Radiosynthesis and in vivo evaluation of the pseudopeptide δ-opioid antagonist [125I]-ITIPP(ψ)

Nuclear Medicine and Biology 28 (2001) 375–381

Radiosynthesis and in vivo evaluation of the pseudopeptide ␦-opioid antagonist [125I]-ITIPP(␺) T.L. Colliera,1,*, P.W. Schillerb, R.N. Waterhousea,2 a

Department of Radiology, Duke University Medical Center, Durham, NC 27710, USA b Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7

Received 30 January 2000; received in revised form 8 August 2000; accepted 2 December 2000

Abstract The radioiodinated tetrapeptide ␦-opioid antagonist [125I]ITIPP(␺) [H-Tyr(3⬘I)-Tic␺[CH2NH]Phe-Phe-OH] (Ki(␦) ⫽ 2.08 nM; Ki(␮)/ Ki(␦) ⫽ 1280) has been synthesized and evaluated as a potential lung tumour imaging agent. [125I]ITIPP(␺) was obtained, via electrophilic iodination, in 46% yield (⬎44,000 MBq/␮mol) from the parent TIPP(␺). The biodistribution of [125I]ITIPP(␺) in nu/nu mice bearing SCLC-SW210.5 xenographs revealed good uptake and prolonged retention of radioactivity in organs known to possess ␦-opioid receptors. Metabolite analysis showed that [125I]ITIPP(␺) was largely unmetabolized at 25 min PI and blocking studies showed significant reduction of uptake of the tracer in the brain, liver, intestine and tumor indicating that the iodinated tetrapeptide binds to ␦ opioid receptors in vivo. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Delta-opioid antagonist; TIPP(␺); Iodine-125; Radiotracers

1. Introduction Early detection and staging are essential in the management of lung cancer and detection of the recurrence of lung cancer. It has been stated that the monitoring of patients following treatment of a primary lung neoplasm would allow for the earlier detection of locally recurrent disease and timely therapeutic intervention [6]. Positron emission tomography (PET) imaging with [18F]fluoro-2-deoxy-Dglucose (FDG) has recently become an important modality to evaluate pulmonary abnormalities, which may be indeterminate by conventional imaging studies. Diagnosis based on histopathology requires bronchoscopy, percutaneous biopsy or open lung biopsy in order to differentiate benign from malignant lesions [16]. PET has a relatively high sensitivity for detecting malignant lesions of the lung, al* Corresponding author. Tel.: ⫹1-212-305-2267; fax: ⫹1-212-3054648. E-mail address: [email protected] (T.L. Collier). 1 Current address: Division of Cardiology, Department of Medicine, College of Physicians and Surgeons, Columbia University, PH 3–342, 630 W. 168th St., New York, New York 10032. 2 Current address: Brain Imaging Division, Department of Neuroscience, New York State Psychiatric Institute, Columbia University, New York, New York 10032.

though its specificity is less than optimal [3,9,10,23]. Thallium-201 has also been used successfully to identify patients with lung carcinoma [16], although lesions less than 1.0 cm in diameter have been falsely determined to be negative [21]. Thus the search continues for a more accurate SPECT imaging agent for differentiating benign from malignant lesions, detecting smaller lesions and also for determining the stage of disease. Due to the accessibility of single photon emission computed tomography (SPECT) to a large number of nuclear medicine centers, a lung cancer imaging agent labelled with gamma emitting isotope, such as 123I, 111 In, or 99mTc would be invaluable. Recent reports have demonstrated high affinity binding sites (10 –300 fmol/mg protein) for delta (␦) opioid receptors on several small cell lung cancer (SCLC) cells lines and their absence normal human lung tissue [2]. It was therefore hypothesised that the ␦-opioid receptor might be an appropriate target for SCLC lung cancer imaging. Currently several radioiodinated peptides targeted against the ␦-opioid receptor are commercially available for in vitro studies, however, they are relatively expensive and are poor candidates for SPECT studies due to their rapid in vivo metabolism [18]. In addition, non-peptidic ligands directed against the same receptor, including radioiodinated analogs of naltrindole [11] and (⫹)-4-[(␣R)-␣-[(2S,5R)-4-(allyl)-

