Distribution,Elimination,and Renal Handling of 99m Technetium-Demogastrin 1

Abstract

Radiolabeled cholecystokinin/gastrin (CCK) receptor-targeting peptides are promising compounds for radiodiagnosis and radiotherapy of certain malignancies. This study evaluated the pharmacokinetic profile of a CCK-2 receptor-specific peptide, Demogastrin 1, labeled with technetium-99m (^99mTc-Demogastrin 1), in rats. To investigate the fate of ^99mTc-Demogastrin 1 in the rat, biodistribution and elimination studies in vivo were performed, and elimination parameters in perfused rat liver and kidney were determined. Biodistribution studies showed that ^99mTc-Demogastrin 1 was rapidly cleared from the blood and most organs. A significant amount of radioactivity was detected in the CCK-2 receptor-rich organs, such as the stomach. Low radioactivity was found in the CCK-1 receptor-rich organs. Radioactivity in bowels and stomach declined relatively slowly. High and long-term retention of radioactivity in the kidneys was observed. Elimination of ^99mTc-Demogastrin 1 via the bile was negligible. A high and rapid renal excretion was observed in elimination experiments in vivo. In the perfused kidney, glomerular filtration was found to be the main renal excretion mechanism of ^99mTc-Demogastrin 1. Demogastrin 1 was distributed preferentially to the organs expressing CCK-2 receptors. The decisive elimination route of ^99mTc-Demogastrin 1 in rats was urinary excretion. A high and prolonged renal retention may limit potential clinical use of the compound.

Introduction

Radiolabeled stable analogs of selected regulatory peptides can be used for scintigraphic imaging^1,2 or, more recently, for treatment of some malignancies.^3,4 Somatostatin receptor-specific peptides have gained an important place in clinical practice, because the somatostatin analog ¹¹¹In-DOTA-octreotide (Octreoscan^®) is used routinely in the diagnosis of somatostatin-positive neuroendocrine tumors.⁵ In addition, results of preclinical and clinical studies have shown that somatostatin analogs labeled with radionuclides emitting alpha or beta particles, or Auger or conversion electrons could be useful in radiotherapy of some tumors.⁶

Other receptor types, however, seem to be the target for diagnosis and therapy of some oncologic diseases. One group of candidates potentially useful in oncology may be compounds with affinity to cholecystokinin/gastrin (CCK) receptors. A high interest was stimulated by reports on high and frequent expression of CCK-2 receptors in human medullary thyroid cancer.⁷ In addition, the expression of CCK-2 receptors has been also documented in small-cell lung cancers, stromal ovarian cancers, astrocytomas,^8,9 and in several other types of human tumors.^10,11

To develop CCK-2-selective radiopharmaceuticals suitable for in vivo targeting of tumors (especially medullary thyroid cancer) in patients, two groups of radiolabeled compounds have been tested. One group is based on CCK octapeptide (CCK-8) analogs.^12
–14 The second group, named minigastrins, is based on diethylenetriaminepentaacetic acid (DTPA)- or DOTA-linked derivatives of gastrin.^15
–17 Many compounds from both groups have been shown to be useful for tumor targeting because of their high CCK-2 receptor selectivity, high affinity, and favorable pharmacokinetic profile.^14,16

Many structure modifications in the CCK-2 receptor-specific peptide group were aimed at conjugation of the receptor-specific part to an optimal chelator enabling effective labeling with technetium-99m (^99mTc) because this radionuclide is very useful for targeted diagnosis in nuclear medicine. Besides hydrazinonicotinamide (HYNIC) derivatives,^18,19 conjugates of [(D)-Glu¹]minigastrin and an open-chain tetraamine chelator have been developed by Nock and associates²⁰ to achieve stable binding of ^99mTc. The authors coupled a tetraazaundecane chelator system to the N-terminus of minigastrin either directly or via different spacers. The developed minigastrin analogs showed a high affinity to CCK-2 receptors and were rapidly internalized in CCK-2 receptor-expressing cells. Furthermore, they were effective to specifically target CCK-2 receptor positive tumors in mice and man.²⁰

