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Original Article
Technetium-99m-labeled lapachol as an imaging probe for breast tumor identification
Lapachol marcado con tecnecio 99m como sonda de imágenes para la identificación de tumores de mama
Sued E.M. Mirandaa, Janaína A. Lemosa, Renata S. Fernandesb, Flaviano Melo Ottonib, Ricardo J. Alvesb, Alice Ferrettic, Domenico Rubelloc,
Corresponding author
domenico.rubello@libero.it

Corresponding authors.
, Valbert N. Cardosoa, André L.B. de Barrosa,
Corresponding author
a Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Minas Gerais, Brazil
b Department of Pharmaceutical Products, Faculty of Pharmacy, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Minas Gerais, Brazil
c Department of Nuclear Medicine, Radiology, NeuroRadiology, Medical Physics, Clinical Laboratory, Microbiology, Pathology, Santa Maria della Misericordia Hospital, Rovigo, Italy
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          "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Radiochemical purity of <span class="elsevierStyleSup">99m</span>Tc-LAP over time in saline&#44; at room temperature&#44; and in the presence of plasma&#44; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>6&#41;&#46;</p>"
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Cancer is a major health issue worldwide&#46; In 2015&#44; 8&#46;8 million people died as a consequence of this disease&#46; Breast cancer is among the most common types of tumors with high mortality&#46;<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">1</span></a> It is well known that the development of more sensitive and specific diagnostic methods is of great importance since an early diagnosis is essential to successfully treat tumors&#46;<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">2</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">The field of nuclear medicine can provide noninvasive techniques for early diagnosis&#44; by visual assessment and quantitative measurement of molecular and biochemical properties of cells&#44; including tumor cells&#46; These techniques are based on physiological and biochemical changes instead of structural alterations&#46;<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">3</span></a> The major advantages of these approaches over others include high sensitivity&#44; quantitative measurements&#44; and the absence of a tissue penetration limit&#46; Technetium-99m &#40;<span class="elsevierStyleSup">99m</span>Tc&#41; has mostly been used to label radiopharmaceuticals&#44; because of its suitable physical and chemical characteristics&#44; large availability in nuclear medicine laboratories&#44; and relatively low isotope cost&#46;<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">4</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">Efforts have been made in order to develop new radiopharmaceuticals to investigate tumors&#46;<a class="elsevierStyleCrossRefs" href="#bib0120"><span class="elsevierStyleSup">2&#44;5&#44;6</span></a> In this context&#44; the use of natural products is an interesting alternative&#46; Natural compounds correspond to 75&#37; of new drugs designed for cancer treatment&#46; Moreover&#44; there are currently more than 30 naturally substances in different stages of clinical studies for cancer therapy and diagnosis&#46;<a class="elsevierStyleCrossRefs" href="#bib0145"><span class="elsevierStyleSup">7&#44;8</span></a></p><p id="par0020" class="elsevierStylePara elsevierViewall">Lapachol &#40;LAP&#41; is a natural compound &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#44; belonging to the naphthoquinone group that has been widely used in traditional medicine to treat various illnesses&#44; including cancer&#46; It was isolated for the first time in 1882 by E&#46; Paternal from the core and bark of <span class="elsevierStyleItalic">Tabebuia avellanedae</span> tree&#46; A myriad of biological effects is related to lapachol&#44; such as antibiotic&#44; analgesic&#44; antiulcerogenic&#44; antioxidant&#44; antiviral&#44; trypanocidal&#44; and anticancer action&#46;<a class="elsevierStyleCrossRefs" href="#bib0155"><span class="elsevierStyleSup">9&#8211;12</span></a> Several studies have been carried out with lapachol in order to evaluate their antitumor activity being considered a promising drug for tumor therapy&#46;<a class="elsevierStyleCrossRefs" href="#bib0160"><span class="elsevierStyleSup">10&#44;13</span></a></p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0025" class="elsevierStylePara elsevierViewall">Therefore&#44; the aim of this study was to evaluate technetium-99m-labeled lapachol as an imaging probe for 4T1 breast tumor identification&#46; To achieve this purpose&#44; lapachol was labeled with technetium-99m&#44; radiochemical purity and <span class="elsevierStyleItalic">in vitro</span> stability were determined&#46; We also evaluated blood