Radiolabeling of Bleomycin-Glucuronide with 131 I and Biodistribution Studies Using Xenograft Model of Human Colon Tumor in Balb/C Mice

Radiolabeling of Bleomycin-Glucuronide with 131Iand Biodistribution Studies Using Xenograft Model

of Human Colon Tumor in Balb/C Mice

Hasan Demiroglu,1 Ugur Avcıbasxı,1 Perihan Unak,2 Fazilet Zumrut Biber Muftuler,2 Cx.A. _Ichedef,2

Fikriye Gul Gumusxer,3 and Serhan Sakarya4,5

Abstract

Bleomycin-glucuronide (BLMG) is the glucuronide conjugate of BLM. In the present study, BLMG was primarilyenzymatically synthesized by using a microsome preparate separated from rat liver, labeled with 131I by iodogenmethod with the aim of generating a radionuclide-labeled prodrug, and investigated its bioaffinities with tumor-bearing Balb/C mice. Quality control procedures were carried out using thin-layer radiochromatography andhigh-performance liquid chromatography. Tumor growing was carried out by following Caco-2 cell inoculationinto mice. Radiolabeling yield was found to be about 65%. Results indicated that 131I-labeled BLMG (131I-BLMG)was highly stable for 24 hours in human serum. Biodistribution studies were carried out with male AlbinoWistar rats and colorectal adenocarcinoma tumor-bearing female Balb/C mice. The biodistribution results in ratsshowed high uptake in the prostate, the large intestine, and the spinal cord. In addition to this, scintigraphicresults agreed with those of biodistributional studies. Xenography studies with tumor-bearing mice demon-strated that tumor uptakes of 131I-BLM and 131I-BLMG were high in the first 30 minutes postinjection. Tumor-bearing animal studies demonstrated that 131I-BLMG was specially retained in colorectal adenocarcinoma withhigh tumor uptake. Therefore, 131I-BLMG can be proven to be a promising imaging and therapeutic agent,especially for colon cancer in nuclear medical applications.

Key words: Balb/C mice, biodistribution, bleomycin, bleomycin-glucuronide, Caco-2, 131I, glucuronidation,scintigraphy, xenography

Introduction

Bleomycin (BLM) is a group of water- and methanol-soluble basic glycopeptide antibiotics isolated from fer-

mentation products of the Streptomyces verticillus in 1966 byUmezawa in Japan.1 The cytotoxic effect of BLM depends onthe blocking of DNA synthesis, and in the presence of iron,oxygen, and a reducing agent, BLM breaks the DNA chains.2

It is conventionally used for the treatment of squamous cellcarcinomas, combined either with other chemotherapy or

with radiation therapy3 or as palliative treatment4, and also inthe treatment of testicular cancers5 and lymphomas.6,7 Theclinically used BLM is a mixture of three distinct isomers: A2(the most abundant, 65%), B2 (30%), and DM, which is a de-methylated form of A2 (5%) (Fig. 1).

Certain enzymes in tissues and body fluids may, throughreversal of the detoxification process, influence the compo-sition and availability of steroid hormones, toxins, and car-cinogens. The enzyme b-glucuronidase, which hydrolyzesglucuronide conjugates, thereby reversing one of the main

1Department of Chemistry, Faculty of Art and Science, Celal Bayar University, Manisa, Turkey.2Department of Nuclear Applications, Institute of Nuclear Sciences, Ege University, Bornova, Izmir, Turkey.3Department of Nuclear Medicine, School of Medicine, Celal Bayar University, Manisa, Turkey.4Department of Infectious Diseases and Clinical Microbiology, Adnan Menderes University School of Medicine, Aydin, Turkey.5ADUBILTEM Science and Technology Research and Development Center, Adnan Menderes University, Aydin, Turkey.

Address correspondence to: Ugur Avcıbasxı; Department of Chemistry, Faculty of Art and Science, Celal Bayar University,45030 Manisa, TurkeyE-mail: [email protected]

CANCER BIOTHERAPY AND RADIOPHARMACEUTICALSVolume 27, Number 6, 2012ª Mary Ann Liebert, Inc.DOI: 10.1089/cbr.2011.1157

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detoxification and excretion pathways, was found to varyin concentration in different cysts and tumor tissues over a300-fold range.8 It is known that breast cancer cells have highamounts of b-glucuronidase. Glucuronides of drugs oftenaccumulate during a long-term therapy. The hydrolysis ofglucuronides can be catalyzed by the enzyme b-glucuronidase,which has already been proven to be useful in tumor-specificbioactivation of glucuronide prodrugs of anticancer agents.9–11

Therefore, antibody-directed enzyme prodrug therapy andgene directed-enzyme prodrug therapy using glucuronideprodrugs, activated by the human enzyme b-glucuronidase,have been developed as an experimental approach to en-hance tumor selectivity and to reduce systemic toxicity ofanticancer agents.

