed high levels of ProGRP, which were 88.63% and 77.95%, respectively. However, Patu8988 (pancreatic cancer cell line) and A549 (lung adenocarcinoma cell line) cells expressed low levels of ProGRP, which were 40.83% and 15.56%, respectively.

Pharmacokinetic study of ¹³¹I-D-D3 in healthy Kunming mouse

The %ID/g of ¹³¹I-D-D3 in healthy Kunming mouse for various organs at different times is summarized in Table 1. ¹³¹I-D-D3 was distributed mainly in the liver, lung, and kidney, but not in the intestine, bone, muscle, and brain. The metabolism of ¹³¹I-D-D3 in healthy Kunming mice was consistent with a two-compartment model with first-order absorption. The metabolism formula c = 50.234^e–2.793t + 26.128^e–0.018t was calculated through regression analysis. The half-life of fast-phase and slow-phase clearance was 0.25 hours (T _1/2α), and 37.89 hours (T _1/2β), respectively.

Table 1.

Biodistribution Data for ¹³¹ I-D-D3 in Healthy Kunming Mice

	Time after injection of ¹³¹I-D-D3
Organs (%ID/g)	5 minutes	15 minutes	30 minutes	1 hour	2 hours	4 hours	8 hours	12 hours	24 hours	48 hours
Heart	12.4 ± 2.3	9.6 ± 2.5	7.8 ± 2.2	6.8 ± 2.1	6.5 ± 2.4	6.6 ± 1.9	6.4 ± 1.2	5.8 ± 2.1	3.4 ± 1.7	2.8 ± 1.0
Liver	23.1 ± 2.6	20.8 ± 4.4	17.3 ± 4.7	9.4 ± 2.8	9.7 ± 2.8	7.8 ± 2.9	6.4 ± 1.5	6.1 ± 1.6	4.6 ± 1.2	3.8 ± 0.8
Spleen	16.5 ± 3.6	12.7 ± 2.8	12.5 ± 2.2	9.7 ± 2.8	9.1 ± 1.8	6.1 ± 1.2	4.9 ± 1.1	3.8 ± 0.9	3.7 ± 1.1	2.8 ± 1.1
Lung	24.8 ± 7.3	23.5 ± 6.5	19.8 ± 4.2	10.7 ± 3.8	9.2 ± 2.2	7.8 ± 2.3	6.4 ± 1.7	5.2 ± 1.3	3.8 ± 1.0	2.6 ± 1.1
Kidney	23.9 ± 3.1	21.6 ± 2.7	19.1 ± 2.1	13.9 ± 2.9	12.2 ± 2.6	11.8 ± 2.1	10.3 ± 1.5	8.5 ± 1.7	7.0 ± 1.6	5.7 ± 1.1
Stomach	10.4. ± 2.2	9.2 ± 1.8	8.7 ± 1.9	7.5 ± 1.8	6.8 ± 1.3	6.2 ± 1.5	5.9 ± 1.3	5.1 ± 1.6	4.6 ± 1.4	4.2 ± 1.5
Intestine	8.3 ± 2.2	6.7 ± 2.0	6.5 ± 1.5	6.5 ± 1.6	6.1 ± 2.4	5.8 ± 1.9	5.2 ± 1.4	4.8 ± 1.3	4.0 ± 1.4	2.6 ± 0.7
Bone	12.8 ± 2.6	11.7 ± 2.7	9.9 ± 2.4	9.6 ± 2.3	8.3 ± 2.1	7.8 ± 2.3	7.3 ± 2.1	6.6 ± 1.8	5.4 ± 1.4	4.2 ± 1.0
Muscle	6.3 ± 1.4	4.8 ± 1.5	3.8 ± 0.9	3.6 ± 0.7	2.9 ± 1.-	2.6 ± 1.1	2.4 ± 0.9	1.6 ± 0.4	1.5 ± 0.6	1.4 ± 0.3
Brain	5.7 ± 1.8	3.6 ± 1.3	3.6 ± 1.2	3.1 ± 0.7	2.6 ± 1.0	2.5 ± 1.1	2.3 ± 0.8	2.3 ± 0.3	1.9 ± 0.5	1.3 ± 0.4
Blood	64.9 ± 10.1	54.3 ± 7.7	34.2 ± 6.6	31.4 ± 8.6	25.6 ± 4.8	24.2 ± 3.7	21.5 ± 3.4	20.3 ± 2.9	17.6 ± 2.3	10.9 ± 2.2

