preparation and biodistribution of [67ga]-dtpa-gonadorelin in normal rats

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DOI: 10.1007/s10967-007-7241-9 Journal of Radioanalytical and Nuclear Chemistry, Vol. 278, No.1 (2008) 123–129 0236–5731/USD 20.00 Akadémiai Kiadó, Budapest © 2008 Akadémiai Kiadó, Budapest Springer, Dordrecht Preparation and biodistribution of [ 67 Ga]-DTPA-gonadorelin in normal rats A. R. Jalilian, 1 * S. Shanehsazzadeh, 2 M. Akhlaghi, 1 J. Garousi, 1 S. Rajabifar, 1 M. B. Tavakoli 2 1 Nuclear Medicine Group, Agriculture, Medicine and Industrial Research School (AMIRS), Karaj, P.O. Box, 31485-498, Iran 2 Department of Biomedical Physics and Engineering, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran (Received December 20, 2007) Gonadorelin was successively labeled with [ 67 Ga]-gallium chloride after residulation with freshly prepared cyclic DTPA-dianhydride. The best results of the conjugation were obtained by the addition of 1 mg of a gonadorelin to a glass tube pre-coated with DTPA-dianhydride (0.33 mg) at 25 °C with continuous mild stirring for 1 hour. Radio thin layer chromatography showed an overall radiochemical purity of >90% at optimized conditions after labeling. HPLC showed a radiochemical purity more than 95% (specific activity = 400–450 GBq/M). The stability of the radioconjugate was tested in presence of human serum at 37 °C. Preliminary in vivo studies in normal rats were performed to determine the biodistribution of the conjugate up to 48 hours. The breast and ovaries uptakes were significantly high in first 15-minute post injection which is in agreement with the other reports regarding the presence of specific GnRH receptors. This tracer can be used in detection of GnRH receptor biodistribution in various diseases and malignancies. Introduction GnRH is the central regulator of the reproductive hormonal cascade and was first isolated from mammalian hypothalamus as the decapeptide (pGlu-His- Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.NH 2 ). 1–3 GnRH is processed in hypothalamic neurons from a precursor polypeptide by enzymic processing and packaged in storage granules that are transported down axons to the external zone of the median eminence. 4,5 Various studies have shown that synthetic analogues of the hypothalamic hormone luteinizing the hormonereleasing hormone (LH-RH), namely gonadorelin, have a therapeutic effect on sex steroid-dependent tumors such as carcinoma of the prostate 6 and breast. 7,8 High affinity GnRH receptors (GnRH-R) have also been demonstrated in different normal and human cancer tissues including breast cancer, 9,10 pancreatic, 11 endometrial 12 and ovarian carcinomas. 13 A high percentage of estrogen receptor-negative breast tumours has been shown to be positive for the GnRH receptor 14 and this, together with data on the anti-proliferative effect of GnRH agonists on breast cancer cells in culture 15,16 could propose the use of radiolabeled GnRH agonists as tumor imaging agents. Few reports on the production and use of radiolabeled gonadorelin analogs are available. 125 I- Gonadorelin has been prepared and used in study of receptor affinity of mutated GnRH receptors, 17 permeability of LHRH into the arterial blood, 18 protein binding determination, 19 detection of pituitary and testis GnRH receptors 20 and the study of receptor stimulation mechanisms. 21 The only reported attempt to design and synthesis a GnRH analog for tumor imaging was made to create a suitable probe labeled with 99m Tc. 22 * E-mail: [email protected] Gonadorelin is a synthetic GnRH agonist used in the induction of the pituitary gland to release other hormones including luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Gonadorelin is also used to evaluate the hypothalamus and pituitary glands function. In order to obtain a gonadorelin radiolabeled conjugate for use in imaging/biodistribution studies of GnRH receptors in normal and malignant models, 67 Ga- labeled gonadorelin was prepared. A precise labeling strategy was employed using DTPA cyclic dianhydride, with various gonadorelin concentrations and used the available 67 Ga for optimization of radiolabeling conditions. Preliminary biodistribution studies were performed using scarification methods. Experimental Production of 67 Ga was performed at the Agriculture, Medicine and Industrial Research School (AMIRS), 30 MeV cyclotron (Cyclone-30, IBA). Enriched 68 Zn chloride with an enrichment of >95% was obtained from Ion Beam Separation Department at AMIRS. Sephadex G-50, sodium acetate, phosphate buffer components, methanol and ammonium acetate were purchased from Sigma-Aldrich Chemical Co. (U.K.). Gonadorelin was a pharmaceutical sample generously provided by Cinnagen Co, Tehran, Iran. Radio thin layer chromatography (RTLC) was performed by counting different 5 mm slices of polymer-backed silica gel paper and/or C 18 thin layer sheets using a thin layer chromatography scanner, Bioscan AR2000, Bioscan Europe Ltd., Paris, France. To determine the specific activity analytical HPLC was performed by a Shimadzu LC-10AT, armed with two

