European Journal of
`
`Nuclear
`Medicine
`
`Original article
`
`Radioiodinated somatostatin analogue RC-160:
`preparation, biological activity,
`in vivo application in rats
`and comparison with [ml-Tyr3]octreotide
`
`W.A.P. Breeman‘, L.J. Hoflancl2, W.H. Bakkerl, M. van der Pluijm‘, P.M. van Koetsveldg, M. de Jong‘,
`B. Setyono-Han3, D.J. Kwekkebooml, T.J. Visserg, S.W.J. Lambertsz, E.P. Krennmgl-2
`1 Department of Nuclear Medicine, University Hospital Dijkzigt. Rotterdam. The Netherlands
`2 Department of Internal Medicine ill, University Hospital Dijkzigi, Rotterdam, The Netherlands
`3 Dr. Daniel den Hoed Cancer Centre, Rotterdam, The Netherlands
`
`Received 1 March and in revised form 20 June 1993
`
`Abstract. We have evaluated the potential usefulness
`of the radioiodinated octapeptidc RC-160, a somatosta-
`tin analogue, which might serve as a radiopham1aceu-
`tical for the in vivo detection of somatostatin receptor-
`positive tumours. For this purpose, iodine-123 and io-
`dine-l25 labelled RC-160 was tested for biological ac-
`tivity and applied in vivo in rats bearing the transplan-
`table rat pancreatic tumour CA20948, which expresses
`somatostatin receptors. Our group has recently de-
`scribed the in vivo visualization of such tumours in rats
`and in humans with the radjoiodinated somatostatin ana-
`
`logue ['l‘yr3]octreotidc. Like [ 1231-Tyr3]octreotidc, ml-
`RC-l60 showed uptake in and specific binding in vivo
`to somatostatin receptor-positive organs and tumours.
`However, blood radioactivity (background) was higher,
`resulting in a lower tumour to blood (background) ratio.
`We therefore conclude that in this animal model
`‘Z31-
`
`RC-l60 has no advantage over [ml-Tyr3]octreotide as
`a radiopharrnaceutical for the in vivo use as a so1nato-
`statin receptor imager, although, like [”3l-Tyr3]octreo-
`tide, ml-RC-160 shows specific binding to different so-
`matostatin recepto1‘—positive organs. Recently differ-
`ences were reported in affinity between sornatostatin
`and its analogues for somatostatin receptors expressed
`in different human cancers, like those Quf the breast,
`ovary, exocrine pancreas, prostate and colon. Therefore
`ml-RC-l60 might be of interest for future use in hu-
`mans as a radiopharrnaceutical for imaging octreotide
`receptor-negative tumours.
`
`Key words: Radioiodinated RC-160 — Somatostatin ~
`Specific binding — Tumour imager — Peptide
`
`Eur J Nucl Med (1993) 20:10S9—1094
`
`Correspondence to: W.A.P. Breeman, Department of Nuclear
`Medicine, University Hospital Dijkzigt, Dr. Molewaterplein 40,
`NL—30l5 GD Rotterdam, The Netherlands
`
`introduction
`
`High numbers of high~affinity somatostatin receptors
`for both native somatostatin (for structure, see Fig.1)
`and the synthetic octapeptide octreotide (Sandostatin)
`have been detected on most endocrine tumours, such as
`endocrine pancreatic tumours and carcinoids [I-4]. We
`have recently described the visualization of sornatosta—
`tin receptor-positive tumours in vivo after the intrave-
`nous administration of [ml-Tyr3Joctreotide [5—-9], and
`these results have been confirmed by others [l0—l2].