0969-8051/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 1 ) 0 0 1 9 3 - 7

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Table 1 In vitro binding properties of selected ␦ opioid ligands Ligands

Ki ␦ (nM)

Ki ␮ (nM)

Ki ␬ (nM)

Selectivity

Reference

TIPP (⌿) [Tyr(3⬘-I)] 1 TIPP(⌿) TIPP-NH2 [D-Pen2,D-Pen5]enkephalin (DPDPE) (⫹)SNC 80 BW373U86 Naltrindole 7-Iodonatrindole C6-O-iodoallyldiprenorphine [D-Ala2]deltophin II [Leu5]enkephalin

0.308 2.08 3.00 2.9 2.88 1.49 0.182 0.22 1.1 6.43 2.53

3230 2660 78.8 82 2467 9.71 3.86

⬎5000 ⬎5000 ⬎1000 ⬎350

10500 1280 ⬃26 ⬎28 856 6.5 21.2

1.9 3930 9.43

0.9

[26] [26] [26] [26] [1] [1] [26] [14] [15] [26] [26]

2,5-dimethyl-1-piperazinyl]-3-[123I]iodo-benzyl]-N,N-diethylbenzamide [7], have also been reported as potential SPECT imaging agents (Table 1). However, to date there have been no reports describing clinically effective ␦-opioid radiopharmaceuticals for SPECT. To provide an effective ␦-opioid-selective SPECT radioligand, we have evaluated the iodinated derivative of the pseudopeptide TIPP(␺) [27]. The parent compound TIPP(␺) is a highly ␦-selective opioid peptide when examined in traditional binding assays and was completely stable for up to 24 hours in enzymatic degradation studies. Therefore, it was thought that ITIPP(␺) might be useful for in vivo studies [26]. The initial report of the binding properties and pharmacological activities of H-Tyr(3⬘-I)-Tic-Phe-Phe-OH (ITIPP(␺)) lead to the hypothesis that ITIPP(␺) would still possess sufficient binding to be useful as an imaging agent. In addition, the data showed that monoiodination at the 3⬘-position of the N-terminal tyrosine aromatic ring of TIPP converted it from a potent and selective antagonist to a full agonist at delta-opioid receptors. ITIPP(␺) was reported to retain its activity as a potent and selective antagonist [25]. Herein is reported the radiosynthesis of [125I]ITIPP(␺) [H-Tyr(3⬘I)-Tic␺[CH2NH]Phe-Phe-OH] 2 and its characterisation in mice bearing SCLC SW210.5 tumor xenographs.

2. Materials and methods 2.1. Syntheses All reagents were purchased from commercial sources and were used without further purification. Sodium 125Iiodide was obtained from MDS Nordion, Kanata, Canada as a solution in sodium hydroxide (0.1 N). TIPP(␺) was obtained from the National Institute for Drug Abuse. HPLC analysis of the radioligand was performed using a Waters 515 HPLC pump, a Linear 210 UV detector, and a radiation detector consisting of an EG&G Acemate amplifier, sodium iodide photomultiplier. The columns used included a semipreparative reversed-phase column (Alltima C-18, 10 ⫻