Several clinical studies have demonstrated that a majority of medullary thyroid cancer primary tumors and metastases are visualized in vivo with CCK-2 receptor scintigraphy.^20
–22 In addition, radiotherapy of CCK-2 receptor expressing medullary thyroid cancers with a radiolabeled minigastrin has been reported in a small number of patients.²³ These findings give support to the proposal that CCK-2 receptors overexpressed in tumors represent a useful target for clinical application.

In this work, we evaluated two important pharmacokinetic characteristics—distribution and elimination—of the minigastrin-conjugate CCK-2 receptor-specific peptide, [N₄ ⁰,(D)Glu¹]-minigastrin (Demogastrin 1), labeled with ^99mTc (^99mTc-Demogastrin 1). Special attention was paid to renal handling of the radiopeptide under study, because renal excretion seems to be the main elimination pathway. A significant renal uptake of the radiolabeled peptides, including the minigastrin analogs, may result in radiotoxicologic injury of the kidney and limit their possible clinical use.^14,24 To investigate the fate of ^99mTc-Demogastrin 1 in the organism, biodistribution and elimination studies in vivo were performed. In addition, the renal and liver excretion of the compound was studied at the organ level using perfused rat liver and kidney.

All the experiments with animals were approved by the Ethical Committee of the Pharmaceutical Faculty, Charles University in Prague, and were performed in compliance with the respective Czech laws concerning animal protection.

Materials and Methods

Chemicals

Synthesis of Demogastrin 1, [N₄ ⁰,(D)Glu¹]-(Glu)₅-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH₂, has been reported.²⁰

Polyfructosan (Inutest, Laevosan, Linz, Austria) was used as a marker of glomerular filtration rate and sodium salt of bromosulfophthalein (Sigma, St. Louis, MO) as the marker of the liver function. All the other chemicals used were of analytical grade.

Radiolabeling and quality control

For radiolabeling of Demogastrin 1 with ^99mTc and for quality control, the procedures described by Nock and colleagues²⁰ were used. Shortly, to 10 μL 0.5 M phosphate buffer, 1 μL of citrate buffer, 81 μL of freshly eluted pertechnetate solution of activity about 2.5 to 5 mCi/mL, and 3 μL peptide solution (in 10 mM acetic acid/ethanol 1:1) corresponding to 7.5 μg of the peptide, were added. Subsequently, 5 μL of freshly prepared ethanol suspension (1 mg/mL) of stannous chloride dihydrate was added and, after stirring, left for 15 minutes at room temperature. After that, additional 2 μL of the same stannous chloride suspension was added. After a total of 30 minutes of incubation, the pH of the solution was neutralized by adding 2 μL 1M HCl. For biologic experiments, labeled peptide was dissolved in physiologic solution to a concentration of 1 μg/mL. For high performance liquid chromatography (HPLC) analysis, 2 μL of preparation was dissolved to 100 μL of mobile phase A.

Quality control of the labeled peptide was performed using gradient HPLC analysis on Pharmacia LKB system with Waters SymetryShield RP₁₈, 3.9mm×15 mm, 5 μm column. The mobile phase comprised solvent A: 0.2% H₃PO₄ neutralized by 25% ammonium hydroxide solution to pH 7, and solvent B: acetonitrile. Gradient elution was conducted as follows: 0 to 5 minutes 0%B; 5 to 35 minutes 0% to 30%B; 35 to 55 minutes 30%B, at a flow rate of 1 mL/min.

Distribution and elimination in vivo

The radiolabeled peptide (1 μg/kg) was administered to male Wistar rats (weight 180–220 g) into the tail vein in a volume of 0.2 mL (dose of radioactivity at time of administration 1.0–2.1 MBq/kg). The animals were then housed in individual cages. At selected time points after administration (5 minutes, 1 hour, 2 hours, 24 hours, and 48 hours), the carotid artery of each rat was exposed under total anesthesia and a blood sample was collected. After exsanguination, selected organs were taken out to determine the distribution of ^99mTc-activity.