clearance&#44; in healthy mice&#44; and biodistribution and scintigraphic images&#44; in 4T1 tumor-bearing mice&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Material and methods</span><p id="par0030" class="elsevierStylePara elsevierViewall">Lapachol and SnCl<span class="elsevierStyleInf">2</span>&#183;2H<span class="elsevierStyleInf">2</span>O were purchased from Sigma-Aldrich &#40;S&#227;o Paulo&#44; Brazil&#41;&#46; RPMI 1640 Medium&#44; fetal bovine serum&#44; penicillin&#44; streptomycin&#44; and trypsin EDTA 0&#46;25&#37; were purchased from Gibco-Invitrogen &#40;Waltham&#44; MA&#44; USA&#41;&#46; <span class="elsevierStyleSup">99m</span>Tc was obtained from an alumina-based <span class="elsevierStyleSup">99</span>Mo&#47;<span class="elsevierStyleSup">99m</span>Tc generator &#40;IPEN&#44; S&#227;o Paulo&#44; Brazil&#41;&#46; All other chemicals were of analytical grade&#46; The subcutaneous tumor model was established in 6&#8211;8 week female BALB&#47;c mice purchased from CEBIO-UFMG &#40;Belo Horizonte&#44; Brazil&#41;&#46; All animal studies were approved by the local Ethics Committee for animal experiments &#40;CEUA&#47;UFMG&#41; under protocol 283&#47;2017&#46;</p><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Radiolabeling of Lapachol</span><p id="par0035" class="elsevierStylePara elsevierViewall">A solution containing lapachol &#40;LAP&#41; in PBS &#40;pH 7&#46;4&#41; was prepared &#40;1<span class="elsevierStyleHsp" style=""></span>mg&#47;ml&#41;&#46; In a sealed vial containing 0&#46;2<span class="elsevierStyleHsp" style=""></span>ml of this solution was added 50<span class="elsevierStyleHsp" style=""></span>&#956;g of SnCl<span class="elsevierStyleInf">2</span>&#183;H<span class="elsevierStyleInf">2</span>O solution in 0&#46;25<span class="elsevierStyleHsp" style=""></span>mol&#47;L HCl &#40;1&#46;0<span class="elsevierStyleHsp" style=""></span>mg&#47;ml&#41;&#46; Then&#44; the pH was adjusted to 7&#46;4 using NaOH solution &#40;1<span class="elsevierStyleHsp" style=""></span>mol&#47;L&#41;&#44; and vacuum was performed to the vial&#46; An aliquot of 0&#46;1<span class="elsevierStyleHsp" style=""></span>ml of Na<span class="elsevierStyleSup">99m</span>TcO<span class="elsevierStyleInf">4</span> &#40;3&#46;7<span class="elsevierStyleHsp" style=""></span>MBq&#41; was added to the flask&#46; The solution was kept at room temperature for 15<span class="elsevierStyleHsp" style=""></span>min&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Radiochemical purity</span><p id="par0040" class="elsevierStylePara elsevierViewall">Radiochemical yield was determined by thin layer chromatography &#40;TLC-SG&#44; Merck&#44; Darmstadt&#44; Germany&#41; using acetone as the mobile phase to quantify <span class="elsevierStyleSup">99m</span>TcO<span class="elsevierStyleInf">4</span><span class="elsevierStyleSup">&#8722;</span>&#46; The radioactivity of the strips was determined by a gamma counter &#40;Wallac Wizard 1470-020 Gamma Counter&#44; PerkinElmer Inc&#46;&#44; Waltham&#44; Massachusetts&#44; USA&#41;&#46; <span class="elsevierStyleSup">99m</span>TcO<span class="elsevierStyleInf">2</span> was removed from the preparation using a 0&#46;22<span class="elsevierStyleHsp" style=""></span>&#956;m syringe filter&#46;<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">2</span></a></p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085"><span class="elsevierStyleItalic">In vitro</span> stability</span><p id="par0045" class="elsevierStylePara elsevierViewall">Tests in saline 0&#46;9&#37; &#40;w&#47;v&#41; and in mouse plasma were performed to evaluate the stability of the radiolabeled complex&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Saline stability</span><p id="par0050" class="elsevierStylePara elsevierViewall">TLC-SG was used to evaluate the stability of the radiolabeled complex&#46; The labeled solution was kept at room temperature and aliquots were taken at 1&#44; 2&#44; 4&#44; 6&#44; 8 and 24<span class="elsevierStyleHsp" style=""></span>h for determination of radiochemical purity&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Plasma stability</span><p id="par0055" class="elsevierStylePara elsevierViewall">A volume of 90<span class="elsevierStyleHsp" style=""></span>&#956;l of the <span class="elsevierStyleSup">99m</span>Tc-LAP solution was incubated&#44; under agitation&#44; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C &#40;Dubnoff Bath MA-095&#47;CF&#41; with 1&#46;0<span class="elsevierStyleHsp" style=""></span>ml of fresh mouse plasma&#46; Radiochemical stability was determined by TLC-SG from samples taken at 1&#44; 2&#44; 4&#44; 6&#44; and 8<span class="elsevierStyleHsp" style=""></span>h after incubation&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Partition coefficient</span><p id="par0060" class="elsevierStylePara elsevierViewall">Aliquots of 0&#46;1<span class="elsevierStyleHsp" style=""></span>ml