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Page 1: Preparation and biodistribution of [67Ga]-DTPA-gonadorelin in normal rats

DOI: 10.1007/s10967-007-7241-9 Journal of Radioanalytical and Nuclear Chemistry, Vol. 278, No.1 (2008) 123–129

0236–5731/USD 20.00 Akadémiai Kiadó, Budapest © 2008 Akadémiai Kiadó, Budapest Springer, Dordrecht

Preparation and biodistribution of [67Ga]-DTPA-gonadorelin in normal rats

A. R. Jalilian,1* S. Shanehsazzadeh,2 M. Akhlaghi,1 J. Garousi,1 S. Rajabifar,1 M. B. Tavakoli2 1 Nuclear Medicine Group, Agriculture, Medicine and Industrial Research School (AMIRS), Karaj, P.O. Box, 31485-498, Iran

2 Department of Biomedical Physics and Engineering, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

(Received December 20, 2007)

Gonadorelin was successively labeled with [67Ga]-gallium chloride after residulation with freshly prepared cyclic DTPA-dianhydride. The best results of the conjugation were obtained by the addition of 1 mg of a gonadorelin to a glass tube pre-coated with DTPA-dianhydride (0.33 mg) at 25 °C with continuous mild stirring for 1 hour. Radio thin layer chromatography showed an overall radiochemical purity of >90% at optimized conditions after labeling. HPLC showed a radiochemical purity more than 95% (specific activity = 400–450 GBq/M). The stability of the radioconjugate was tested in presence of human serum at 37 °C. Preliminary in vivo studies in normal rats were performed to determine the biodistribution of the conjugate up to 48 hours. The breast and ovaries uptakes were significantly high in first 15-minute post injection which is in agreement with the other reports regarding the presence of specific GnRH receptors. This tracer can be used in detection of GnRH receptor biodistribution in various diseases and malignancies.

Introduction

GnRH is the central regulator of the reproductive hormonal cascade and was first isolated from mammalian hypothalamus as the decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.NH2).1–3 GnRH is processed in hypothalamic neurons from a precursor polypeptide by enzymic processing and packaged in storage granules that are transported down axons to the external zone of the median eminence.4,5 Various studies have shown that synthetic analogues of the hypothalamic hormone luteinizing the hormonereleasing hormone (LH-RH), namely gonadorelin, have a therapeutic effect on sex steroid-dependent tumors such as carcinoma of the prostate6 and breast.7,8

High affinity GnRH receptors (GnRH-R) have also been demonstrated in different normal and human cancer tissues including breast cancer,9,10 pancreatic,11 endometrial12 and ovarian carcinomas.13 A high percentage of estrogen receptor-negative breast tumours has been shown to be positive for the GnRH receptor14 and this, together with data on the anti-proliferative effect of GnRH agonists on breast cancer cells in culture15,16 could propose the use of radiolabeled GnRH agonists as tumor imaging agents.

Few reports on the production and use of radiolabeled gonadorelin analogs are available. 125I-Gonadorelin has been prepared and used in study of receptor affinity of mutated GnRH receptors,17 permeability of LHRH into the arterial blood,18 protein binding determination,19 detection of pituitary and testis GnRH receptors20 and the study of receptor stimulation mechanisms.21 The only reported attempt to design and synthesis a GnRH analog for tumor imaging was made to create a suitable probe labeled with 99mTc.22

* E-mail: [email protected]

Gonadorelin is a synthetic GnRH agonist used in the induction of the pituitary gland to release other hormones including luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Gonadorelin is also used to evaluate the hypothalamus and pituitary glands function.