For an elimination study in vivo, the peptide was administered to rats as described for the biodistribution study. The animals were separately placed in glass metabolic cages where separation of urine and faeces is possible. For the urine collection, the rats were forced to empty their urinary bladder by handling (immobilization) 2 hours after injection and urine and feces were taken. The animals were placed into the same cages again and urine and feces were repeatedly taken out at 24-hour and 48-hour intervals using the same withdrawal procedure.

Perfused rat kidney

Male Wistar rats weighing 280 to 330 g were used for perfusion experiments. The perfusion was performed according to the procedure described previously.²⁵ Shortly, the rat kidney was perfused with 100 mL of Krebs-Henseleit buffer 7.4 containing glucose (5.6 mmol/L), bovine serum albumin, fraction V (6%), washed rat erythrocytes (5%–6%), and a mixture of amino acids and metabolic substrates. The kidney was perfused via the renal artery under a recirculation regimen at 37°C and a constant pressure of 14.5 kPa (110 mm Hg). After an equilibration period, ^99mTc-Demogastrin 1 (0.2 μg; radioactivity 0.1–0.3 MBq) was added into the perfusion circuit as a single dose, and the kidney was perfused for an interval of 60 minutes. Urine samples were collected every 10 minutes, and midpoint samples of perfusate were obtained. As a marker of glomerular filtration rate (GFR), polyfructosan renal clearance was used. The concentrations of polyfructosan in the perfusate and urine were determined colorimetrically.²⁶

Rates of elimination of the peptide in the perfused rat kidney were characterized by total renal clearance (CL_TR=urinary activity×volume of urine/activity in perfusate), and by the filtered amount of the compounds (CL_GF=GFR×free fraction in the perfusate).

Binding of the peptide to the proteins in the perfusion medium was determined by ultrafiltration across a semipermeable membrane (Priesvit, Chemosvit, Slovak Republic) for 20 minutes at 2000 g.²⁷

Perfused rat liver

A perfused rat liver technique has been described in detail.²⁸ After midline incision of the animal, the bile duct, the portal vein, and the inferior vena cava were cannulated and ligated. Krebs-Henseleit buffer pH 7.4 with glucose in a single-pass mode was used to release the blood from the liver. The liver was perfused at 37°C with the perfusate consisted of a heparinized Krebs-Henseleit bicarbonate buffer pH 7.4 with 10 mM glucose, 4% bovine serum albumin, and 10% (v/v) bovine erythrocytes oxygenated with a 95% O₂ and 5% CO₂. After an equilibration period (15 minutes), the flow of the perfusate was kept at 25 mL/min, and the peptide under study was added to a 150 mL reservoir. The samples of the input and outflow perfusate were repeatedly removed at 10-minute intervals in the middle of the 10-minute periods of bile collection. The experiments were performed for 90 minutes after the peptide loading. Bromosulfophthalein was added in an amount of 30 nmol/mL perfusate as the marker of the liver function.

Radioactivity measurement

The ^99mTc-activity in biologic samples was measured by a gamma-counter Wallac 1480 Wizard 3 (Wallac, Turku, Finland) in a comparison with three standard radioactivity samples.

Results

Radiolabeling and quality control

HPLC analysis of the radiolabeled peptide showed radiochemical purity higher than 99%. Because of the high yield, no other purification step was needed. Specific activity of the prepared and checked radiopeptide was 0.03 to 0.06 mCi/μg.