`Radioiodinatcd [Tyr3]octIcotidc is frequently used for
`the in vitro determination of the presence of somatosta-
`tin receptors [l3]. Recently, several reports have been
`published on the in vitro binding to somatostatin recep-
`tors of another sornatostatin analogue, the octapeptide
`RC-160 [1-4--16]. It has been reported that RC-160 has
`a higher affinity than octreotide for the somatostatin re-
`ceptor in human breast, ovarian, exocrine pancreatic,
`prostatic and colonic cancers [l4—l6J. Aphase 1 clinical
`trial with RC-160 in patients with advanced exocrine
`pancreatic cancer is being performed, and it appears that
`RC-160 is well tolerated at doses up to 1500 ug/day
`[17, l8]. The possibility of RC-160 binding to a soma-
`tostatin receptor subtype on human exocrine pancreatic
`adcnocarcinoma, which does not bind octreotide [19],
`offers a potential advantage for RC-160 over octreotide
`as a radiolabelled tumour tracer. RC-160 may, in con-
`trast to octreotide [20], also pass the blood-brain barrier
`[21]. This could represent a benefit in visualizing so-
`matostatin receptor-positive brain tumours with an in-
`tact blood-brain barrier. We investigated radioiodinated
`RC-160 (for structure, see Fig. l) for potential use in
`scintigraphy in normal rats and in rats bearing the trans-
`plantable pancreatic somatostatin receptor-positive tu-
`mour CAZO9-48 [13, 19, 22]. A comparison was made
`with [ml-Tyr3]octreotide, and the possible additional
`value of ml-RC-160 as
`a radiopharrnaceutical was
`evaluated.
`
`European Journal of Nuclear Medicine
`Vol. 20, No. 11, November 1993 - © Springcr—Verlag 1993
`
`Roxane Labs, Inc.
`Exhibit 1029
`
`Page 001
`
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`Exhibit 1029
`Page 001
`
`

`
`1090
`
`Somatostatin
`
`I
`I
`Ala-Gly-Cys-Lys~Asn—Phe~Phe—Trp—Lys-Thr—Phe-Thr~Ser—Cys
`
`Octreotide
`
`r*‘—?“"‘——‘s
`D—Phe-Cys-Phe-D—Trp-Lys—Thr—Cys-Thr(ol)
`
`[‘“I—Tyr’]ocI:reotide
`
`r"—*—“‘—“—I
`H-D—Phe—Cys—Tyr—D—Trp-Lys-Thr—Cys-Thr(ol)
`
`1:31
`
`ml-RC—l60
`
`l""‘”j-‘—"‘_-l
`H-D-Phe-Cys-Tyr—D—Trp»Lys—Val~Cys~T1p—NH2
`123
`
`I
`
`Fig. 1. Stuctural formulae of native somatostatin, octreotide, [ml-
`Tyr3]octreotide and ml—RC-160.
`In [Tyr’]octreotide the amino
`acid Tyr replaces Phe to make radioiodination possible. In RC—l60
`Tyr is naturally present
`
`Materials and methods
`
`Radiopharmacemicnls. RC-160 was obtained from Peninsula
`Laboratories (Belmont, Calif, USA). Radioiodination and purifi-
`cation was performed using the technique described by Bakkcr
`[6].
`
`[Tyr3]octreotide and RC-160 were labelled with 1231- (specific
`activity 3.7 'l‘Bq 1231:/mg, Medgenix, Belgium) and 1251 (specific
`activity 0.62 TBq ml/rug, Arnersham, UK). For the in vivo stud-
`ies We used the sornatostatin analogues labelled with ml. The in
`vitro binding studies were performed with (l-IPLC)-high—perform-
`ance liquid chromatography purified rnono—iodinated somatostatin
`analogues, since cam'er—free radioligands are required for these
`assays. The radioiodination was carried out by adding 2.5 pg RC-
`l6O in 35 ttl 0.05 M acetic acid and 1.6 ug chloramine—T in 20
`in 0.05 M phosphate buffer (pH 7.5) to 200 MBq (=60 pl) ‘Z31
`or SO Ml-Sq (20 1.11) 1251 in the form of sodium iodide. The reaction
`was started by the addition of chloramine-T, representing an only
`2.5-fold molar excess over peptide in order to prevent oxidation
`of the disulphide-bridge of RC-160. The mixture was then vor-
`tcxed for l min. The radioiodination was stopped by adding 1 ml
`10% human semm albumin (lvlerieux, Lyon, France). After vor-
`texing for 30 3, 20 ml 5 mM ammonium acetate was added. Pu-
`
`rification was performed using a SEP-PAK C13 reversed-phase
`extraction cartridge (Waters Associates, Milford, Mass, USA),
`which was washed with 5 ml 70% ethanol, 5 ml ’.Z»propanol and
`5 ml distilled water. After application of the sample, the SEP-PAK
`cartridge was washed with 5 ml distilled water and radioiodinated
`RC-160 was eluted with 5 ml 96% ethanol. The solvent was
`evaporated at 40°C under a gentle stream of nitrogen. The residue
`(approximately 0.5 ml) was diluted with 2.5 ml 154 mM NaCl
`and 0.05 M acetic acid (pH 3), and the mixture was passed
`through a low protein-binding 0.22—ttm Mi.llex—GV filter (Milli-
`pore, Milford, Mass., USA). The labelling of [Tyr3]octreotide and
`the measurements of radioactivity in all fractions were carried out
`as described previously [6]. The 1231-labelled somatostatin ana-
`logues for in vivo use were not purified by HPLC and, hence,
`consisted of mixtures of mono— and di~radioiodinated and non-
`iodinated peptides (see Results, Radiolabelling of RC~J60). All
`chemicals used were of the highest purity available.