BLM has been widely studied as a G-quadruplex interac-tive compound and telomerase inhibitor.12–20 G-quadruplexesare unusual DNA secondary structures based on planes offour guanines (G-tetrads) stabilized by Hoogsteen G–Gpairings and monovalent cations. The central aromatic coreof the perylene diimides is suitable for p–p stacking inter-actions with the terminal G-tetrad of DNA G-quadruplex,whereas the hydrophilic side chains interact with the DNAgrooves. By means of these two kinds of interactions, thesemolecules are able to induce and stabilize G-quadruplexstructures in G-rich single-stranded oligonucleotides. This isof the great pharmaceutical interest, since the terminal endsof eukaryotic chromosomes (telomeres) are characterizedby the presence of a single-stranded G-rich overhang thatrepresents the substrate of a reverse transcriptase enzyme,ribonucleoprotein telomerase, which is involved in themaintenance of telomere length.21,22 This enzyme is not ac-tive in most somatic cells, but is active in most human tu-mors, and is therefore considered as being of high potentialas a selective target for different antitumor strategies.21

The aim of the current study was to synthesize a novelglucuronide derivative of BLM (BLMG) (Fig. 2) as a ra-diopharmaceutical that is able to be labeled with 131I, andto investigate its radiopharmaceutical potential usingmale Albino Wistar rats and adult male Albino rabbit inbiodistribution and scintigraphic studies, respectively,and then to evaluate its accumulation in the human co-lorectal adenocarcinoma tumor- bearing female Balb/Cmice.

Materials and Methods

BLM was purchased from Sigma Chemical Co. Radio-iodination and its preliminary biological activities inthe rabbit metabolism were examined using the gammacamera-imaging technique and biodistribution studies.Na131I (74 MBq) was obtained from the Department of Nu-clear Medicine of Celal Bayar University. Iodogen (1,3,4,6-tetrachloro-3a,6a-diphenylglycouril), 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid, sodium salt (HEPES), Trisbuffer, UDP-glucuronic acid (UDPGA), and Triton X-100were purchased from Sigma, and all other chemicals werepurchased from Merck Co. Caco-2 (human colorectal ade-nocarcinoma) was obtained from American Type CultureCollection. Primary human intestinal epithelial cells (ACBRI519) were purchased from Applied Cell Biology ResearchInstitute. Minimum essential medium (MEM EAGLE) fetalbovine serum (FBS), Tripan blue (BIO. IND), phosphate-buffered saline (PBS), and Tripsin–EDTA were supplied fromBIO. IND. Thin-layer radiochromatography (TLRC) andhigh-performance liquid chromatography (HPLC) chro-matograms were obtained using a Cd(Te) detector equippedwith a RAD 501 single-channel analyzer and HPLC (an LC-10ATvp quaternary pump and an SPD-10A/V UV detectorand a syringe injector equipped with a 20-lL loop and 5-lmreversed-phase (RP)-C-18 column 250 · 4.6 mm I.D.; Ma-cherey-Nagel), respectively. Liquid chromatography massspectrometry (LC/MS/MS) chromatograms were taken us-ing an LC/MS/MS instrument (Agilent HPLC 1100 binarypump, degasser, autosampler, and column oven) in EgeUniversity ARGEFAR (Research and Application Center ofDrug Development and Pharmocokinetics), and scintigramswere obtained using a double-headed gamma camera (In-finia, GE) in the Department of Nuclear Medicine of CelalBayar University.

Synthesis of BLMG

Preparation of microsomal fraction from rat livers. Twomale Albino Wistar rats were sacrificed by cervical dislo-cation. Microsomal fractions from the rat livers were pre-pared according to the procedure previously described byZihnioglu.23 Briefly, the liver was excised and placed in cold(0�C–4�C) 250 mM sucrose/5 mM HEPES 99%. It was then

FIG. 1. Chemical structuresof BLM A2 and BLM B2

isomers.

372 DEMIROGLU ET AL.

chopped using scissors and was blended and homogenizedafter adding 35 mL of 250 mM sucrose/5 mM HEPES, pH 7.4, ina teflon/glass homogenizer at 1700 g (1500 rpm) for 30 minutes.

Purification of UDP-glucuronyl transferase from rats.Homogenates were then centrifuged at 12,000 g (10,500 rpm)for 10 minutes, and the resulting supernatant was decantedthrough glass wool to trap fat particles and centrifuged at105,000 g (31,500 rpm) for 1 hour. All further operations wereperformed at + 4�C. Microsomal pellets were solved by re-suspension in a volume (in 2 mL) of 0.2 M potassium phos-phate, 2 mM mercaptaethanol, and 0.4% Triton X-100 (pH 7)buffer, equal to twice the wet weight (in grams) of the tissue.The suspension was stirred on ice for 30 minutes and centri-fuged for 1 hour at 105,000 g to remove the insoluble material.The resulting supernatant was stored at - 80�C until use.