of the <span class="elsevierStyleSup">99m</span>Tc-LAP &#40;0&#46;37<span class="elsevierStyleHsp" style=""></span>MBq&#41; were added to tubes containing 2&#46;0<span class="elsevierStyleHsp" style=""></span>ml of n-octanol&#47;water &#40;1&#58;1&#41;&#46; The tubes were stirred vigorously for 5<span class="elsevierStyleHsp" style=""></span>min&#44; and then the mixture was left to rest for 120<span class="elsevierStyleHsp" style=""></span>min for phase separation&#46; Afterward&#44; aliquots of 0&#46;5<span class="elsevierStyleHsp" style=""></span>ml from each phase were collected&#46; Partition coefficient was determined using radioactivity measured in each aliquot by an automatic scintillation apparatus&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Blood clearance</span><p id="par0065" class="elsevierStylePara elsevierViewall">Aliquots of 3&#46;7 MBq of <span class="elsevierStyleSup">99m</span>Tc-LAP were injected intravenously into healthy BALB&#47;c mice&#46; An incision was made in the tail of the animals and blood was collected in pre-weighed tubes at times of 1&#44; 5&#44; 10&#44; 15&#44; 30&#44; 45&#44; 60&#44; 90&#44; 120&#44; 240&#44; 480 and 1440<span class="elsevierStyleHsp" style=""></span>min after administration&#46; The tubes were weighed and their radioactivity determined by a gamma counter&#46; These data were used to plot a percentage of dose injected per gram tissue &#40;&#37; ID&#47;g&#41; <span class="elsevierStyleItalic">versus</span> time&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Cell culture</span><p id="par0070" class="elsevierStylePara elsevierViewall">4T1 cell line was grown in RPMI 1640 medium&#44; supplemented with 10&#37; &#40;v&#47;v&#41; of fetal bovine serum&#44; penicillin &#40;100<span class="elsevierStyleHsp" style=""></span>IU&#47;ml&#41;&#44; and streptomycin &#40;100<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;ml&#41;&#46; Cells were kept in humidified air containing 5&#37; CO<span class="elsevierStyleInf">2</span> at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The cells were grown to con&#64258;uence and harvested by trypsinization&#46;</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Tumor cell inoculation</span><p id="par0075" class="elsevierStylePara elsevierViewall">Aliquots of 2&#46;5<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span> 4T1 cells in RPMI medium was injected &#40;SC&#41; into the right tight of female BALB&#47;c mice&#46; The mice were kept in an area with light control&#44; with free access to water and food&#46; Tumor cells were allowed to grow <span class="elsevierStyleItalic">in vivo</span> for 7 days&#44; once the tumor volume reached about 100<span class="elsevierStyleHsp" style=""></span>mm<span class="elsevierStyleSup">3</span>&#46;</p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Scintigraphic images</span><p id="par0080" class="elsevierStylePara elsevierViewall">Aliquots of 18 MBq of <span class="elsevierStyleSup">99m</span>Tc-LAP were injected intravenously into tumor-bearing BALB&#47;c mice&#46; At 1&#44; 4&#44; 8 and 24<span class="elsevierStyleHsp" style=""></span>h after administration the animals were anesthetized with a mixture of ketamine &#40;80<span class="elsevierStyleHsp" style=""></span>mg&#47;kg&#41; and xylazine &#40;15<span class="elsevierStyleHsp" style=""></span>mg&#47;kg&#41; and placed horizontally under a gamma camera &#40;Nuclide TM TH 22&#44; Mediso&#44; Hungary&#41;&#46; The images were obtained with a LEHR &#40;Low Energy High Resolution&#41; collimator and 128<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>128<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>32 dimension matrices with a 300<span class="elsevierStyleHsp" style=""></span>s acquisition time&#44; using a 20&#37; symmetrical window with a fixed energy peak at 140<span class="elsevierStyleHsp" style=""></span>keV&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Biodistribution studies</span><p id="par0085" class="elsevierStylePara elsevierViewall">Aliquots of 3&#46;7<span class="elsevierStyleHsp" style=""></span>MBq of <span class="elsevierStyleSup">99m</span>Tc-LAP were injected intravenously into tumor-bearing BALB&#47;c mice&#46; At 1&#44; 4&#44; 8 and 24<span class="elsevierStyleHsp" style=""></span>h post-injection organs such as spleen&#44; heart&#44; stomach&#44; liver&#44; small and large intestine&#44; muscle&#44; bone&#44; lungs&#44; kidneys&#44; thyroid&#44; and tumor were removed&#44; dried on filter paper and weighed&#46; The radioactivity in each organ was determined by a gamma counter&#46; A standard dose containing the same injected amount was counted simultaneously in a separate tube&#44; which was defined as 100&#37; radioactivity&#46; The results were expressed as the percentage of injected dose per gram of tissue &#40;&#37;ID&#47;g&#41;&#46;</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Statistical