`
`Biological activity. The biological activity of HPLC—purified
`mono- 125l—RC-160 and [1251-Ty13]octreotide was assessed by
`measuring their potency to inhibit the secretion of rat growdt hor-
`mone (rGH) from cultured rat pituitary cells as described pre-
`viously [23].
`
`Animals and tumours. Twenty—four male Lewis rats (250—300 g)
`were inoculated in both upper hind legs with the transplantablc
`rat pancreatic tumour CA20948, which has previously been
`shown to possess somatostatin receptors [19]. Sixteen male Lewis
`rats (250—300 g) without tumour were used as controls. The rats
`were anaesthetized with ether. The radiopharmaccuticals were in-
`jected into the dorsal vein of the penis, using siliconized syringes
`(Sigmacoat, Sigma, St. Louis, Mo., USA). The dose was 18.5
`MBq (0.5 ttg) for the ”31—ahaiogue. The radioactivity of the syr-
`inges was measured in a dose calibrator (VDC—2(]2, Veenstra,
`Ioure, The Netherlands) in a standard geometry before and after
`the injection.
`In order to study the organ distribution of 1231-RC—l60 and
`[1231-Tyr3]octreotide in the 16 control rats, the rats were allocated
`to two groups for each radiopharmaceutical. Four rats were in-
`jected subcutaneously between the scapulae with ml 0.01 M acetic
`acid containing 154 mM NaCl, and four other rats were injected
`with 1 mg RC—l6O in the same solvent in order to saturate the
`somatostatin receptors. Forty—five minutes later the rats were in
`jected with 123I—RC~l60. Similarly, the two other groups of four
`rats were pretreated with vehicle or l mg octreotide and were
`subsequently injected intravenously with [1231-Ty13]octreotjde.
`The rats were killed 4 h after administration of '23I—RC—l6O or
`[I23I«Ty13]octre0tide.
`The 24 tumounbearing Lewis rats were divided into three
`groups of eight rats. In cach group four rats were injected sub-
`cutaneously with 1 mg RC~l60 in order to saturate the so1nato—
`statin receptor, as mentioned above. The three groups of eight
`rats were killed at 30 min. 4 h or 24 h after administration of
`1231-RC-160. The radioactivity concentration in various tissues
`was subsequently measured.
`The specific binding was defined as the difference between
`the individual uptake in the non-saturated tissues and the mean
`uptake in the saturated tissues, which are expressed as percentages
`of the injected radioactivity per gram tissue in the respective or-
`gans (mean i SD) after administration of 123I—RC-160 or [1231-
`Tyr3] octrcotide.
`
`Data acquisition and statistical analysis. All results are expressed
`as the mean i SD. Tissue-binding values and effects on rat growth
`
`European Journal of Nuclear Medicine Vol. 20, No. ll, November 1993
`
`Roxane Labs, Inc.
`Exhibit 1029
`
`Page 002
`
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`Exhibit 1029
`Page 002
`
`

`
`percentage of maximum
`120—
`l
`
`1091
`
`100-
`
`‘
`
`0 e
`
`lution volume (ml)
`Fig. 2. HPLC-elution pattern of the ethanol fraction of SEP—PAK
`C13 purification. Non-radioiodinated RC-160 is measured by UV
`absorbance (K2254 rim, broken line), radioiodinated RC—l60 and
`free radioiodide by gamma detection (solid line). The gamma de-
`tected peak at 27 min is the di—iodinated compound
`
`Table 1. Effects of125I—RC-160 and ['35I—Tyr3]octreotide on se-
`cretion of rGH from cultured rat pituitary cells (n=4)
`
`Peptide
`
`Control
`
`'25I-RC-160
`
`[l25I—Tyr3]octreotide
`
`* P< 0.05 vs control
`
`Concentration
`(nM)
`
`rGH
`(nglnil : so) (via)
`
`0.01
`0.1
`1
`
`0.01
`0.1
`1
`
`39.1 : 9.0 (100)
`
`91.7 : 4.6 (103)
`95.5 : 3.0 (107),
`77.2 ;: 11.1 (87)"
`
`90.0 : 4.8 (101)
`79.9 i 11.3 (90)
`64.8 —_~ 4.5 173)‘
`
`as measured above the heart with the gamma camera,
`decreasedin less than 2 min to 50% of the highest mea-
`surcd radioactivity. This rapid fall in the blood activity
`can be explained by the phenomenon of distribution of
`the activity over the whole blood and the interstitial
`space.