In vivo biodistribution of ¹³¹I-D-D3 in nude mice bearing SCLC xenografts

At 48 hours after injection of ¹³¹I-D-D3, the %ID/g of tumor mass in SCLC-bearing nude mice was much higher than that of other organs/tissues significantly, which reached the peak level at 72 hours (Table 2). The T/NT ratio of tumor to other organs/tissues gradually increased with time, and the T/NT was more than 1.0 at 48 hours for most organs except tumor/blood (Table 3).

Table 2.

Biodistribution Data for ¹³¹ I-D-D3 in Nude Mice Bearing Small Cell Lung Cancer Xenografts

	Time after injection of ¹³¹I-D-D3
Organ (%ID/g)	1 hour	24 hours	48 hours	72 hours	96 hours
Heart	11.2 ± 2.6	7.3 ± 2.3	5.7 ± 1.7	3.6 ± 1.4	3.5 ± 1.1
Liver	21.1 ± 5.5	12.5 ± 3.8	9.6 ± 2.4	5.5 ± 1.2	4.7 ± 1.1
Spleen	16.3 ± 4.3	11.7 ± 3.1	8.6 ± 2.9	6.5 ± 2.3	5.3 ± 1.8
Lung	14.6 ± 3.9	7.6 ± 2.4	6.1 ± 2.1	5.4 ± 1.2	3.5 ± 2.2
Kidney	14.9 ± 4.6	6.9 ± 1.4	6.2 ± 1.2	4.4 ± 1.8	3.1 ± 1.3
Stomach	7.2 ± 2.7	4.8 ± 1.6	4.1 ± 1.2	2.7 ± 0.9	2.4 ± 0.9
Intestine	10.9 ± 2.8	9.2 ± 3.7	7.6 ± 2.5	5.3 ± 1.8	3.8 ± 1.0
Brain	4.7 ± 1.1	3.4 ± 1.4	3.1 ± 1.1	3.2 ± 1.4	3.1 ± 1.1
Bone	22.9 ± 7.2	12.4 ± 5.4	9.9 ± 2.3	6.8 ± 1.9	6.3 ± 2.2
Muscle	6.5 ± 2.1	5.8 ± 1.7	5.5 ± 1.2	5.6 ± 1.6	5.5 ± 1.9
Tumor	5.9 ± 1.3	9.7 ± 2.1	15.3 ± 3.3^a	21.6 ± 4.5^a	15.7 ± 3.2^a
Blood	56.6 ± 8.8	18.1 ± 6.2	9.6 ± 3.8	7.7 ± 2.2	6.9 ± 1.8

Compared with other organs, p < 0.01.

Table 3.

Tumor/Nontumor Tissue Ratio in Nude Mice Bearing Small Cell Lung Cancer Xenografts