Distribution and elimination in vivo

Distribution of radioactivity after intravenous (i.v.) administration in selected organs at 5 minutes, 1 hour, 2 hours, 24 hours, and 48 hours after ^99mTc-Demogastrin 1 administration to rats is presented in Table 1. Whereas rapid disappearance of radioactivity from the blood and most organs was found, the activity in bowels and stomach lowered relatively slowly. A relatively high activity level was found in the kidney. The maximum was achieved between 1 and 2 hours after injection, and radioactivity remained high even 48 hours after administration.

Table 1.

Distribution Of Radioactivity (%ID/g) After Intravenous Administration of ^99M Technetium-Demogastrin 1 at 1 μg/kg Dose (Mean±Standard Deviation; N=4)

Organ	5 minutes	60 minutes	120 minutes	24 hours	48 hours
Blood	1.03±0.21	0.12±0.04	0.03±0.01	<0.01	0
Plasma	1.99±0.41	0.22±0.08	0.04±0.01	<0.01	0
Pancreas	0.34±0.09	0.05±0.01	0.02±0.01	0	0
Liver	0.29±0.05	0.1±0.02	0.07±0.02	0.03±0.01	0.02±0.01
Adrenals	0.37±0.08	0.06±0.02	0.02±0.01	0	0
Kidney	12.77±2.11	16.93±1.75	17.03±2.66	9.91±1.89	5.67±0.52
Lung	0.62±0.03	0.09±0.03	0.05±0.04	0.01±0.01	0
Heart	0.43±0.09	0.06±0.02	0.01±0.01	0	0
Spleen	0.26±0.03	0.06±0.01	0.03±0.01	0.01±0.01	0.01±0.01
Stomach	0.50±0.11	0.41±0.10	0.51±0.71	0.12±0.03	0.07±0.03
Intestine	0.29±0.04	0.17±0.12	0.22±0.31	0.06±0.02	0.02±0.02
Colon	0.13±0.04	0.03±0.01	0.03±0.02	0.56±0.12	0.30±0.18
Testes	0.11±0.01	0.05±0.01	0.01±0.01	0	0
Skin	0.42±0.06	0.09±0.03	0.02±0.01	0.01±0.01	0
Muscle	0.22±0.04	0.03±0.01	0.01±0.01	<0.01	<0.01
Thyroid	0.78±0.04	0.11±0.04	0.03±0.01	0.01±0.01	0.02±0.02
Brain	0.04±0.01	0.01±0.01	0	0	0
Fat	0.40±0.15	0.07±0.02	0.01±0.01	0.01±0.01	0.03±0.03
Femur	0.22±0.04	0.08±0.02	0.04±0.01	0.02±0.01	0.01±0.01

Results of the elimination study in rats are summarized in Table 2, where the data are given as percentages of radioactivity excreted in urine and feces after i.v. administration of the labeled peptide up to the selected interval. The results show that ^99mTc-Demogastrin 1 is eliminated in rats predominantly via the urine. The small part of the activity found in feces documents the minor role of the hepatobiliary excretion route.

Table 2.

Cumulative Excretion of Radioactivity (% ID) After Administration of 99M-Technetium-Demogastrin 1 (Mean±Standard Deviation; N=4)

0–2 hours		0–24 hours		0–48 hours
Urine	Feces	Urine	Feces	Urine	Feces
50.8±9.0	-	67.8±7.7	3.0±1.3	73.1±7.3	6.5±4.5

Handling in perfused rat kidney

Renal clearance parameters of ^99mTc-Demogastrin 1 in the perfused rat kidney are presented in Table 3. No significant changes in renal clearance of the radiotracer during 60 minutes of perfusion were found (not shown). More than 50% of the agent was unbound to proteins in the perfusion medium (Table 3). The renal clearance corrected to protein binding was nonsignificantly lower, but comparable to the total renal clearance of the agent. Table 3 also presents renal retention parameters as the radioactivity remaining in the kidney and the ratio of kidney/perfusate determined at the end of the perfusion.

Table 3.