`
`Thirty minutes after injection static images showed
`a clear uptake of radioactivity in the liver and intestines.
`The left kidney was seen, as well as excreted activity
`in the urinary bladder. The right kidney was overpro-
`jected by the liver.
`Dynamic tumour uptake in situ in the rat during the
`first 30 min after injection of ml-RC-160 was analysed
`with the gamma camera. After background correction,
`using adjacent tissue as reference, no increased uptake
`in the tumour was found, whereas specific binding in
`the isolated tumour became statistically significant at 4
`hi (Table 2). As can be seen in Table 2, there was sig-
`
`honnone secretion for both radiolabelled peptides were evaluated
`using Student’s t-test. A P value of <0.05 was considered signif-
`icant. The tissue distribution and metabolism of 1331-RC-160 and
`[ml-Tyr3loctreotide in vivo were studied by gamma camera
`(Rota~1l, Siemens) scintigraphy and measurement of isolated or-
`gans in a LKB-1282-Compugammasystem [6]. The radioactivity
`in blood and urine was analysed as described previously [6].
`
`Results
`
`Radiqltzbellirlg of RC-I60
`
`The efficiency of labelling of RC-160 was 40%—60%
`for ‘Z51 and 70%-90% for 1231,
`in agreement with the
`radioiodination data for [Tyr7‘]octreotide, as described
`previously [6]. Purification of the iodination mixture us-
`ing the SEP-PAK C13 reversed-phase cartridge resulted
`in mainly non-peptide—bound radioiodine in the water
`fraction and more than 99% peptide-bound radioiodine
`in the ethanol fraction, revealed by HPLC. In Fig. 2 a
`typical HPLC elution pattern of the peptides eluted in
`the ethanol fraction is show, indicating a radiochemical
`purity of more than 95% of mono—radioiodinated RC-
`160. The simultaneously measured absorbance at 254
`um and radioactivity Show a clear separation between
`radioiodinated RC-160 and non-radioiodinated RC—l60.
`The acetic acid wash, included in the SEP-PAK pu-
`rification of [ml-Tyr‘]octreotide, was omitted from the
`isolation procedure for ml-RC-160, since this wash was
`found already to contain a substantial fraction (-—5%—
`10%) of the radioiodinated RC-160, while only negli-
`gible amounts of free radioiodide were detected.
`Since the radiolabelling and the SEP-PAK C15 sepa-
`ration technique appeared adequate (more than 95% ra-
`diochemical purity of mono-radioiodinated [Ty13]oc-
`treotide and RC-160), HPLC purification of the radio-
`labelled somatostatin analogues was not performed.
`These results were in agreement with the radiolabclling
`results of [Tyr3]octreotide [6].
`
`Receptor binding and specific biological activity
`
`Table I shows the effects of ‘Z51-RC-l60 and [1251-
`Tyr3]octreotide on the secretion of rGH by cultured rat
`pituitary cells. Both iodinated somatostatin analogues
`significantly inhibited rGH secretion at 1 nM. Both ra-
`dioiodinated analogues caused a similar dose-response
`as the non-radioiodinated counterparts (data not shown).
`
`Animal studies
`
`Dynamic scintigraphy of tumour-bearing and control
`rats after i.V. administzration of ”3l—RC-160 and [ml-
`Tyr3]octre0tide showed a fast disappearance of the ra-
`dioactivity from the circulation. With both radioiodi—
`nated analogues radioactivity in the blood circulation,
`
`European Journal of Nuclear Medicine Vol. 20, _\’o. 11, November 1993
`
`Roxane Labs, Inc.