In order to obtain a gonadorelin radiolabeled conjugate for use in imaging/biodistribution studies of GnRH receptors in normal and malignant models, 67Ga-labeled gonadorelin was prepared. A precise labeling strategy was employed using DTPA cyclic dianhydride, with various gonadorelin concentrations and used the available 67Ga for optimization of radiolabeling conditions. Preliminary biodistribution studies were performed using scarification methods.

Experimental

Production of 67Ga was performed at the Agriculture, Medicine and Industrial Research School (AMIRS), 30 MeV cyclotron (Cyclone-30, IBA). Enriched 68Zn chloride with an enrichment of >95% was obtained from Ion Beam Separation Department at AMIRS. Sephadex G-50, sodium acetate, phosphate buffer components, methanol and ammonium acetate were purchased from Sigma-Aldrich Chemical Co. (U.K.). Gonadorelin was a pharmaceutical sample generously provided by Cinnagen Co, Tehran, Iran. Radio thin layer chromatography (RTLC) was performed by counting different 5 mm slices of polymer-backed silica gel paper and/or C18 thin layer sheets using a thin layer chromatography scanner, Bioscan AR2000, Bioscan Europe Ltd., Paris, France. To determine the specific activity analytical HPLC was performed by a Shimadzu LC-10AT, armed with two

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detector systems, flow scintillation analyzer (Packard-150 TR) and UV-visible (Shimadzu) using Whatman Partisphere C-18 column 250×4.6 mm2, Whatman, NJ, USA. Calculations were based on the 184 keV peak for 67Ga. All values were expressed as mean ± standard deviation (Mean ± SD) and the data were compared using student T-test. Statistical significance was defined as P<0.05. Animal studies were performed in accordance with the United Kingdom Biological Council's Guidelines on the Use of Living Animals in Scientific Investigations, 2 nd ed.

Production of 67Ga

68Zn(p,2n)67Ga was used as the best nuclear reaction for the production of 67Ga. Impurities could be removed by the radiochemical separation process. After the target bombardment process, chemical separation was carried out in no-carrier-added form. The irradiated target was dissolved in 10M HCl (15 mL) and the solution was passed through a cation exchange resin (AG 50W, H+ form, mesh 200–400, h: 10 cm, Ø: 1.3 cm) which had been preconditioned by passing 25 mL of 9M HCl. The column was then washed by 25 mL of 9M HCl at a rate of 1 mL/min to remove copper and zinc ions. To the eluent 30 mL water plus about 100 mL of a 6M HCl solution was added. The latter solution was loaded on another exchange resin (AG1X8 Cl– form, 100–200 mesh, h: 25 cm, Ø: 1.7 cm) pretreated with 6M HCl (100 mL). Finally, the 67Ga was eluted as [67Ga]GaCl3 using 2M HCl (50 mL); the whole process took about 60 minutes.

Quality control of the product

Control of radionuclide purity: Gamma-spectroscopy of the final sample was carried out by counting by a HPGe detector coupled to a Canberra™ multi-channel analyzer for 1000 seconds.

Chemical purity control: The presence of zinc and copper cations were checked by polarography. Even at 1 ppm of standard zinc and copper concentrations, the area under the curve of the polarogram of the test samples were lower than the standards.

Conjugation of cyclic DTPA di-anhydride with gonadorelin

The chelator diethylenetriamine penta-acetic acid dianhydride was conjugated to gonadorelin using a small modification of the well-known cyclic anhydride method.23 Conjugation was performed at a 1 : 1.1 (peptide : ccDTPA) molar ratio. In brief, 20 µL of a 1 mg.mL–1 (0.033 mg, 92 µM) suspension of DTPA anhydride in dry chloroform (Merck, Darmstadt, Germany) was pipetted under ultrasonication and

transferred to a glass tube. The chloroform was evaporated under a gentle stream of nitrogen. Commercially available gonadorelin (0.1 mg, 85 µM 0.5 mL, pH 7.5) was subsequently added and gently mixed at room temperature for 60 minutes. Conjugation reaction was checked using TLC with methanol:chloroform mixture as eluent.