Estimation of protein in microsomal samples. The pro-tein content in microsomal samples was estimated using theBradford method. This method is based on the observation ofmaximum absorption for a solution of Coomasie Blue G-250at acid pH shifts from 465 to 595 nm when binding to proteinoccurs. Using this method, standard curves covering a rangeof protein concentration were constructed as follows: 0.02,0.05, 0.01, 0.12, 0.15, 0.20, and 0.25 mg/mL. The Bradfordreagent consists of 40 mg Coomasie Brilliant Blue and 55 mL88% w/v phosphoric acid dissolved in 50 mL ethanol that wasdiluted to 1 L and filtered off. Standard curves, using bovineserum albumin (BSA), plotted by linear regression analysis,were prepared and protein concentrations of appropriatelydiluted samples calculated from the relevant standard curve.The protein content was found to be *8.22 mg/mL, whichwas similar to the value reported by Bradford.24

Glucuronidation reaction. Reactions were performedsimilar with other reports.25–27 Microsomal enzyme pre-parate (0.98 mg protein/119 lL) was added to 5 mL of 50 mMTris buffer (pH 8.0) containing 6 mM CaCl2, 10 mM UDPGA,and 1 mM dithiothreitol at a temperature of 37�C. The reac-tion mixture (total volume 5 mL) containing UDP-glucuronyltransferase (UDPGT) was stirred at 37�C in a water bath for10 minutes. The contents were then sonicated in an ultra-sonic bath for 30 seconds to disperse the microsomes, and thereactions were started by the dropwise addition of 0.5 mg/0.5 lL BLM in water, with stirring. Slow stirring at 37�C wascontinued for 18 hours. The reaction was terminated after 18

hours by the addition of 300lL of acetonitrile, and the precip-itated protein was removed by centrifugation at 5200 rpm for 10minutes by using a microcentrifuge. The supernatant was thenanalyzed by reversed-phased HPLC (Shimadzu 10 AVp). TheHPLC analysis indicated that the glucuronidation yield wasabout 100%, and one peak was obtained for BLMG.

HPLC studies

Cold iodinated compounds of BLM and BLMG [127I-BLM and 127I-BLMG] were investigated in HPLC. Table 1shows chromatographic conditions used for analytical ex-periments in HPLC. For analytical experiments, a 5-lmRP-C18 column (250 · 4.6 mm I.D.; Macharey-Nagel) and asyringe injector equipped with a 20-lL loop were used. Theflow rate was set at 1 mL/min. UV was detected at 260 nm.The mobile phase and UV detection are given in Table 1.

Liquid chromatography mass spectrometry

Chromatographic conditions for LC/MS/MS experimentsare given in Table 2.

Inactive iodination of BLMG

BLMG was iodinated with inactive iodine under the sameconditions as previously described by Avcıbasxı et al.11

Structural parameters were obtained by the LC/MS/MSspectrometry system instrument (Agilent HPLC 1100 binarypump, degasser, autosampler, and column oven) in the EgeUniversity ARGEFAR Center.

Radioiodination procedure

Radioiodination procedure of BLMG with 131I. BLMGwas first radioiodinated with 131I using the iodogen method.To label BLMG with 131I, 25 lg of BLMG was added into theiodogen-coated tube, and then 1 mCi (37 MBq) of Na131I wasadded. This reaction mixture was kept at room temperaturewithout stirring for 15 minutes. At the end of this time, themixture was transferred to another tube by a syringe, andthen quality control was performed.

Quality control studies. For TLRC studies, TLC aluminumsheets (Merck, 20 · 20 cm code: 5552) were used, and citricacid monohydrate 100% was used as the mobile phase. TheTLRC technique was used to determine the Rf values of theradioiodinated products. Each TLRC sheet was covered by an

FIG. 2. Chemical structureof bleomycin-glucuronide(BLMG).

HIGHEST UPTAKES OF 131I-BLMG WAS OBTAINED IN TUMOR 373

adhesive band after its development and was cut into 0.5-cmwidth. Those pieces of TLC were then counted by using a Cd(Te)detector equipped with a RAD 501 single-channel analyzer.

Stability of radiolabeled BLMG in human serum. In vitrostability of 131I-BLMG in human serum was determined byincubating 100 lL (25 lg) of the labeled compound with300 lL of human serum at 37�C. The aliquots were analyzedin time intervals of 30, 60,180, and 1440 minutes by the TLRCtechnique after labeling.

Lipophilicity (partition coefficient)

The lipophilicity (logP) of the radiotracer was measured asfollows: 100 lL of the radiolabeled compound (131I-BLMG) wasadded to a premixed suspension of 200 lL of n-octanol in 200lL,pH 7 buffer. The resulting solution was mixed for 15 minutes atroom temperature and centrifuged for 30 minutes at 2500 rpm.Then, 0.1-mL aliquots of each phase were removed and countedby a Cd(Te) detector equipped with a RAD 501 single-channelanalyzer. Experiments were conducted in triplicate.

Biodistribution studies in rats and tumor xenographymodel in Balb/C mice

Experiments with animals were approved by the Institu-tional Animal Review Committee of Ege University. Thebiodistribution data are expressed as percentage of injectedradioactivity per gram of tissue (%ID/g) for selected organsas the mean value of three rats. The experiments were per-

formed on male Albino Wistar rats weighing *180–200 g.For blocking iodine uptake into the thyroid gland, 10 mg ofpotassium iodide was added to 1 L of the animal’s drinkingwater. The 131I-BLMG in 60% purity was sterilized by amembrane filter and then injected into the tail vein of theanimals ([3.7 MBq (100 lCi)]/1 lg 131I-BLMG per rat). Then,they were sacrificed at 30, 120, and 240 minutes under etheranesthesia, and the tissues of interest were removed. Bloodwas taken, and organs were excised. All tissues wereweighed and counted for radioactivity with a Cd(Te) detec-tor. The percent of radioactivity per gram of tissue weight (in% injected activity/g tissue) was determined.