611 3.7

250 mm, 10 ␮m particle size, 90 Å) and an analytical reversed-phase column (Alltima C-18, 4.6 ⫻ 250 mm, 5 ␮m particle size, 90 Å). The mobile phases used are indicated in the text below. 2.2. Radiolabeling [125I][H-Tyr(3⬘-125I)-Tic(␺)[CH2NH]Phe-Phe-OH], [125I]TIPP(␺), [125I]TIPP(␺), [125I]-2: Four IODOBEADS® were added to a 3.0 ml Wheaton vial followed by 500 ␮l of phosphate buffer (0.1 M, pH ⫽ 7.02) [24]. This solution was allowed to stand for 5 minutes and then it was removed and 0.5 ml of fresh phosphate buffer was added and the cap applied to the vial. This was immediately followed by the injection of 25 ␮l of [125I]iodide in 0.1 M NaOH (300 ␮Ci), which was allowed to stand for 2 minutes. After two minutes, a solution of TIPP(␺) 䡠 bis(trifluoroacetate) 1 in distilled water (100 ␮l, 1 ␮g/␮l), was injected to the reaction vial and was gently agitated for 10 minutes. 1–2 ␮L aliquots of the reaction mixture were removed at 1 minute intervals and were analysed by thin layer chromatography (silica gel, 0.1% trifluoroacetic acid in acetonitrile: 0.1% trifluoroacetic acid in water (10:1 V/V)) to determine the % radiochemical incorporation. 5 ␮L aliquots were removed at 1 minute intervals and diluted with 50 ␮L of the HPLC mobile phase. The diluted aliquots were then analysed by HPLC to also determine % incorporation and radiochemical purity of the reaction. The reaction mixture was removed using the HPLC injection syringe and a fresh aliquot of phosphate buffer (250 ␮l) was added and the reaction vessel subsequently agitated. This wash was combined with the reaction mixture and was injected onto the HPLC system (mobile phase: 0.1% trifluoroacetic acid in acetonitrile:0.1% trifluoroacetic acid, 30:70, 4.0 ml/min) to provide 138 ␮Ci (46% EOS) of the desired radiotracer. The radiochemical purity of the product was ⬎99% as determined by analytical HPLC analysis. The purified radioligand co-eluted with the standard (ret. time ⬇ 24 min) when a spiked aliquot of the purified product was analysed using identical HPLC conditions.

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vein injection to 10 –14 week old male mice in a total volume of no more than 100 ␮l of sterile saline. During injections, no anaesthesia was used and a perspex rodent restrainer restrained the mice. At selected times post-injection of the radioligand, the mice were sacrificed by an overdose of CO2 at selected timepoints between 30 minutes and 12 hours and various organs (e.g. brain, liver, tumour, muscle, blood, lung, thyroid, kidneys) were weighed and evaluated for the presence of the radioactivity using a gamma counter. The percent injected dose (%ID) for each organ was calculated by comparison of a diluted standard solution of the initial injected dose. The density of radioactivity of each organ (%ID/g) was found by dividing the %ID for each tissue by the weight of the tissue. All mice were allowed food and water ad lib throughout the experiment. 2.6. Statistical analysis

Scheme 1. Scheme for the radiosynthesis of

I-ITIPP(␺) from TIPP(␺).

125

Statistical significance was evaluated using ANOVA and an unpaired students T-test. The criterion for significance was p ⬍ 0.05.

2.3. Specific activity determination The specific activity of the product was determined by HPLC analysis using the analytical column and a mobile phase consisting of 0.1% trifluoroacetic acid in acetonitrile: 0.1% trifluoroacetic acid in water (30:70), with a flow rate of 1.0 ml/min. The limit of detection of 2 was determined by plotting the mass of 2 injected versus UV detector response at 220 nm. The detection limit was determined to be the response of the detector providing a peak height 2.5 times the noise level. From extrapolation, this response corresponded to 60 picomole of 2 injected. Upon analysis of 15 ␮Ci of 125I-2 (in saline), no UV peak was detected and the specific activity of 2 was determined to be ⬎1,200 mCi/ ␮mol (⬎44,000 MBq/␮mol). To obtain suitable preparations of 125I-2 for use in vivo, the eluted radioactive peak corresponding to 125I-2 was collected, the mobile phase removed in vacuo and the product redissolved in saline (0.9% NaCl, sterile). The saline solution was passed through a 0.22 ␮m sterile filter into an evacuated sterile vial and was diluted as required with 0.9% saline to provide approximately 10 ␮Ci of 125I-2 per 100 ␮l solution. 2.4. Biological Biodistribution studies were carried out according to an approved Duke University Medical Center Animal Care and Use Committee (DUIACUC) protocols. 2.5. In vivo tissue distribution studies Briefly, the radioligand (10 ␮Ci–25 ␮Ci) and blocking agents dissolved in sterile saline were administered via tail