Radiolabeling of gonadorelin conjugate with 67Ga

The gonadorelin conjugate was labeled using an optimization protocol according to the literature.24 Typically, 37–40 MBq of 67Ga-chloride (in 0.2M HCl) was added to a conical vial and dried under a flow of nitrogen. To the 67Ga containing vial, conjugated fraction was added in 1 mL of phosphate buffer (0.1M, pH 8) and mixed gently for 30 seconds. The resulting solution was incubated at room temperature for 30 minutes. Following incubation, the radiolabeled conjugate was checked using RTLC method for purity. Control labeling experiments were also performed using 67GaCl3, and DTPA with 67GaCl3.

Quality control of 67Ga-DTPA-gonadorelin

Radio thin layer chromatography: A 5 µL sample of the final fraction was spotted on a chromatography C18 sheet paper, and developed in a mixture of 10 mM DTPA in double-distilled H2O as the mobile phase.

High performance liquid chromatography: HPLC was performed on the final preparation using acetate buffer solution (50 mM pH 5.5) as eluent (flow rate: 1 mL/min pressure: 130 kg/cm2) for 20 minutes in order to elute low molecular weight components. Radiolabeled peptide was eluted using a gradient of the latter solution (100 to 0%) and citrate buffer solution (50 mM, pH 4, 0 to 100%) using reverse stationary phase. Any remaining free Ga3+ cation can be complexed with citrate anion, however, pre-complexed 67Ga-DTPA-gonaderelin was not challenged with the addition of citrate chelate.25

Stability testing of the radiolabeled compound

Stability of 67Ga-DTPA-gonadorelin in PBS was determined by storing the final solution at 4 °C for 7 days and performing RTLC analysis to determine radiochemical purity. Frequent RTLC analysis was performed. Furthermore, the stability of the conjugated DTPA-gonadorelin stored at –20 °C for more than 1 month was investigated. RTLC analysis of the conjugated product was performed to monitor degradation products or other impurities. After subsequent 67Ga-labeling of the stored conjugated product, both labeling efficiency and radiochemical purity were determined.

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Stability testing of the radiolabeled compound in presence of human serum

Labeled compound stability in freshly prepared human serum was assessed by RTLC using the solvent system used for labeling step control.

Biodistribution of 67Ga-DTPA-gonadorelin in normal rats

To determine its biodistribution, 67Ga-DTPA-gonadorelin was administered to normal rats (NMRI) purchased from Razi Institute, Karaj, Iran. A volume (50 µL) of final 67Ga-DTPA-gonadorelin solution containing 4.4–5.2 MBq radioactivity was injected intravenously to rats through their tail vein. The animals were sacrificed at specific time intervals, and the specific activity of different organs was calculated as percentage of injected dose per gram of the tissue (ID/g%) (based on the area under the curve of 184 keV peak using an HPGe detector).

Discussion

Conjugation of gonadorelin with DTPA cyclic di-anhydride and radiolabeling of DTPA-gonadorelin with 67Ga

The labeling yield of 67Ga-DTPA-gonadorelin has been studied in the wide range of gonadorelin/DTPA ratios in order to optimize the process and to improve 67Ga-DTPA-gonadorelin performance in vitro. We could not confirm the real structure of the peptide conjugate by spectroscopic methods such as 1HNMR or 13CNMR, and since there is no lysine moiety in the peptide structure that is being usually considered as a source of nucleophilic NH2 to attack ccDTPA. The only possible explanation would be the conjugation of the peptide via NH2 of arginine moiety, which is not usually considered as a suitable conjugation site.

The other possibility could be an un-specific 67Ga labeling via many nitrogen atoms at the histidyl-guanidyl part. This was tested by the direct addition of unconjugated peptide to 67Ga and incubation at room temperature as a control reaction. An unspecific radiolabeling (5–8%) was observed. The overall radiolabeling efficiency was over 77% and the specific activity was kept in the range of 400–450 MBq/M (Fig. 1).

Small fractions were taken from this mixture and tested by RTLC to find the best time scale for labeling. After one hour, free 67Ga/conjugated 67Ga ratio in the labeled sample remained unchanged. Figure 2 shows the RTLC chromatogram of free 67Ga and 67Ga-DTPA as control experiments and Figure 3 demonstrates the RTLC of final 67Ga-DTPA-gonadorelin sample at the optimized conditions.