In the xenography model studies, 6-week-old femaleBalb/C mice weighing *20–30 g were obtained from De-partment of Pharmacology, the Adnan Menderes University,Aydın, Turkey. The mice were cared for at Ege University,Izmir, Turkey, according to the institutional animal careguidelines. To establish animal tumor models, 1.5 · 107

(cells/mouse) Caco-2, the human colorectal adenocarcinomacell lines, were resuspended in 100 lL of phosphate buffersolution (PBS-R, Gibco-BRL) and injected into the subcuta-neous tissue of the left flank of the mice.

All animals were provided with free access to drinkingwater and a basal diet. The mice were divided into threegroups for the experiments: control, BLMG, and BLM. Eachgroup contained nine mice. All animals were caged andhoused under controlled conditions of humidity (45% – 5%)and temperature (25�C) on a 12-hour light/dark cycle. Tu-mor growth and weight of the mice were checked daily after3 days of Caco-2 cell inoculation into the mice. Biodistribu-tion was performed 10 days post-tumor grafting, when thetumors had reached a measurable size of about 6 · 5 mm.131I-BLMG in 60% purity was sterilized by a membrane filterand then injected into the tail vein of the animals ([3.7 MBq(100 lCi)]/1 lg 131I-BLMG per Balb/C mice).

In the cell culture studies, Caco-2 cells were cultured in theEagle’s minimum essential medium supplemented with 20%FBS, 2 mM glutamine, 1.5 g/L sodium bicarbonate, 0.1 mMnonessential amino acid, and 1 mM sodium pyruvate. In theexperiments, cells were grown at 37�C in an incubator withhumidified air and equilibrated with 5% CO2. The cells weremaintained in exponential growth by subculturing the cellsusing trypsin-EDTA (0.25% by w/v in the Hanks BalancedSalt Solution). The cells were pelleted and resuspended in thecell medium. Before the experiments, cell cultures weretrypsinized and, to remove trypsin, washed once in the re-spective culture medium. The cells were resuspended at aconcentration of 1 · l05 cells/mL in the culture medium,transferred to falcon tubes, and added 1 mL PBS.

Scintigraphic studies

The imaging studies were performed on healthy adult maleAlbino rabbits using a gamma camera (Infinia, GE) in theDepartment of Nuclear Medicine of Celal Bayar University.The 131I-BLMG was intravenously injected into an adult maleAlbino rabbit via the ear vein after anesthetizing by the mix-ture of xylazine and ketamine to determine the dynamic andstatic situations of 131I-BLMG in the metabolism. Dynamic andstatic scintigrams were obtained using a gamma camera,which was adjusted to detect c radiations of 131I. Dynamicscintigrams were obtained over the first half hour with frames

Table 1. Chromatographic Conditions Used

Analytical Experiments in High-Performance

Liquid Chromatography

Column In analytical experiment: RP-C18(250 · 4.6 mm)

Flow speed In analytical experiment: 1.00 mL/minWave length 260 nmTemperature 25�CMobile phase 100% Ammonium acetate buffer (10 mM)

RP, reversed-phase.

Table 2. Chromatographic Conditions in Liquid

Chromatography Mass Spectrometry

Ionization mode ESI positive vs. negativeAPI nebulizing Gas 51 psiColumn Phenomenex 00F-4337-B0Scan time 1.0 sSIM width 0.7 amuNeedle Positive 5000 V

Negative - 4500 VShield Positive 600 V

Negative - 600 VCapillary 50 VDetector 1500 VMobile phase A: 10 mM sodium perchlorate

in 0.1% phosphoric acidB: ACN

Spray chamber temperature 50�CDrying gas temperature 350�CDrying gas pressure 35 psiNebulizing gas pressure 51 psi


Table 3. LC–MS/MS Spectrum (m/z) Values for Compound127I-BLMG and Some Different Fragments

and Proposed Structures of Selected Fragments

Fragment Structure m/z Fragment Structure m/z

1 549 7 1724

2 587 8 1699

3 587 9 387

4 1197 10 1564

5 1465 11 175

6 1851 12 162


of 1 minutes after the administration of the labeled compound.Static images were obtained from posterior projection afterdifferent time intervals up to about 4 hours after the admin-istration of the 131I-BLMG.

Statistical analysis

For in vivo experiments, data were analyzed statistically byusing SPSS statistical software (SPSS for Windows; Release10.0.1 Standard Version). Comparisons between differentgroups were performed by Pearson correlation and one-wayanalysis of variance (ANOVA); if ANOVA revealed significantdifferences, post hoc comparisons were performed by Duncanmultiple range tests. p < 0.05 was considered significant. Studieswere performed six times for each experimental condition.