3. Results 3.1. Radiosynthesis Radioiodination of the tyrosine amino acid of TIPP(␺) was performed with a starting activity of approximately 300 ␮Ci 125I-iodide using IODOBEADS® as the oxidant (Scheme 1). The optimum time for the reaction was determined by the removal of aliquots every minute from the reaction mixture, the % 125I incorporation was determined by TLC and HPLC and the optimum reaction time was determined to be 10 minutes. The reaction was terminated by the removal of the reaction mixture from contact with the IODOBEADS®. The resulting solution was purified by HPLC to provide 125I-2 in yields of 46 ⫾ 5% EOS (n ⫽ 3). The specific activity of the IodoTIPP(⌿), 125I-2, was determined by HPLC to be greater than 1,200 mCi/␮mol (⬎44,000 MBq/␮mol). 3.2. In vivo distribution studies The biodistribution of [125I]ITIPP(␺) in nu/nu mice bearing SCLC-SW210.5 xenographs showed uptake of radioactivity in tissues known to express ␦ opioid receptors, including the intestines, kidney, liver, muscle, lung, blood and tumour. Table 1 summarises the uptake of radioactivity in selected organs at several time points post-injection of the radioligand. In addition, the thyroid activity did not increase significantly from 30 minutes to 12 hours, (⬃0.17– 0.47%ID) indicating that the tracer did not undergo rapid deiodination in vivo.

378 Table 2 Regional biodistribution of

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125

I-ITIPP(␺) in nu/nu mice bearing SCLC-SW210.5 xenographs. Data are means of %ID/g tissue ⫾ S.D.; n ⫽ 3

Organ

30 Minutes

1 Hour

4 Hours

12 Hours

Brain Intestines Lung Liver Tumor Thyroid Muscle Blood Kidney

0.79 ⫾ 0.003 19.083 ⫾ 4.375 1.398 ⫾ 0.012 2.956 ⫾ 0.850 0.623 ⫾ 0.022 0.171 ⫾ 0.055 0.433 ⫾ 0.179 3.803 ⫾ 0.531 4.653 ⫾ 0.463

0.067 ⫾ 0.014 28.323 ⫾ 1.038 0.768 ⫾ 0.066 2.099 ⫾ 0.598 0.454 ⫾ 0.022 0.329 ⫾ 0.048 0.207 ⫾ 0.083 2.459 ⫾ 0.086 2.309 ⫾ 0.160

0.037 ⫾ 0.012 19.285 ⫾ 4.955 0.582 ⫾ 0.133 1.297 ⫾ 0.247 0.329 ⫾ 0.015 0.379 ⫾ 0.191 0.199 ⫾ 0.055 0.719 ⫾ 0.050 0.525 ⫾ 0.070

0.002 ⫾ 0.000 0.076 ⫾ 0.009 0.023 ⫾ 0.002 0.053 ⫾ 0.003 0.019 ⫾ 0.003 0.468 ⫾ 0.243 0.010 ⫾ 0.001 0.022 ⫾ 0.002 0.027 ⫾ 0.001