analysis</span><p id="par0090" class="elsevierStylePara elsevierViewall">Data are expressed as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SD&#46; Statistical analyses were performed using GraphPad PRISM&#44; version 5&#46;00 software &#40;GraphPad Software Inc&#46;&#44; La Jolla&#44; CA&#44; USA&#41;&#46; The difference among experimental groups was tested using the one-way analysis of variance &#40;ANOVA&#41;&#44; followed by Tukey&#39;s test&#46; Differences were considered statistically significant when <span class="elsevierStyleItalic">P</span> values were &#60;0&#46;05&#46;</p></span></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Results</span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Radiochemical purity and partition coefficient</span><p id="par0095" class="elsevierStylePara elsevierViewall">Radiochemical yields of the <span class="elsevierStyleSup">99m</span>Tc-LAP&#44; evaluated by TLC-SG after purification&#44; was 98&#46;9<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;9&#37; &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>6&#41;&#46; The <span class="elsevierStyleSup">99m</span>TcO<span class="elsevierStyleInf">2</span> was removed from the solution through the syringe filter &#40;pore diameter<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;22<span class="elsevierStyleHsp" style=""></span>&#956;m&#41;&#46; By using the filter&#44; the radiocolloids were retained while the other compounds&#44; <span class="elsevierStyleSup">99m</span>TcO<span class="elsevierStyleInf">4</span><span class="elsevierStyleSup">&#8722;</span> and <span class="elsevierStyleSup">99m</span>Tc-LAP&#44; freely pass through the filter pore&#46;<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">2</span></a></p><p id="par0100" class="elsevierStylePara elsevierViewall">The partition coefficient of the <span class="elsevierStyleSup">99m</span>Tc-LAP was determined by the ratio between n-octanol and water&#46; Log<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">P</span> of the <span class="elsevierStyleSup">99m</span>Tc-LAP was &#8722;1&#46;55<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;17&#44; indicating it as hydrophilic complex&#46;</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0145"><span class="elsevierStyleItalic">In vitro</span> stability</span><p id="par0105" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a> shows the radiochemical stability curve for <span class="elsevierStyleSup">99m</span>Tc-LAP in saline&#44; at room temperature&#44; and mouse plasma&#44; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C under stirring at 1&#44; 2&#44; 4&#44; 6&#44; 8 and 24<span class="elsevierStyleHsp" style=""></span>h after the labeling process&#46; Results indicate radiochemical purity values above 90&#37;&#44; for all time-points&#44; both in saline and plasma&#46; These data demonstrate an excellent stability over time&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0150">Blood clearance</span><p id="par0110" class="elsevierStylePara elsevierViewall">Blood clearance curve of <span class="elsevierStyleSup">99m</span>Tc-LAP is demonstrated in <a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#46; The complex&#44; after injection into healthy BALB&#47;c female mice &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>7&#41;&#44; presents blood levels decaying in a biphasic manner&#44; with a fast half-life &#40;<span class="elsevierStyleItalic">t</span><span class="elsevierStyleInf">1&#47;2&#945;</span>&#41; of 3&#46;3<span class="elsevierStyleHsp" style=""></span>min&#44; a slow half-life &#40;<span class="elsevierStyleItalic">t</span><span class="elsevierStyleInf">1&#47;2&#946;</span>&#41; of 50&#46;0<span class="elsevierStyleHsp" style=""></span>min&#44; and area under the curve &#40;AUC&#41; of 1909&#37;ID<span class="elsevierStyleHsp" style=""></span>min<span class="elsevierStyleSup">&#8722;1</span>&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0155">Scintigraphic images and biodistribution studies</span><p id="par0115" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a> shows the scintigraphic images of <span class="elsevierStyleSup">99m</span>Tc-LAP administered intravenously into 4T1-tumor-bearing mice &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>7&#41;&#46; It could be noted a high uptake in kidneys&#44; at 1<span class="elsevierStyleHsp" style=""></span>h after injection&#44; and high uptake in intestines at longer time-points&#46; That could be related to both renal and hepatobiliary elimination-route of the radiolabeled complex&#46;<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">14</span></a> In addition&#44; no significant uptake was observed in other organs&#44; mainly the thyroid&#44; which confirms the <span class="elsevierStyleItalic">in vitro</span> stability data&#44; since it is well known that