`Exhibit 1029
`
`Page 003
`
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`Exhibit 1029
`Page 003
`
`

`
`1092
`
`Table 2. Tissue distribupon (% of injected dose/g tissue) and
`specific binding (A) of “31-RC-160 in tumour-bearing rats after
`intravenous aclininistration (mean i SD)*
`
`Table 4. Tissue distribution (% of injected dose/g tissue, mean
`it 5D) in tUm0IJr—bea.I'ing rats (21:41) 24 h after intravenous injec-
`tion of 0.5 ttg radioiodinated sornatostatin analogue
`
`Tissue
`
`0.5 11 p.i.
`
`4 11 p.i.
`
`24 11 p.i.
`
`Tissue
`
`1231-R0160
`
`[1231-Tyl-3]gctreo{jde
`
`it 0.38
`0.91
`Pancreas -
`+ 0.10 i 0.01 p
`A 0.31
`1 0.38‘
`
`0.32 i 0.08
`0.07 i 0.02‘
`0.25 : 0.03‘
`
`1' 0.02
`0.06
`0021 i 0.002
`0040 i 0.019“
`
`:1: 0.06
`1.17
`Adrenals ~
`0.20 :r 0.06
`+
`A 0.97 i 0.060
`
`0.54 i 0.09
`0.07 : 0.01
`0.47 1- 0.090
`
`i 0.03
`0.06
`0.033 : 0.009
`0.022 i 0.028
`
`0.16 i 0.06
`Pituitary —
`+ 0.021 : 0.003
`A 0.13 : 006*
`
`i 0.05
`0.05
`0.08 i 0.02
`0.004 : 0.000
`0.013 i 0.002
`0.054 =_r 0020* 0.044 i 0.048
`
`—
`
`0.014 : 0.002
`
`0.008 i 0.002
`
`0.0019 : 0.0009
`
`Spleen
`Kidneys
`Liver
`Intestines
`Thyroid
`Thymus
`Lungs
`Blood
`Pancreas
`Adrenals
`Pituitary
`Brain cortex
`TUTHOUT
`
`i 0.03
`0.06
`i 0.03
`0.12
`: 003*
`0.17
`: 00*
`0.2
`i 70
`320
`0.068 : 0032*
`0.12
`i 0.040
`0.13
`i 003*
`0.06
`i 0.02
`0.06
`i 0.03
`0.05
`3: 005*
`0.0019 i 0.0009
`0.037 i 0.021;’
`
`0.028 i 0.021
`0.13
`i‘ 0.01
`0.027 : 0.005
`0.03
`i 0.01
`180
`i 50
`0.016 : 0.007
`0.033 :1: 0.006
`0.006 : 0.000
`0.11
`i 0.04
`0.04
`i 0.01
`0.17
`1 0.02
`0002 i 0.001
`0.007 i 0.001
`
`Brain
`
`cortex
`
`0.0013 i 0.0003
`0.003 : 0.000
`+ 0008 i 0.001
`A 0.007 : 0.003“ 0.005 4; 0.002" 0.0006 : 0.0009
`
`$
`
`7
`P< 0.01, 0-‘I~RC—160 vs [“3I-Tyrhoctreotide
`
`'
`
`"
`
`Tumour —
`
`0.23 $0.07
`
`0.20 : 0.05
`
`0.037 t 0.021
`
`+ 0-“ i 0-02
`A 0-“ i 0-07
`
`_
`Kldneys ' 0'7? i 0'13
`+ 0.5.) i 0.04
`A 0.21 i (M3
`
`0-08 i 0-02*
`0-12 i 0-05
`
`0-035 5? 0-003
`0-001
`1-' 0-021
`
`0'43 i 0'1?