3.3. Blocking studies Pharmacological blocking studies were performed to examine the degree of in vivo specific binding of [125I]ITIPP(␺) in SCLC-SW210.5 xenographs. For these studies, TIPP(␺) (␦, Ki ⫽ 0.308 nM; ␮, Ki ⫽ 2660 nM; ␬, Ki ⫽ ⬎5000 nM) at a dose of either 0.1 mg/Kg or 3 mg/Kg was administered via tail vein injection five minutes prior to the injection of the radioligand. The results of the blocking study are shown in Table 2. A significant reduction of radioactivity was observed, with the 0.1 mg/kg pre-injection, in the intestine and tumour ( p ⬍ 0.003 and 0.028 respectively) which are known to possess ␦ receptors. However, the pre-administration of a larger dose, 3 mg/kg, of TIPP(␺) resulted in a significant decrease in the activity in the brain, tumour, liver and intestine ( p ⬍ 0.014, 0.048, 0.01, and 0.008 respectively). The activity in the lung was observed to decrease by 25%, however this decrease was not significant at the 0.05 level. The activity in the blood increased with the preadministration of a 3 mg/Kg dose of TIPP(␺), however this increase was not determined to be significant and perhaps this increase in blood activity resulted from the displacement of activity from other organs. 3.4. Metabolite studies The tissue was mechanically homogenised and the resulting mixture was extracted with a solution of 0.5 mg/ml TIPP(␺) in acetonitrile. This mixture was centrifuged and the supernatant fraction was separated from the pellet. Both fractions were counted to determine the efficiency of the extraction process. The extract was concentrated under a stream of air to approximately 25 ␮l. A solution of 1 mg/ml of ITIPP(␺), 10 ␮l, was applied to the TLC plate at the origin, followed by the organ metabolite extract to be analysed. This method was found to minimise the streaking of the ITIPP(␺) under the TLC conditions used in this study. The TLC plate was then developed using a solution of 0.1% trifluoroacetic acid in acetonitrile:0.1% trifluoroacetic acid in water (10:1 V/V), allowed to dry and the TLC plate, between the origin and the solvent front, was cut into five

equal sections. The top of the plate, above the solvent front was used as a background control and all of the sections were counted. The results of the metabolite study (Table 4) indicated that in all organs studied the ITIPP(␺) was ⬎73% intact. For the liver, only 51% of the activity was derived from intact ITIPP(␺).

4. Discussion Successful imaging of delta opioid receptors in the human brain has been accomplished by the use of positron emission tomography (PET) imaging agents such as [11C]methylnaltrindole ␦-opioid receptor [18,17] and [11C]-diprenorphine which bind ␮, ␬, and ␦-opioid receptors [19]. Iodinated analogues of naltrindole and N1-methylnaltrindole have been reported, however, the selectivity of these ligands was not as high as that seen for the ITIPP(␺). The reported stability of the psuedopeptide TIPP(␺) [H-TyrTic␺[CH2NH]Phe-Phe-OH] against chemical and enzymatic degradation [22] and its high selectivity for the ␦-opioid receptor over all other opioid receptors, lead us to investigate the potential of the monoiodinated derivative as a SPECT imaging agent [22]. Radioiodination of TIPP(␺) at the 3⬘ position of the terminal tyrosine was easily performed by classical electrophilic iodination methods using Iodobeads®. This method provides ITIPP(␺) in good yield without adversely affecting binding affinity. The high selectivity of this iodinated ligand for the ␦-opioid receptors over the ␮ and ␬-opioid receptors along with its high binding makes it an excellent candidate for use as an imaging agent. Schiller et al. reported that the unreduced form of the peptide, TIPP, Tyr-Tic-Phe-Phe-OH, when iodinated at the 3⬘-position of the Tyr1 aromatic ring converted the highly selective peptide ␦-opioid receptor antagonist into an agonist [13]. The psuedopeptide, TIPP(␺), when iodinated maintained its activity as an antagonist (K e ⫽ 19.8 nM), although with about a seven-fold reduction in affinity (Table 1). The synthesis of radioiodinated ITIPP(␺), 2, conducted under no-carrier-added conditions using IODO-BEADS® and, following the basic conditions outlined in the instructions for use of this iodination reagent [24], provided the