free technetium is&#44; preferentially&#44; taken up by this organ&#46;<a class="elsevierStyleCrossRefs" href="#bib0125"><span class="elsevierStyleSup">3&#44;5</span></a></p><elsevierMultimedia ident="fig0020"></elsevierMultimedia><p id="par0120" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a> shows the biodistribution profile of <span class="elsevierStyleSup">99m</span>Tc-LAP after administration into 4T1-tumor-bearing mice &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>7&#41;&#46; These results confirm scintigraphic images data&#44; once could be noted a high uptake in kidneys&#44; liver and intestines&#44; and no significant uptake by the other organs&#46; Tumor uptake is also higher than the contralateral muscle at all evaluated times&#46; It is interesting to mention that this uptake increases over time&#44; reaching the highest value at 24<span class="elsevierStyleHsp" style=""></span>h post-injection &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a> &#8211; inset&#41;&#46;</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia><p id="par0125" class="elsevierStylePara elsevierViewall">Tumor-to-muscle ratios obtained from biodistribution studies are shown in <a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>&#46; Worth mentioning that ratios have increased over time&#44; reaching 3&#46;3<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;3 at 4<span class="elsevierStyleHsp" style=""></span>h&#59; 4&#46;1<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;6 at 8<span class="elsevierStyleHsp" style=""></span>h&#59; and 4&#46;4<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;2 at 24<span class="elsevierStyleHsp" style=""></span>h post-injection&#46; These findings suggest that <span class="elsevierStyleSup">99m</span>Tc-LAP specifically accumulates in the tumor tissue&#46;</p><elsevierMultimedia ident="fig0030"></elsevierMultimedia></span></span><span id="sec0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0160">Discussion</span><p id="par0130" class="elsevierStylePara elsevierViewall">Cancer is the second cause of death worldwide&#44; being responsible for one in every six deaths&#46; Breast cancer is the leading cause of death among women&#44; and it is the most frequently diagnosed type of tumor&#46;<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">15</span></a> An early diagnosis is crucial to a better prognostic and a successful treatment&#46;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">16</span></a> The need for a specific tumor probe&#44; led to the research in the field of natural products&#44; in order to discover new substances with tumor affinity&#46;<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">17</span></a> Therefore&#44; we aimed to radiolabel lapachol with technetium-99m in order to develop a new probe for breast tumor identification&#46;</p><p id="par0135" class="elsevierStylePara elsevierViewall">In this study&#44; we achieved a high labeling efficiency&#44; with radiochemical yields above 95&#37;&#46; American Pharmacopeia recommends that impurities should not exceed 10&#37; since a high content of impurities might lead to a low-quality image&#44; in addition&#44; to exposing the patient to unnecessary level of radioactivity&#46;<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">18</span></a> The filtration step is also an advantage in the process since the radiolabeled solution is purified from <span class="elsevierStyleSup">99m</span>TcO<span class="elsevierStyleInf">2</span>&#46;<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">2</span></a> Besides&#44; the labeled solution at physiological pH is optimal for intravenous injection&#46; Therefore&#44; the proposed protocol could be used for further assays&#46;</p><p id="par0140" class="elsevierStylePara elsevierViewall">Radiolabeling stability is also essential to the development of a new radiotracer&#46; Weak bond stability between <span class="elsevierStyleSup">99m</span>Tc and the molecule of interest may lead to metal detachment over time&#44; which can compromise biodistribution and scintigraphic images&#44; generating erroneous interpretations&#46;<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">5</span></a> An excellent stability &#40;above 90&#37;&#41;&#44; even over long periods&#44; was observed &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; This finding guarantees the use of the <span class="elsevierStyleSup">99m</span>Tc-LAP in further <span class="elsevierStyleItalic">in vivo</span> assays since the radiometal will remain stable in the complex throughout the study&#46;</p><p id="par0145" class="elsevierStylePara elsevierViewall">Blood levels of the <span class="elsevierStyleSup">99m</span>Tc-LAP &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>&#41; decline in a biphasic manner &#40;&#945; half-life<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>3&#46;3<span