Results

Enzymatic synthesis mechanism

To obtain the enzyme UDPGT, microsomal fractions ofrat livers were separated, and BLMG was enzymaticallysynthesized using this enzyme. Glucuronidation consistsof transfer of the glucuronic acid component of UDP-glucuronic acid to a substrate by any of several types of UDP-glucuronosyltransferase. Thus, drug molecules and othersmall lipophilic molecules are converted into hydrophilic

conjugates and excreted from the body. The enzyme UDPGTcatalyzes the reaction. Thus, many endogenous and exoge-nous compounds, food molecules, hormones, and drugs maybe glucuronidated with this way similar with in vivo. Enzy-matic glucuronidation is run according to the SN2 reaction(second-degree nucleophilic reaction), and at the end of thereaction, glucuronic acid is converted to a beta anomer fromalpha. The reason is the nucleophilic attack from behind thesubstrate in the SN2 reactions.28 UDP glucuronosyltransfer-ase-rich microsome preparates were extracted with a goodyield and high purity from rat livers. BLMG can be deglu-curonidated by the b-glucuronidase enzyme, which has anactivity that is considerably high in certain kinds of cancercells. Owing to this enzyme activity, BLMG can be consideredas a potential anticancer drug.

HPLC chromatograms showed that a BLMG derivativeprobably occurred as N-glucuronide. LC/MS/MS results of127I-BLMG given in Table 3 supported this idea, since m/zvalues of fragments are 162, 175, 387, 549, 587, 1197, 1465,1564, 1699, 1724, and 1851.

Radioiodination

An optimum amount of iodogen, 1 mg, was fixed, sinceincreasing amount of iodogen caused decreasing iodinationyield. The reason may be the oxidative effect of iodogen to

FIG. 3. High-performanceliquid chromatography chro-matograms of BLMG and127I-BLMG (first peak belongsto 127I-BLMG and the secondpeak is BLMG) monitored at260 nm: linear gradient 100%Ammonium acetate buffer(10 mM) over 10 minutes.

FIG. 4. Stability of 131I-BLMG in serum.


the substrates. On the other hand, it was reported that ahigh concentration of the iodogen may have resulted withself-iodination of iodogen.29 Iodogen (1,3,4,6-tetrachloro-3a,6a-diphenylglucoluril) is an oxidizing agent commonlyused for the radioiodination of proteins. The oxidativemechanism of iodogen is not clear, but the two carbonylgroups in its structure probably play an essential role.Iodogen has been used for radioiodination of some com-pounds and drug molecules such as monoclonal anti-bodies,30 anti-inflammatory drugs,31 and flavonoids.32

Radioiodinated glucuronides were produced by differentways previously, such as metabolically29,33–36 or enzymati-cally.11,37 99mTc- and 18F-labeled glucuronide derivativeswere also synthesized.38–41

Optimum radioiodination conditions for BLM and BLMGwere as follows: pH 6, 1 mg of iodogen, 15 minutes of reac-tion time, and 25 lg of substrate. The results showed that theradioiodination of BLMG was successfully realized, and itsyield was found to be about 65%. Also, the results of TLRCstudies showed that citric acid monohydrate, 100%, was themost suitable developing solvent to establish the Rf valueobtained as 0.04 for 131I-BLMG.

HPLC studies

HPLC chromatograms confirmed that 127I-BLMG was dif-ferent from its noniodinated derivative. It was detected as onlyone peak for BLMG and three peaks for 127I-BLMG in the

FIG. 5. Dynamic scintigrams of 131I-BLMG, which was administered to a rabbit via the ear vein in 30 minutes. (The mixtureof xylazine and ketamine anesthesia was used in the scintigraphy studies. Dynamic scintigrams were obtained over the firsthalf hour with frames of 1 minute following the administration of the labeled compound.)


HPLC studies, and also, the retention times of related com-pounds were different from each other as seen in Figure 3.Beside, the results obtained from this study clearly showed thatBLMG could be successfully radioiodinated using iodogen asan oxidation agent. HPLC conditions are given in Table 1.

Lipophilicity (partition coefficient)

The n-octanol/water partition coefficient (lipophilicity) of131I-BLMG was determined, and the lipophilicity was foundto be - 0.68 – 0.1 (n = 3). It was reported that the n-octanol/water partition coefficient of BLM was - 0.52 according tothe ACD/lopP algorithm program.42 It is known that logPhas been calculated for the uncharged molecule theoretically.Theoretical lipophilicity of 131I-BLMG could not be calcu-lated due to the charge of this molecule.

Stability studies

Stability in the human serum was investigated at 0, 30, 60,180, and 1440 minutes after radiolabeling. The results of theserum stability experiments demonstrated that *50%–60%

of 131I-BLMG existed as an intact complex in the human se-rum within 1440 minutes as seen in Figure 4. Hence, theperiod of stability of 131I-BLMG is sufficient for imagingprocedures.

Results of scintigraphic studies

Figure 5 shows the dynamic image corresponding to 30minutes after the administration of 131I-BLMG. Figure 6 in-dicates the static scintigram corresponding to 30 minutesafter the administration of 131I-BLMG. As seen on this scin-tigram, 131I-BLMG was eliminated via kidneys and accu-mulated in the stomach and the bladder within 30 minutes,and some thyroid uptake was observed. This also indicates aradiolabeling yield of about 60% for this compound. After 4hours, the activity was completely cleared from the bladder.On the other hand, it was also observed that 131I radioac-tivity remained for a sufficiently long time in the metabolismand was not rapidly cleared.