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Table 3 Blocking data on TIPP(␺) on the uptake of 125I-ITIPP(␺) in various mice organs. Injection of 0.1 and 3 mg/Kg of TIPP(␺) five minutes prior to injection of radiotracer. Data are means of from %ID/g tissue ⫾ S.D.; n ⫽ 3. * p ⬍ 0.05 Organ

3 mg/Kg blocking

Brain Tumor Blood Muscle Lung Liver Intestine

0.1 mg/Kg blocking

% Change in uptake with blocking agent

t-test (2 sided)

% Change in uptake with blocking agent

t-test (2 sided)

⫺37.5 ⫺40.1 19.6 ⫺7.1 ⫺25.8 ⫺52.6 ⫺63.4

0.014 0.048 0.721 0.738 0.104 0.010 0.008

⫺11.1 ⫺50.9 ⫺1.5 ⫺2.2 23.4 ⫺3.9 ⫺63.5

0.994 0.003 0.962 0.896 0.712 0.938 0.028

product with a radiochemical yield was 46 ⫾ 5% (Scheme 1). The purity and specific activity of the [125I]-labelled ligand was determined by analytical HPLC. The radiochemical purity was typically ⬎99%, and the specific activity was ⬎44 GBq/␮mol for all experiments. The biodistribution [125I]ITIPP(␺) in male nu/nu mice bearing SCLC-SW210.5 xenographs showed uptake and retention of radioactivity in rodent tissues known to express ␦ opioid receptors, including the intestines, lung, liver, kidney, muscle, blood and tumour. Uptake in these tissues at 30 min post-injection (PI) was 19.1, 1.4, 3.0, 0.6, 0.4, 3.8, and 4.7% ID/g, respectively. There was also a rapid accumulation in the tumor which peaked at 0.6% ID/g at 30 minutes. The blood uptake of the radiotracer peaked at 3.8% ID/g at 30 minutes, and slowly washed out over time. It has been shown that ␦-opioid receptors are present in the blood on immune cells [8,12], and human mononuclear cells (B and T lymphocytes and monocytes) [20] and this may explain the high blood uptake observed. The maximum uptake in the intestine was 28.3% ID/g at one hour, and this washed out over time to 0.076% ID/g at 12 hours. All other organs studied, except the thyroid, decreased to less than 0.053% ID/g at 12 hours. Blocking studies were performed at 30 minutes using two blocking doses of TIPP(␺) (0.1 mg/kg and 3 mg/kg), to determine the amount of specific binding. The blocking agent was dissolved in 0.9% saline and injected 5 minutes prior to the injection of the radiotracer. The effect of the blocking agents on the radiotracer uptake is shown in Table

3. At 0.1 mg/kg the blocking of the radiotracer was significant ( p ⬍ 0.05) for the intestine and tumour only, at 3 mg/kg the blocking of the radiotracer was significant additionally in the brain ( p ⬍ 0.014) and liver ( p ⬍ 0.010). It has been shown that all three opioid receptor subtypes are widely expressed in several peripheral tissues of the rat, and the ␦ receptor was detected in spleen, kidney, liver, adrenal, testis, ovary, non-pregnant uterus, stomach, small and large intestine, lung and heart [28]. The opioid receptor present in skeletal muscle fibers of the mouse was found to be of the delta subtype and the number of these receptors is increased in type II diabetes in the mouse [5]. The results of the metabolite studies (Table 4) at 25 minutes PI indicated that the radioactivity in the blood, intestine, tumour, muscle and brain was essentially from the intact ITIPP(␺) and the activity in the liver was mainly from polar metabolites. Specific binding for [125I]ITIPP(␺) in selected tissues was estimated by subtracting the non-specific binding (results of the 3 mg/kg ITIPP(␺)) from the total binding. Comparison of the %ID/g and the specific binding of [125I]ITIPP(␺), for organs which both specific binding and expression of ␦-opioid receptor mRNA in rat tissue was known [24], showed a correlation for the liver, lung, and intestine (Fig. 1). The brain did not show a correlation, however this may be attributed to the small amount of tracer which was able to cross the blood-brain-barrier.