Excretion Parameters and Retention of 99M-Technetium-Demogastrin 1 In the Perfused Rat Kidney (Mean±Standard Deviation; N=4)

Parameter	Value
Renal clearance (mL/min/g)	0.398±0.056
Glomerular filtration rate (mL/min)	0.769±0.086
Free fraction in perfusate	0.557±0.057
Filtration clearance (mL/min/g)	0.438±0.052
Radioactivity of the kidney at the end of the perfusion (% dose)	23.5±2.7
Ratio of kidney/perfusate radioactivity (g/mL)	35.2±1.7

Handling in perfused rat liver

Bile clearance of radioactivity in the perfused rat liver was very low (0.0011±0.0003 mL/min) in comparison with the perfusate flow (25.0 mL/min). This indicated negligible elimination of the agent (and/or its metabolites) via the hepatobiliary route.

Discussion

^99mTc-Demogastrin 1 belongs to peptides with high affinity to CCK-2 receptors.²⁰ Because of its high affinity to CCK-2 receptors, it is expected to accumulate predominantly in CCK-2 receptor-rich organs. ^99mTc-Demogastrin 1 exhibited rapid clearance from blood and from most organs and tissues. We detected, however, a significant amount of radioactivity in the CCK-2 receptor-rich organs, such as the stomach. More than 1% of radioactivity was further found only in liver. Negligible radioactivity in the brain, which belongs to another CCK/gastrin receptor-rich organ,¹⁵ can be explained by the inability of the radiotracer to cross the blood-brain barrier. Low radioactivity found in the pancreas confirms that ^99mTc-Demogastrin 1 has low affinity to CCK-1 receptors. Especially important is the low uptake of ^99mTc-Demogastrin 1 in normal thyroid, which is a prerequisite for imaging of medullary carcinoma lesions in the thyroid gland. A pattern of distribution similar to that of ^99mTc-Demogastrin 1 was observed in [¹¹¹In-DTPA⁰]minigastrin in tumor-bearing mice.¹⁶ [¹¹¹In-DTPA⁰]minigastrin had a rapid clearance from the blood and blood-pools organs with mainly renal and some biliary excretion. In contrast to ^99mTc-Demogastrin 1, a prolonged retention in the adrenals was observed in the [¹¹¹In-DTPA⁰]minigastrin.¹⁶

The total contribution of the hepatobiliary route to ^99mTc-Demogastrin 1 excretion in rats is low. In the perfused rat liver, elimination of ^99mTc-Demogastrin 1 via the bile was negligible. Contrary to these results, in the biodistribution studies, we observed radioactivity uptake in the liver and intestine immediately after administration. Some radioactivity was detected also in the feces of rats. A contribution of secretion of the agent into the intestine might be considered.

The decisive elimination route for ^99mTc-Demogastrin 1 in vivo was urinary excretion. The ^99mTc-Demogastrin 1 renal excretion can be characterized as almost exclusive and rapid, because only a very small part was excreted in feces and more than 50% of radioactivity was excreted via the urine within the first 2 hours after administration. As documented in the biodistribution study, unexcreted radioactivity was predominantly found in the kidneys, and its elimination was much slower. The dominant role of renal excretion of ^99mTc- Demogastrin 1 is consistent with its hydrophilic nature.

To analyze the renal handling of ^99mTc-Demogastrin 1, the method of the perfused rat kidney was used. This method enables analysis of kidney elimination mechanisms under the conditions when the effect of extrarenal organs and tissues is eliminated and the kidney function characteristics are only slightly altered.²⁹ The filtration clearance of ^99mTc-Demogastrin 1 found in the perfused rat kidney was nonsignificantly lower than the total renal clearance. It means that glomerular filtration is the decisive renal excretion mechanism in the agent under study, and any substantial contribution of tubular secretion to renal excretion is not probable. Any comparable experimental data on renal excretion parameters and excretion mechanisms in the kidney for other gastrin analogs are not available. In smaller peptides, such as ^99mTc-labeled tetrapeptides, net tubular secretion has been demonstrated.²⁵ On the other hand, any significant tubular secretion in a series of radiolabeled peptides from the group of somatostatin analogs was found.^28,30