Biodistribution studies

131I-BLMG at 60% radiochemical purity [specific activity:3.7 MBq (100 lCi)/lg radiolabeled compound per rat] wasinjected to rats (for each time intervals, n = 3). Figure 7 rep-resents the biodistribution results of 131I-BLMG obtained at30, 120, and 240 minutes after its administration to the rats.The %ID/g values of the radiolabeled BLMG in the stomach,bladder, prostate, and spinal cord were 1.16, 3.32, 0.39, and0.43 at 120 minutes, respectively. It is obvious that the ac-tivity in none of the followed organs at none of the measuredtime points exceeded 3.5% of the injected activity. Very lowaccumulation in the liver and the spleen is in particular fa-vorable, since accumulation in the liver and the spleen isoften considered as a sign of poor biocompatibility. No sig-nificant deposition of the released activity in the thyroid isimportant, but indirect demonstration of the 131I-BLMGstability in vivo is. The uptake in these organs decreased withtime. Significant amount of radioactivity was also seen in theprostate, the spinal cord, and the large intestine. Radio-activity in these organs was not cleared after 240 minutes.Biodistribution results agreed well with that of scintigraphicresults obtained with this compound. In this study, a sig-nificantly positive correlation was shown in lung–kidney(r = 0.805, p < 0.02), lung–spinal cord (r = 0.920, p < 0.04), kid-ney–spleen (r = 0.867, p < 0.03), thyroid–blood (r = 0.978,p < 0.02), stomach–prostate (r = 0.842, p < 0.04), blood–kidney(r = 0.940, p < 0.02), and prostate–stomach (r = 0.891, p < 0.02)in rat’s organs.

FIG. 6. Static scintigram of 131I-BLMG, which was ad-ministered to a rabbit via the ear vein in 30 minutes. (Themixture of xylazine and ketamine anesthesia was used inthe scintigraphy studies. Static image was obtained fromposterior projection following the administration of the131I-BLMG.)

FIG. 7. Activity (%ID/g) of 131I-BLMG in major organs of the rats inthe given time intervals (n = 3); errorbars mean standard deviation; %ID/g = percent of the injected dose pergram tissue. All the presented data aredecay corrected. S.I., small intestine;L.I., large intestine.


In vivo targeting activities of 131I-BLMG and 131I-BLM

Efficiencies of 131I-BLMG and 131I-BLM to target humancolon carcinoma xenographies (Caco-2 cell line xenografts)were explored in biodistribution studies. At various timesafter 131I-BLMG and 131I-BLM injection, blood, the tumor,and the normal organs were analyzed to determine theamount of each radionuclide retained per gram of tissue.Biodistribution results (given as %ID/g vs. organ, organ-to-muscle ratio vs. organs) of 131I-BLM and 131I-BLMG in Balb/C mice tissues are given in Figures 8–11. The high values oforgan-to-muscle ratio for 131I-BLM were observed within 30minutes after the administration of 131I-BLM in the tumor,the bladder, the breast, the spleen, and the lung. Someamount of the radioactivity was seen in the stomach. Sto-mach-to-muscle ratios were 1.17, 0.51, and 1.19 at 30, 120,and 240 minutes, respectively. In addition to this, the blad-der-to-muscle ratio was highest at 30 minutes. The liver-to-

muscle ratios of 131I-BLM were 0.64, 0.26, and 0.66 in the timeintervals. Activity uptake in the thyroid and, evenmore, inthe stomach is a (well-known) strong indication for thepresence of radioiodide. Therefore, it is to be assumed thatelimination of radioactivity is mainly due to the clearance ofradioiodide beside the metabolism of 131I-BLM. Radioactivityin this organ was not cleared after 240 minutes. After intra-venous (i.v.) administration of 131I-BLMG, radioactivity waswidely distributed into most tissues. Blood radioactivity ki-netics displayed a maximum with levels of around 1.3%ID/gat 240 minutes postinjection (p.i.). After administration of131I-BLMG, radioactivity was widely distributed into mosttissues. All tissues showed high uptake rapidly achieved 30minutes p.i. The highest concentrations were found in thelungs, the spinal cord, and the stomach. The highest valuesof 131I-BLMG in the organ-to-muscle ratio were observedin the bladder, the stomach, the spinal cord, the lung,and the tumor within 30 minutes after the administration of

FIG. 8. Activity (%ID/g) of 131I-BLMin major organs of the Balb/C mice inthe given time intervals (n = 3); errorbars mean standard deviation;%ID = percent of the injected dose pergram tissue. All the presented data aredecay corrected.

FIG. 9. Activity (%ID/g) of 131I-BLMG in major organs of the Balb/Cmice in the given time intervals (n = 3);error bars mean standard deviation;%ID = percent of the injected dose. Allthe presented data are decay corrected.

FIG. 10. Organ-to-muscle ratio of131I-BLM in the Balb/C mice in thegiven time intervals (n = 3); error barsmean standard deviation; %ID = per-cent of the injected dose per gram tis-sue. All the presented data are decaycorrected.