	Time after injection of ¹³¹I-D-D3
Organ	1 hour	24 hours	48 hours	72 hours	96 hours
Tumor/heart	0.95 ± 0.14	1.78 ± 0.46	2.86 ± 0.53	4.78 ± 1.1	3.50 ± 1.12
Tumor/liver	0.45 ± 0.10	1.77 ± 0.31	3.88 ± 0.72	7.19 ± 1.27	5.84 ± 1.82
Tumor/spleen	0.56 ± 0.08	1.50 ± 0.25	3.60 ± 0.46	6.60 ± 1.47	6.31 ± 0.64
Tumor/lung	0.36 ± 0.15	1.09 ± 0.20	1.77 ± 0.22	3.09 ± 0.66	2.96 ± 0.58
Tumor/kidney	0.31 ± 0.12	1.94 ± 0.45	2.87 ± 0.46	4.34 ± 0.33	3.69 ± 1.1
Tumor/stomach	0.59 ± 0.11	0.88 ± 0.30	1.23 ± 0.61	2.08 ± 0.38	1.77 ± 0.62
Tumor/intestine	0.49 ± 0.11	1.93 ± 0.40	3.96 ± 0.51	7.01 ± 1.89	6.26 ± 1.18
Tumor/brain	2.03 ± 0.55	9.29 ± 1.08	12.65 ± 1.17	21.79 ± 2.70	18.94 ± 3.01
Tumor/bone	0.45 ± 0.07	1.63 ± 0.45	2.46 ± 0.30	4.09 ± 1.26	2.99 ± 0.87
Tumor/muscle	1.03 ± 0.13	2.01 ± 0.39	3.13 ± 0.62	5.64 ± 0.86	5.16 ± 0.79
Tumor/blood	0.19 ± 0.02	0.62 ± 0.16	0.95 ± 0.15	2.10 ± 0.48	1.58 ± 0.39

Radioimmunoimaging study

As demonstrated in Figure 1, at 1 hour after injection of ¹³¹I-D-D3, the tumor mass in nude mice bearing SCLC xenografts started to show up on the imaging study. For the time being, the radioactivity uptake gradually increased at the tumor mass and the resolution with the surrounding normal tissue also gradually increased. At 24 hours, the tumor mass was apparent on imaging and reached the best resolution at 72 and 96 hours. This result is consistent with that of the in vivo biodistribution of ¹³¹I-D-D3 in nude mice bearing SCLC xenografts.

FIG. 1.

Sequence image of mouse in the prone position with NCI-H446 small cell lung cancer tumors at 1, 4, 8, 12, 24, 48, 72, and 96 hours after injection of 7.4 MBq ¹³¹I-D-D3 (arrows indicate the location of tumor mass). Preset 1000 kcts, zoom 1.6, matrix 64 × 64.

Similarly, the imaging result of the nude mice bearing gastric cancer xenografts was comparable to that of SCLC-bearing nude mice (Fig. 2).

FIG. 2.

Sequence image of mouse in the prone position with SGC-7901 small cell lung cancer gastric cancer at 1, 4, 8, 12, 24, 48, 72, and 96 hours after injection of 7.4 MBq ¹³¹I-D-D3 (arrows indicate the location of tumor mass). Preset 1000 kcts, zoom 1.6, matrix 64 × 64.

No apparent accumulation of radioactivity was found in the tumor mass of the nude mice bearing pancreatic cancer xenografts or lung adenocarcinoma xenografts from 1 to 96 hours after injection of ¹³¹I-D-D3 (Figs. 3 and 4).

FIG. 3.

Sequence image of mouse in the prone position with Patu8988 pancreatic cancer at 1, 4, 8, 12, 24, 48, 72, and 96 hours after injection of 7.4 MBq ¹³¹I-D-D3 (arrows indicate the location of tumor mass). Preset 1000 kcts, zoom 1.6, matrix 64 × 64.

FIG. 4.

Sequence image of mouse in the prone position with A549 lung adenocarcinoma at 1, 4, 8, 12, 24, 48, 72, and 96 hours after injection of 7.4 MBq ¹³¹I-D-D3 (arrows indicate the location of tumor mass). Preset 1000 kcts, zoom 1.6, matrix 64 × 64.

The T/B ratios of tumor mass to the corresponding contralateral site at various times are summarized in Table 4. The T/B ratios of SCLS and gastric cancer xenografts progressively increased with time, whereas that of pancreatic cancer or lung adenocarcinoma xenografts remained unchanged. The T/B ratios of SCLS and gastric cancer xenografts were much higher than that of pancreatic cancer or lung adenocarcinoma xenografts from 4 hours after injection (p < 0.05), with the ratio of SCLC being higher than that of gastric cancer from 8 hours (p < 0.05).

Table 4.