class="elsevierStyleHsp" style=""></span>min and &#946; half-life<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>50&#46;0<span class="elsevierStyleHsp" style=""></span>min&#41;&#46; A fast blood clearance is important to support early imaging since a minimal level of background radiation will be present resulting in higher signal-to-noise ratios&#46;<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">19</span></a></p><p id="par0150" class="elsevierStylePara elsevierViewall">From biodistribution studies and scintigraphic images in tumor-bearing mice could be noted high uptake in the kidneys&#44; suggesting that <span class="elsevierStyleSup">99m</span>Tc-LAP is eliminated mainly through the renal system&#46; This is in accordance with partition coefficient results since renal route eliminates hydrophilic molecules preferentially&#46;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">20</span></a> It was observed some uptake in liver and intestines&#44; indicating that fecal route also contributes for <span class="elsevierStyleSup">99m</span>Tc-LAP elimination&#46; Noteworthy is the low uptake in the thyroid that confirms the high labeling stability observed in <span class="elsevierStyleItalic">in vitro</span> assays&#46;<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">5</span></a></p><p id="par0155" class="elsevierStylePara elsevierViewall">Regarding the tumor uptake&#44; <span class="elsevierStyleSup">99m</span>Tc-LAP showed higher tumor accumulation compared with muscle&#44; used as a control tissue&#44; throughout the experiment&#46; Tumor-to-muscle ratios &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>&#41; evidenced the affinity for the tumor area of <span class="elsevierStyleSup">99m</span>Tc-LAP&#46; The ratios increase over time reaching &#8764;4&#46;5 within 24<span class="elsevierStyleHsp" style=""></span>h post-injection&#46; It is important to mention that tumor-muscle ratio greater than 1&#46;5 is required for a potential candidate as an imaging probe&#46;<a class="elsevierStyleCrossRefs" href="#bib0215"><span class="elsevierStyleSup">21&#44;22</span></a></p></span><span id="sec0105" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0165">Conclusion</span><p id="par0160" class="elsevierStylePara elsevierViewall">Lapachol was successfully labeled with technetium-99m&#44; showing high radiochemical yield and prolonged stability <span class="elsevierStyleItalic">in vitro</span>&#46; <span class="elsevierStyleSup">99m</span>Tc-LAP accumulated in 4T1 tumor tissues leading to high tumor-to-muscle ratios&#46; Therefore&#44; results obtained in this study suggest that <span class="elsevierStyleSup">99m</span>Tc-LAP can be used as a radiotracer for breast tumor identification&#46; Besides that&#44; the radiocomplex might be applied as a complementary tool to monitor treatment regimens in pre-clinical studies using 4T1 tumor models&#46;</p></span><span id="sec0110" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0170">Funding</span><p id="par0165" class="elsevierStylePara elsevierViewall">This research did not receive any specific grant from funding agencies in the public&#44; commercial&#44; or not-for-profit sectors&#46;</p></span><span id="sec0115" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0175">Conflict of interest</span><p id="par0170" class="elsevierStylePara elsevierViewall">None declared&#46;</p></span></span>"
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          "titulo" => "Resumen"
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          "titulo" => "Introduction"
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          "titulo" => "Material and methods"
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              "identificador" => "sec0015"
              "titulo" => "Radiolabeling of Lapachol"
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              "titulo" => "Radiochemical purity"
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              "titulo" => "In vitro stability"
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              "titulo" => "Saline stability"
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              "titulo" => "Plasma stability"
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              "titulo" => "Partition coefficient"
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              "titulo" => "Cell culture"
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              "titulo" => "Tumor cell inoculation"
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              "titulo" => "Scintigraphic images"
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              "identificador" => "sec0065"
              "titulo" => "Biodistribution studies"
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              "titulo" => "Statistical analysis"