131I-BLMG. The lowest radioactivity levels were observed inthe head and the muscles. At 240 minutes p.i., most of theradioactivity has been eliminated from the tissues, except forthe stomach, the liver, and the spinal cord. In contrast, tumoruptake did not vary significantly over the time frame stud-ied, and remained at around 2–3%ID/g globally. Moreover,radioactivity accumulated in the thyroid and the stomach asgenerally observed with molecules directly radiolabeled withiodine. Stomach-to-muscle ratios of 131I-BLMG were ob-tained as 3.32, 11.22, and 19.12 at 30, 120, and 240 minutes,respectively. The highest value of the bladder-to-muscle ratiowas observed at 30 minutes as *24. The ratio in the tumordecreased with time.

There were also significant relations between breast andheart ( p < 0.01); breast and large intestine ( p < 0.05); bloodand large intestine ( p < 0.05); blood and kidney ( p < 0.05);spinal cord and blood ( p < 0.01); muscle and small intes-tine ( p < 0.01); muscle and fat ( p < 0.05); breast and tumor( p < 0.01); testis and large intestine ( p < 0.01); testis and blood( p < 0.05); and testis and spinal cord ( p < 0.05).

Figure 12 represents the tumor-to-large intestine ratio of131I-BLMG, and 131I-BLM was obtained 240 minutes aftertheir administration to mice. High levels of tumor-to-largeintestine ratios of 131I-BLMG and 131I-BLM were found as7.81 and 2.29 at 30 minutes, respectively. These resultsshowed that 131I cleared away from the large intestinequickly, but accumulated gradually in the tumor.

Discussion

In a recent study, BLMs were separately labeled with 131I,and radiopharmaceutical potentials were investigated usinganimal models by Avcıbasxı et al.43 In this respect, they havereported that these labeled compounds showed high uptakein the stomach, the bladder, the prostate, the testicle, and thespinal cord in rats, and the scintigraphies of radiolabeledisomers (131I-A2 and 131I-B2) were similarly found with thatof 131I-BLM.

Pharmacokinetics of BLM was compared with those of99mTc-, 111In-, and 57Co-BLM, and 67Ga citrate in mice bear-ing a transplanted KHJJ tumor by Krohn et al.44 It wasindicated that the in vivo kinetics and stability of 123I- and57Co-BLM were similar: both were acceptable, although notequivalent, tag for BLM and, along with 67Co citrate, bothhad biologic properties suitable for tumor detection. Both99mTc- and 111In-BLM dissociated rapidly in vivo and hencedid not represent legitimate tags for BLM. Tumor-to-bloodand tumor-to-liver ratios were higher for 123I-BLM thanfor 67Ga- or 57Co-BLM. Antunes et al. have described their firstPET tracer for extracellular b-glucuronidase (b-GUS), fluor-oethylamine glucuronic acid labeled with 18F ([18F]-FEAnGA),which consists of an [18F]-fluoroethylamine ([18F]-FEA) groupbound to a glucuronic acid via a self-immolative nitrophenylspacer. [18F]-FEAnGA was synthesized by alkylation of itsimidazole carbamate precursor with [18F]-FEA, followed bydeprotection of the sugar moiety with NaOH in the 10–20%overall radiochemical yield. At the end of this study, theycarried out a micro-PET study in mice bearing tumor. In apreliminary micro-PET study, in mice bearing both CT26 andCT26m_GUS tumors, [18F]-FEAnGA exhibited a 2-fold higherretention of radioactivity in the tumor expressing b-GUS thanin the control tumor. [18F]-FEA did not show any difference intracer uptake between tumors.38

In the presence of BLMG, electrophilic aromatic substitu-tion reactions might probably take place in the 2 position ofthe imidazole ring on the compound. This is supported bythe given m/z values of fragment 587, 1197, 1465, 1851, 1724,1699, and 1564. The stability of radioiodinated compoundswas long enough to be able to complete the scintigraphicstudies. For these reasons, the 131I-BLMG was directly in-jected to the experimental animals without needing anyseparation or purification procedures.

FIG. 11. Organ-to-muscle ratio of131I-BLMG in the Balb/C mice in thegiven time intervals (n = 3); error barsmean standard deviation; %ID = per-cent of the injected dose per gram tis-sue. All the presented data are decaycorrected.

FIG. 12. Tumor to large intestine ratio of 131I-BLM and131I-BLMG.


It is well known that many aromatic amines, such as b-naphthylamine and 4-aminobiphenyl, are known to inducetumors, most notably in the urinary bladder.45 N-hydroxyl-ation of arylamines has been demonstrated to be involved inproducing toxicity from these compounds.46,47 Another ma-jor metabolic pathway for aromatic amines, which maycompete with N-hydroxylation and thereby influence carci-nogenic potential, is N-glucuronidation.48,49 Glucuronidationof xenobioics is generally considered to be a significant stepin their detoxification and elimination from the body.However, N-glucuronides are labile and may be easily hy-drolyzed to the parent amine derivative under weakly acidicconditions, which exist generally in the urinary bladder.50 Asis expected, the scintigrapic image of the glucuronide de-rivative of 131I-BLM (131I-BLMG) supported this hypothesis.Therefore, activity in the bladder was probably due to ac-cumulation of the 131I-BLMG. In addition to this, high ac-tivity in the urogenital zone was not only due to uptake ofthe 131I-BLMG in the bladder but also due to the accumula-tion of a radiolabeled glucuronide derivative of BLM in theprostate and the testis.