In HPLC studies using reverse phase column HPLC chromatogram using a gradient of acetate/citrate buffer, the fast eluting component (2.62 min) was shown to be a mixture of free 67Ga and 67GaDTPA. Both compounds are ionic, so they are eluted at the same retention time. The radiolabeled peptide was finally washed out at 18.31 minutes (Fig. 4).

Stability of radiolabeled peptide in vitro

The stability of the radiolabeled peptide in vitro was determined after challenge with phosphate-buffered saline and serum. TLC analysis showed that the peptide retained the radiolabel over a period of one hour, indicating that the Ga-peptide chelate was of low stability.

There was no evidence for either degradation or transchelation of 67Ga to other serum peptide over a time period (1 hour) consistent with the normal blood clearance time of gonadorelin. However, this data cannot demonstrate the real biological half life of the tracer since the enzymatic degradation of the tracer cannot be studied by this method.

Fig. 1. Peptide sequence of gonadorelin

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Fig. 2. RTLC of 67Ga and 67GaDTPA in 10 mM DTPA in DDH2O as mobile phase and C18 stationary phase

Fig. 3. RTLC of 67GaDTPA-gonadorelin in 10 mM DTPA in DDH2O as mobile phase and C18 stationary phase

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Fig. 4. HPLC chromatogram of final radiolabeled solution on a reversed phase column using a gradient of acetate/citrate buffer

Fig. 5. Bio-distribution of 67Ga-DTPA-gonadorelin in normal rats 15 minutes to 48-hour post-injection

Biodistribution studies

The distribution of [67Ga]-GaCl3 and 67Ga-DTPA-gonadorelin among tissues was determined for untreated rats. A volume (0.1 mL) of tracer solutions containing 4.4–5.2 MBq radioactivity was injected into the dorsal tail vein. The total amount of radioactivity injected into each rat was measured by counting the

1-mL syringe before and after injection in a dose calibrator with a fixed geometry. The accumulation of radioactivity in case of Ga3+ cation is mostly incorporated in long bones such as femor (>40%), while sternum, lung and stomach show low but significant uptakes (about 5% each), the accumulation in other organs are not significant (data not shown).

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Fig 6. Bio-distribution of 67Ga-DTPA-gonadorelin in other organs (excluding lung and urine) in normal rats 15 minutes to 48-hour post-injection

In the case of 67Ga-DTPA-gonadorelin, a

significantly different pattern is observed, the accumulation of the tracer in ovaries and breast is significant (>5%) at 15 minutes which is in agreement with the natural distribution of the GnRH receptors. After 20 minutes, the uptakes of urinary excretion increased rapidly. This behavior is not in agreement with the distribution of radiolabeled peptides and is believed to be mostly related to gallium containing peptide metabolites or complexes such as 67GaDTPA. Since the biological half life of gonadorelin is very short, (4 minutes after IV injection), the degradation of the peptide by serum peptidases can result in free forms of gallium leading to urinary tract accumulation of the activity. However, there are reports from the accumulation of radiolabled gonadorelin in the liver and kidney. The peptide is rapidly metabolized into smaller inactive peptides and amino acids and excreted through kidneys26 (Figs 5 and 6).

Conclusions

The total labeling and formulation of 67Ga-DTPA-gonadorelin took about 70 minutes. A suitable specific activity product was formed via insertion of 67Ga cation. No other labeled conjugates were observed upon RTLC and/or HPLC analysis of the final preparations. The radio-labeled complex was stable in human serum for at least one hour and no significant amount of free 67Ga as well as 67Ga-DTPA was observed. A radiochemical purity of higher than 95% was detected by HPLC with a specific activity of 400–450 MBq/M. Fifteen minutes after administration of radiolabeled gonadorelin, the

activity is accumulated in ovaries, breast as well as kidneys, all in accordance with reported GnRH receptor biodistribution and metabolized peptides. This tracer can be used in various malignancies associated with over-expression of gonadorelin receptors 15 minutes post injection.

*

Authors wish to thank Ms. S. MORADKHANI and Mr. S. DANESHVARI for conducting animal studies. Authors also would like specially thank to IAEA for technical supports under IRA-2007 project. Finally special thanks to Dr. Hale HAMEDIFAR and Cinnagen Company, Tehran, Iran, for their generous gift.

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