`0.22 i 0.0:»
`0.2} i 0.13
`
`0'“
`0.10
`0.0]
`
`i 003
`i 0.02
`i 003
`
`* P< 0.01, specific binding significantly different from zero
`
`" Each group contained four rats: —, no pretreatment; +, pretreat-
`ment with 1 mg unlabelled RC-160 subcutaneously 45 min prior
`to the injection of ml-RC-160
`
`Table 3. Tissue distiibutioii [with (+) and without (—-) pretreat-
`ment of rats with unlabelled R0160] and specific binding (A) in
`somatostatin receptor—positive organs (mean 1 SD) in non—tu-
`mour-bearing rats (n=4), 4 h after injection of ml-RC—l60. The
`rats in the parallel experiment with {ml—Tyr3]octretide were pre-
`treated with 1 mg octreotide
`
`Tissue
`
`Pancreas
`
`Adrenals
`
`Pituitary
`
`Brain
`Cortex
`
`123l—RC-160
`
`[1231-Tyr3]oc1:reotide
`
`-
`+
`A
`
`-
`+
`A
`
`—
`+
`A
`
`—
`+
`A
`
`1: 0.05‘
`0.21
`0.025 : 0.006
`0.17
`i 0.05*=**
`
`-: 003*
`0.19
`0.042 5-. 0.010 _
`0.15
`: 0.03‘-*‘
`
`: 0.15
`1.03
`0.023 2 0.002
`1.01
`: 0.15”
`
`: 0.02
`0.26
`0.027 : 0.002
`0.23
`: 0.02”
`
`0.066 :r 0007*
`0.0078 i 0.0020
`0.063 : 0007*-"”
`
`: 0.09
`0.32
`0.0067 : 0.001
`0.31
`-_ 0.09“
`
`0.0052 : 0.0014*
`0.0019 : 0.0003
`0.0033 i 0.00141“-**
`
`0.0019 : 0.0002
`0.0012 : 0.0002
`0.0007 : 0.0002
`
`“ P< 0.01. mI—RC-160 vs [1231-'l‘yr3]octreotide
`** P< 0.01, specific binding significantly different from zero
`
`nificant specific binding in all the somatostatin recep-
`tor- positive tissues analysed, i.e. pancreas, adrenals, pi-
`tuitary and brain cortex, at 0.5 and 4 h, and in the tu-
`mour at 4 h after injection. After 24 h there was still
`.
`.f.
`.f.
`.
`.
`.
`srgni icant speci 1c binding in the pancreas. From 30
`min after the administration of ‘Z31-RC-160, total radio-
`activity disappeared rapidly from the measured organs
`and tumours. Statistically significant specific binding
`was still present 24 h after [”3I—Tyr3]octreotide injection
`in the pancreas. adrenal and pituitary gland (data not
`shown).
`In Table 3 the tissue distributions, 4 h after injection,
`are compared between pretreated and non-pretreated,
`non-tumounbearing rats. We found a higher uptake of
`[031-Tyi-3]octreot-ide in the adrenals, the pancreas and the
`pituitary gland, than was found when using ml-RC-160.
`The effect of pretreatment with unlabelled RC-160 or
`octreotide is evident in the somatostatin receptor-posi-
`tive organs for both 1231-RC-160 and [ml-Tyr3]octreo—
`tide. Significantly higher specific binding of radioactiv-
`ity was found after administration of [”3l-Tyr3]octreo-
`tide in the adrenals, the pancreas and the pituitary gland
`than after administration of ml-RC-160. In the rat brain
`cortex ‘Z31-RC-160 has a higher uptake and specific
`binding than [‘231—'l‘yr3]octreotide.
`Urine samples were obtained 30 min p.i. from tu-
`mour-bearing rats (n:4), showing 13% i 1% of the total
`radioactivity in the form of peptide-bound radioiodine.
`TWenty—four hours p.i. the percentage of peptide—bound
`radioiodine in urine and blood had dropped to 1.3% i
`0.2% and 1.1% i 0.3%, respectively, and more than
`95% of the radioactivity in the urine was free radiolo-
`dine, which is comparable with the results obtained us-
`ing [1231-Tyr3]oetreot1'de as radioligand (data not shown).
`A comparison of the tissue radioactivity concentra-
`tions 24 h after injection of ‘Z31-RC—160 and [ml-
`"1‘y13]octreotide is presented in Table 4. [”3l—Tyr3]octreo-
`
`European Journal of Nuclear Medicine Vol. 20, No. 11, November 1993
`
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`
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`
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`Page 004
`
`

`
`tide had a higher clearance of radioactivity from soma-
`tostatin receptor-negative tissues, such as liver, thymus,
`blood and lungs, and a significantly higher binding in
`the somatostatin receptor-positive pituitary than ‘23I-RC-
`160. Only in the tumour was there significantly higher
`binding of radioactivity for ‘Z31-RC-160 compared to
`[ml-Tyr3]octreotide, but as can be seen from Table 2,
`this was not specific binding.