T/B Ratio in Nude Mice Bearing Various Tumor Xenografts

	Time after injection of ¹³¹I-D-D3
Types of xenograft	1 hour	4 hours	8 hours	12 hours	24 hours	48 hours	72 hours	96 hours
Small cell lung cancer	1.71 ± 0.37	2.63 ± 0.25^a	3.58 ± 0.37^b	4.79 ± 0.49^b	5.97 ± 0.69^b	6.56 ± 0.46^b	6.76 ± 0.55^b	7.61 ± 0.49^b
gastric cancer	1.39 ± 0.19	2.51 ± 0.23^a	2.37 ± 0.34^a	2.58 ± 0.47^a	2.99 ± 0.63^a	4.61 ± 0.57^a	4.75 ± 0.63^a	5.35 ± 0.58^a
Pancreatic cancer	1.13 ± 0.27	1.12 ± 0.22	1.24 ± 0.32	1.32 ± 0.18	1.29 ± 0.33	1.34 ± 0.29	1.40 ± 0.31	1.33 ± 0.27
Lung adenocarcinoma	1.02 ± 0.25	1.10 ± 0.37	1.03 ± 0.32	1.22 ± 0.46	1.27 ± 0.43	1.19 ± 0.22	1.25 ± 0.31	1.30 ± 0.15

At the same time, when compared with the group of pancreatic cancer and lung adenocarcinoma, p < 0.05.

At the same time, when compared with the group of gastric cancer, pancreatic cancer, and lung adenocarcinoma, p < 0.05.

Discussion

The common methods of labeling antibody include chloramine-T, lipoperoxidase (LPO), and iodogen iodination method. Chloramine-T method is the most widely used method to label antibody because of its high labeling efficiency, good repeatability, and low cost of reagents. Therefore, chloramine-T method was used to label D-D3. The present study showed that the labeling rate of ¹³¹I-D-D3 was 86.56% ± 3.8% and the radiochemical purity was 99.27% ±0.6%. The radiochemical purity of ¹³¹I-D-D3 was 97.64% ± 0.5% at 12 hours after incubation in 37°C water bath and it remained at 88.38% ± 0.4% after 48 hours. After incubation with healthy human serum in 37°C water bath for 24 hours, the radiochemical purity was more than 64%, indicating that ¹³¹I-D-D3 was stable.

The metabolism of ¹³¹I-D-D3 in healthy Kunming mice was consistent with a two-compartment model with first-order absorption. The half-lifes of fast-phase and slow-phase clearance were 0.25 hours (T _1/2α), and 37.89 hours (T _1/2β), respectively. ¹³¹I-D-D3 was distributed in all the tested tissues. Initially, that is, 5 minutes after injection, ¹³¹I-D-D3 was mainly distributed in the liver, lung, and kidney, and the ID%/g of these three organs gradually reduced with time. The quickest reduction occurred at 1 hour after injection, with the ID%/g of the liver, lung, and kidney dropping to 40.69%, 43.14%, and 58.16% of the original level, respectively. Then, the reduction speed was slowed down and maintained at a low level. The radioactivity in intestine, bone, muscle, and brain was maintained at very low levels throughout the entire study. This result indicated that D-D3 antibody was mostly metabolized in liver and kidney, with a quick clearance from the serum and a low uptake by organs/tissues such as the intestine, skeletal, and muscle. These properties are beneficial to radioimmunological imaging studies and other related research for the diagnosis and treatment of diseases.

In vivo biodistribution study of ¹³¹I-D-D3 in nude mice bearing SCLC xenografts demonstrated that the %ID/g of tumor and the T/NT ratio increased with time. At 48 hours, the %ID/g of tumor was much higher than other organs/tissues. The T/NT ratio was more than 1.0 except for tumor/blood. At 72 hours, the %ID/g of tumor and the T/NT ratio reached the peak level, with the T/NT ratio more than 2.0 and the T/NT of tumor to muscle as high as 5.64. Although this ratio slightly reduced after 72 hours, the reduction was not statistically significant (p > 0.05).