We observed high and long-term retention of radioactivity in the kidneys in the distribution study. Approximately 30% of injected radioactivity was found in the kidneys 1 hour after administration. Considerable radioactivity administered to rats was retained in the kidneys even after 24 and 48 hours. This is comparable to more than 20% of the added dose accumulated in the perfused kidney during 60 minutes of perfusion. The mechanism of renal accumulation is usually explained as a process including several transport and metabolic steps. The accumulated radiolabeled compounds are primarily filtered in the glomerulus and subsequently partly reabsorbed into the cells of proximal tubules. In the next step, the agents are transferred into lysosomes by means of pinocytosis and degraded by proteolytic enzymes. Breakdown products, namely radiolabeled chelate-conjugated amino acids, cannot leave the lysosomes and remain trapped in the proximal tubular cells.^24,31 The renal retention of ^99mTc-Demogastrin 1 is a feature similar to other minigastrins such as [¹¹¹In-DTPA⁰,(D)Glu¹]-minigastrin.^32,33 In addition, some derivatives of CCK such as ¹¹¹In-labeled or radioiodinated nonsulfated CCK-2 receptor-specific CCK-8 analogs also exhibit various levels of long-term renal retention.¹⁶ Conversely, some radioiodinated gastrin derivatives including minigastrins exhibited relatively lower renal retention. They had very high uptake in the gallbladder of mice, however.¹⁶ Newly synthetized cyclic minigastrin analogs labeled with ^99mTc exerted relative lower renal retention¹⁹ than in the case of ^99mTc-Demogastrin 1. The in vivo stability of the cyclic minigastrin derivatives, however, seems to be suboptimal.¹⁴

A prolonged renal retention of a radiolabel is one of the main problems in the therapeutic application of radiolabeled receptor-specific peptides, because nephrotoxicity represents a serious health hazard. High renal uptake of labeled minigastrins may be compared with the group of labeled somatostatin analogs in which undesirable high renal retention of ⁹⁰Y-labeled compounds resulting in radionephrotoxicity has been observed.^34,35 Similarly, nephrotoxicity developed in patients who were treated with [⁹⁰Y-DTPA⁰,(D)Glu¹]-minigastrin after the radiopeptide therapy.^{14,23 99m}Tc-labeled minigastrins for diagnostic purposes are probably not as radiotoxic to the kidney as the beta-emmiters. Rather, the high renal retention may cause difficulties in detection of oncologic lesions in the abdominal area. Minimalization of the renal accumulation of radiolabeled minigastrins, however, seems to be an important prerequisite for their potential use in receptor-based radiotherapy.

Conclusions

Distribution studies confirmed preferential distribution of ^99mTc-Demogastrin 1 to the organs expressing CCK-2 receptors in comparison with the CCK-1 receptor-rich organs. The peptide did not enter the brain in a significant amount. The experiments in vivo and in the perfused rat liver showed very low excretion of the studied radiopeptide via the liver. The decisive elimination route of ^99mTc-Demogastrin 1 in rats was urinary excretion. In the perfused kidney, glomerular filtration was found to be the main renal excretion mechanism of ^99mTc-Demogastrin 1. A significant participation of reabsorption or secretion in the renal tubules was not proved. The observed high and prolonged renal retention may limit potential clinical use of the compound because of a risk of radionephrotoxicity.

Footnotes

Acknowledgments

The authors thank Mrs. I. Filipova, Mrs. J. Hoderova, and Mrs. Teichmanova for their excellent technical assistance.

The work originated in collaboration within COST BM0607 and was supported by a grant of the Ministry of Education of the Czech Republic No. OC 08006, by the grant of the Czech Science Foundation No. P304/10/1738, and by grant No. 124409/FaF/C-LEK of the Grant Agency of Charles University.

Disclosure Statement

No competing financial interests exist.

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