In case of administration of phenolphthalein (PPH) witha regular dose, it is metabolized as phenolphthalein-O-glucuronide (PPHOG) in the intestinal lumen and in theliver and excreted largely as this glucuronide conjugate.51

The intestine contains significant amounts of the enzymeb-glucuronidase as a result of enzymatical hydrolyses ofthe glucuronide conjugate resulting in the formation offree-131I-PPH. This may then be reabsorbed and transportedto the liver and undergo reconjugation and re-excretion.This behavior is termed enterohepatic recirculation andmakes a significant contribution, prolonging the half-life ofthe drug in the body, with the obvious result of potentialof the pharmacological action of the 131I-PPH. In the bio-distribution results obtained for BLMG, it was shown that131I-BLMG was significantly localized in the large intes-tine within 30 minutes as is shown in Figure 9. Biber et al.observed similar results with the glucuronide derivative of99mTc-labeled b-estradiol (1,3,5,[10]-estratriene-3,17b-diol)attached to diethylenetriamine pentaacetic acid.39,52 Resultssupported that intestines express the enzyme b-glucuronidase,which hydrolyzes the glucuronide bond similar to radioio-dinated PPHG.11

As seen in Figures 8 and 9, 131I-BLM exhibited about 2-foldhigher retention of radioactivity than that of 131I-BLMG inthe tumor. The liver-to-muscle ratios of BLMG were obtainedas high values than those of BLM. This was probably becauseof the significant amounts of b-glucuronidase activity in theliver.

A specific enzyme detoxifying BLM, BLM hydrolase(BLMH), was recently put forward as a plausible candidatefor associations with mutagen sensitivity (Caporaso, 1999).53

BLMH, a neutral cysteine protease with exopeptidase activ-ity that detoxifies BLM through deamination of the beta-aminoalanine moiety,54 has been found in most humantissues, including peripheral blood leukocytes. In fact, resis-tance to BLM chemotherapy, observed in some cancers, hasbeen postulated to result from overactive BLMH. BLM de-toxification may also involve N-acetyltransferases (NATs). InStreptomyces verticillus, the BLM-producing antinomycetebacterium, resistance to BLM is conferred by an NAT en-zyme.55

With respect to the explanations given above about someglucuronide conjugate, Biber et al. synthesized a derivativeof estradiol glucuronide that can be labeled with 99mTc andinvestigated its radiopharmaceutical potential using imag-ing and biodistribution studies. In conclusion, all thesestudies indicated that 99mTc-estradiol–glucuronide conju-gates have not showed estrogen receptors specificity. En-zymatic mechanism seemed more effective than receptorspecificity in tumor uptake. It was suggested that radio-nuclide-labeled estrogen–glucuronide conjugates may bea new series of radiopharmaceuticals for the enzymeb-glucuronidase-rich tissues and tumors.39 Kocan et al. in-vestigated the radiopharmaceutical potential of 99mTc-BLMand 99mTc-BLMG. They found that maximum uptakes of99mTc-BLM and 99mTc-BLMG metabolized as N-glucuro-nide were observed within 2 hours in the liver, the bladder,and the spinal cord for 99mTc-BLM and the lung, the liver,the kidney, the large intestine, and the spinal cord for99mTc-BLMG, respectively.56

In conclusion, 131I-BLMG, which has diagnostic and ther-apeutic application potentials in nuclear medicine, was firstsynthesized and radioiodinated using the iodogen methodand investigated to evaluate its biodistribution in the humancolon cancer xenograph–bearing Balb/C mice. 131I-BLMGwas obtained in high radiochemical purity and high yield bydirect electrophilic iodination of BLMG. Radiolabeling ofBLMG with 131I means that it can also be radioiodinated withother radioiodine isotopes such as 123I, 124I, and 125I undersimilar conditions. 131I-BLMG has shown specifity in theprostate, the large intestine, and the spinal cord in the bio-distribution studies in male rats. Tumor-bearing mice modelstudies demonstrated that the highest uptake of 131I-BLMGwas obtained in the tumor in the first 30 minutes. Radi-olabeled BLMG proved a useful tool for assessing the in vivobehavior of the drug in mice bearing subcutaneous Caco-2human colorectal adenocarcinoma, consequently validatingits potential for the treatment of colorectal cancers.

Acknowledgments

The authors thank Celal Bayar University Research Fund(Contract no. 2007 FEF 007) for financial support. The au-thors also thank Dr. _Ilker Medine; Research Assistant FerayKocan; M.Sc. student Gokcen Topal; and Ph.D. student Ya-semin Parlak for their technical assistance during the animalexperiments and scintigraphic studies.

Disclosure Statement

There is no any conflict of interest among the authors inthis study.

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Documents

Radiolabeling of Bleomycin-Glucuronide with 131 I and Biodistribution Studies Using Xenograft Model of Human Colon Tumor in Balb/C Mice