5. Limitations Table 4 Metabolite analysis of

I-ITIPP(␺) at 30 minutes post injection

125

Organ

% extracted

% intact by tle 30 minutes

Intestines Lung Muscle Blood Tumour Liver Brain

73.6 80 83.1 72.8 86.8 57.6 83.1

75.4 77 73 89.2 77.7 51.4 75

The ligand ITIPP(␺) has been shown to be displaceable by a single selective ␦-opioid receptor ligand. However, furthers studies should be performed to verify that the ligand is not displaced in vivo by ␮- or ␬-opioid ligands or other non-opioid ligands. In the comparison of the known distribution of ␦-opioid receptor and the %ID/g and specific binding of [125I]ITIPP(␺), the only data currently available for the ␦-opioid receptor was for the distribution of transcripts to the ␦-opioid receptor in rat. However, it should be noted that the level of expression of these transcripts in

380

T.L. Collier et al. / Nuclear Medicine and Biology 28 (2001) 375–381

Fig. 1. Plot of %ID/g of 125I-ITIPP(␺) and in vivo specific binding of 125I-ITIPP(␺) versus the distribution of ␦-opioid transcripts in rat brain and selected organs. The specific binding was obtained by multiplying the % specific binding of 125I-ITIPP(␺) and the %ID/g obtained for each organ. The relative intensity of the hybridising band for the ␦-opioid mRNA in the rat for each tissue is indicated by the number of ⫹, and is taken from reference [28].

tissue may not necessarily reflect protein concentrations and may also be species dependent.

␦-opioid receptors in the periphery, the poor brain penetration which is likely due to the high molecular weight of this ligand, may limit its use in imaging disorders of the central nervous system.

6. Conclusions To provide potential probes for the in vivo SPECT examination of ␦-opioid receptor densities, we have prepared H-Tyr(3⬘-125I)-Tic␺[CH2-NH]Phe-Phe-OH ([125I]ITIPP(␺)) as a high affinity selective ␦-opioid receptor ligand and performed an initial in vivo evaluation of the ligand. The biodistribution of [125I]ITIPP(␺) in nu/nu mice bearing SCLC-SW210.5 xenographs showed uptake and prolonged retention of radioactivity in tissues known to express ␦ opioid receptors, including the intestines, lung, liver, kidney, muscle, blood and tumour. Uptake in these tissues peaked at 30 min post-injection (PI) for all organs studied, except the intestines, which peaked at 1 hour. However the high blood uptake in comparison to the tumour (⬃10:1 blood to tumour ratio at 30 minutes), would not allow for the use of this ligand as a tumour imaging agent. Blocking studies with 3 mg/kg TIPP(␺) showed significant reduction of uptake of the tracer in the brain, liver, intestine and tumour (⬃38 – 63%, p ⬍ 0.05), indicating that the iodinated tetrapeptide binds ␦-opioid receptors in vivo. Metabolite studies revealed that [125I]ITIPP(␺) was largely unmetabolized in most organs (⬎73% unchanged) at 30 min PI. In addition, the thyroid activity (0.4% ID) did not increase significantly over the course of the study (12 hours), indicating that the tracer did not undergo rapid deiodination in vivo. While this tracer may be suitable for imaging

Acknowledgments The authors are grateful to the National Institute for Drug Abuse (NIDA) for supplying the researchers with the precursor TIPP(␺). Dr. E.F. Patz Jr and the Department of Radiology, Duke University Medical Center for providing the facilities and for supporting this research and Dr. M.J. Campa for his assistance in the animal distribution studies. Portions of this work were presented at the 13th International Symposium on Radiopharmaceutical Chemistry [4].

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