As demonstrated in Figure 1, the ¹³¹I-D-D3 mostly accumulated at the trunk including the chest and abdomen at 12 hours after injection, with much less accumulation in the tumor. After 24 hours, apparent uptake by the tumor started to show up and steadily increased with time as reflected by the increased T/B ratio. At 72 and 96 hours, the T/B ratio reached the maximum level with the best imaging result. This result was consistent with in vivo biodistribution of ¹³¹I-D-D3 in nude mice bearing SCLC xenografts. ProGRP is the precursor of GRP, and its serum level reflects the GRP level at both transcriptional and translational levels.^8,9 The ProGRP produced by SCLC is normally stored in the Golgi apparatus and will be released into the tumor tissue, where it binds to the ProGRP receptor on the cell membrane, thereby promoting the proliferation and unlimited growth of tumor cells.^20
–22 ¹³¹I-D-D3 could bind directly with the ProGRP released into the extracellular space of the tumor tissue or could possibly bind to the ProGRP in the Golgi apparatus by being uptaken into the cytoplasm after being degraded into small molecules by various enzymes. Accumulation of ¹³¹I-D-D3 in the tumor could thereby create a clear radioimage.

The ¹³¹I-D-D3 was not accumulated in the xenografts of lung adenocarcinoma, which was consistent with the fact that the adenocarcinoma expressed only very low levels of ProGRP. These results indicated that ¹³¹I-D-D3 could potentially benefit the differential diagnosis of SCLC.

Additionally, the ProGRP is also expressed in gastrointestinal tumors. Studies on the colon cancer cell lines have showed that they universally express ProGRP and ProGRP receptor mRNA that leads to the release of the intracellular Ca²⁺ but not the growth of tumor cells.^6,7 In the present study, the radioimaging study of nude mice bearing gastric cancer xenografts demonstrated that prominent accumulation of ¹³¹I-D-D3 in the xenografts did occur consistent with the expression of ProGRP in these cells.

The present study indicated that ¹³¹I-D-D3 could selectively accumulate in the tumor site of nude mice bearing SCLC xenografts with the best resolution, and the best imaging time is 72 and 96 hours after injection. This result will potentially provide an effective differential diagnosis for SCLC. However, the longer duration needed to obtain the best imaging result is a hindrance to the potential clinical application. Smaller fragments of antibody and labeling with shorter half-life radioisotope such as ⁹⁹Tc^m would be the focus of future research.

Footnotes

Acknowledgments

This study was funded by the Science and Technology Development Planning (Suzhou, China, 2009; Grant ID: YJS0926).

Disclosure Statement

No competing financial interests exist.

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Study on Biodistribution and Radioimmunoimaging of 131 Iodine-Labeled Monoclonal Antibody D-D3 Against Progastrin-Releasing Peptide (31–98) in Tumor-Bearing Mouse

Abstract

Introduction

Materials and Methods

Labeling, isolation, purification, and stability of 131I-D-D3

Flow cytometric analysis for ProGRP expression in tumor cells

Pharmacokinetic study of 131I-D-D3 in normal Kunming mice

Establishment of tumor-bearing nude mice

In vivo biodistribution study of 131I-D-D3 in nude mice bearing SCLC xenografts

Radioimmunoimaging study

Statistical analysis

Results

Labeling, isolation, purification, and stability of 131I-D-D3

ProGRP expression ratio in tumor cells

Pharmacokinetic study of 131I-D-D3 in healthy Kunming mouse

In vivo biodistribution of 131I-D-D3 in nude mice bearing SCLC xenografts

Radioimmunoimaging study

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Labeling, isolation, purification, and stability of ¹³¹I-D-D3

Pharmacokinetic study of ¹³¹I-D-D3 in normal Kunming mice

In vivo biodistribution study of ¹³¹I-D-D3 in nude mice bearing SCLC xenografts

Labeling, isolation, purification, and stability of ¹³¹I-D-D3

Pharmacokinetic study of ¹³¹I-D-D3 in healthy Kunming mouse

In vivo biodistribution of ¹³¹I-D-D3 in nude mice bearing SCLC xenografts