`
`Discussion
`
`RC-160 is, like octreotide, a somatostatin analogue with
`potent hormone secretion-inhibiting characteristics in
`vivo and in vitro. However, discrepancies with octreo-
`tide have been described, especially with regard to bind-
`ing to a number of human cancers,
`like those of the
`breast, ovary, exocrine pancreas, prostate and colon
`[l4—18, 21]. Therefore, radioiodinated RC-160 might be
`an important radiopharmaceutical, having potential ad-
`vantages over radioiodinated octreotide for the in vivo
`detection of the aforementioned somatostatin receptor-
`positive tumours. In the literature no data are available
`on tissue distribution of the sornatostatin analogue RC-
`160, either in animals or in humans.
`In the present
`study, therefore, we evaluated the potential use of radi-
`ioiodinated RC—16O for somatostatin receptor scintigra—
`phy. There was significantly higher uptake and specific
`binding in somatostatin receptor-positive organs, such
`as the pancreas, the adrenal and the anterior pituitary
`gland, of ‘:31 after administration of [ml-Tyr’]oct:rcotide
`than after ml-RC-160.
`In brain cortex of control rats we found at 4 h a low,
`but statistically significant specific binding of ”3I-RC-
`l60. However, this was caused by a very low amount
`of tracer (0.0052% of injected dose per gram) in com-
`parison with other somatostatin receptor-positive tis-
`sues, such as the pancreas (O.21% of injected dose per‘
`gram). Since we found a significant difference between
`the uptake of radioactivity in saturated and non-satu-
`rated brain cortex, these data suggest that, in contrast
`to octreotide, RC—l60 and radioiodinated RC-160 are
`able to cross the blood-brain barrier, as has also been
`reported for cold RC-160 and radioiodinated RC-160
`by Banks et al. [21, 24]. The presence of the C-terminal
`amino acid tryptophan in RC-160 (see Fig. l) enhances
`the lipophilicity of the molecule, and this might also
`explain its increased blood-brain barrier permeability
`and reduced clearance from the tissues and blood.
`
`During the first 30 min after the injection of ”3I-RC-
`160 there was no statistically significant uptake in the
`tumour as measured by gamma camera scintigraphy, nor
`was there any after background correction. This finding
`is in contrast with the results of the experiments with
`[ml-Tyr3]octreotide as described by Bakker et al. [6].
`However, 30 min after the injection of ml-RC-160, sta-
`tistically significant specific binding in the isolated tu-
`mour was found. Since there is no significant difference
`
`1093
`
`between the uptake or specific binding after the injec-
`tion of {’23I—Tyr3]octreotide and ml-RC-160 in the iso-
`lated tumour at 30 min, the relatively low tumour to
`blood ratio in these experiments is probably the reason
`for this discrepancy.
`To conclude: mI—RC-160 does not seem to have ad-
`vantages over [1231-Tyr3]octreotide as a radiopharmaceu-
`tical for somatostatin-receptor scintigraphy, despite the
`fact that ‘Z31-RC-160 shows specific hi gh-affinity bind-
`ing to various somatostatin receptor—positive organs. In
`contrast to radioiodinated [Tyfloctreotide and octreo-
`tide, which do not pass the blood-brain barrier, our ex-
`periments continn that RC-160 and radioiodinated RC-
`160 indeed do pass the blood-brain barrier. However,
`this occurs in low quantities, and consequently the ap-
`plication of radioiodinated RC—l60 in nuclear medicine
`for visualizing sornatostatin receptor-positive brain tu-
`mours with an intact blood-brain barrier is hampered.
`In comparison with [”3l—Tyr3]octreotide, the main dis-
`advantage of ”3I—RC-160 is its relatively low tumour to
`blood (background) ratio, implying poorer in vivo tu-
`mour detection.
`
`Apart from the discussed data, it must be emphasized
`that several authors have reported that in comparison to
`octreotide, RC-160 has superior binding characteristics
`in some human tumours. Therefore, RC-160 and,
`in
`spite of its disadvantages, ml-RC-160 could open new
`diagnostic and/or therapeutic applications in patients
`bearing such tumours. Consequently, in analogy to the
`development of the indium-ill
`labelled [DTPA-D-
`Phe’]octreotide
`analogue,
`‘“In-labelled
`[DTPA-D-
`Phe1]RC-160 is being prepared and investigated.
`
`The authors wish to thank Dr. Wil Kort, Ineke Hekking-Weyma,
`Reno Mekes, Marcello Hamis and lna Loeve for their expert as-
`sistance during the experiments.
`
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