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              "titulo" => "Radiochemical purity and partition coefficient"
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              "titulo" => "Scintigraphic images and biodistribution studies"
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          "identificador" => "xack404830"
          "titulo" => "Acknowledgments"
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          "titulo" => "References"
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    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2018-07-31"
    "fechaAceptado" => "2018-10-19"
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          "clase" => "keyword"
          "titulo" => "Keywords"
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          "palabras" => array:5 [
            0 => "Lapachol"
            1 => "Tumor"
            2 => "Technetium-99m"
            3 => "Diagnosis"
            4 => "Radiopharmaceutical"
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          "palabras" => array:5 [
            0 => "Lapachol"
            1 => "Tumor"
            2 => "Tecnecio-99m"
            3 => "Diagn&#243;stico"
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    "resumen" => array:2 [
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        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Aim</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Breast cancer is a health problem worldwide with high incidence and mortality rates&#46; It is well known that the development of more sensitive and specific diagnostic methods is of great importance since an early diagnosis is essential to successfully treat tumors&#46; Lapachol is a natural compound&#44; belonging to the naphthoquinone group that has been widely used in traditional medicine to treat various illnesses&#44; including cancer&#46; The aim of this study was to evaluate technetium-99m-labeled lapachol as an imaging probe for breast cancer identification&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">To achieve this purpose&#44; lapachol was labeled with technetium-99m&#44; radiochemical purity and <span class="elsevierStyleItalic">in vitro</span> stability were determined&#46; Blood clearance&#44; in healthy mice&#44; and biodistribution&#44; in 4T1 tumor-bearing mice&#44; were also evaluated&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Lapachol was successfully labeled with technetium-99m&#44; with high values of radiochemical yield &#40;95&#46;9<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;4&#37;&#41;&#46; <span class="elsevierStyleItalic">In vitro</span> stability showed that the radiolabeled complex remained stable for up to 24<span class="elsevierStyleHsp" style=""></span>h&#44; with values above 90&#37; for both saline and plasma &#40;95&#46;6<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;6&#37; and 96&#46;4<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;7&#37;&#44; respectively&#41;&#46; The radiolabeled complex decays in a biphasic manner&#44; with a half-life of distribution and elimination equal to 3&#46;3 and 50&#46;0<span class="elsevierStyleHsp" style=""></span>min&#44; respectively&#46; Biodistribution and scintigraphic images showed high uptake in organs of excretion &#40;kidneys&#44; liver&#44; and intestine&#41;&#46; It could be also noted that tumor uptake was higher than the muscle at all time points&#46; Tumor-to-muscle ratio reaches &#8764;4&#46;5 at 24<span class="elsevierStyleHsp" style=""></span>h after administration&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusion</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">These findings suggest that <span class="elsevierStyleSup">99m</span>Tc-Lapachol can be a potential diagnostic agent for breast tumors&#46;</p></span>"
        "secciones" => array:4 [
          0 => array:2 [
            "identificador" => "abst0005"
            "titulo" => "Aim"
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          1 => array:2 [
            "identificador" => "abst0010"
            "titulo" => "Methods"
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          2 => array:2 [
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            "titulo" => "Results"
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        "titulo" => "Resumen"
        "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Objetivo</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">El c&#225;ncer de mama es un problema de salud en todo el mundo con altas tasas de incidencia y mortalidad&#46; Es bien sabido que el desarrollo de m&#233;todos de diagn&#243;stico m&#225;s sensibles y espec&#237;ficos es de gran importancia&#44; ya que un diagn&#243;stico precoz es esencial para tratar con &#233;xito los tumores&#46; Lapachol es un compuesto natural&#44; perteneciente al grupo de la naftoquinona&#44; que se ha utilizado ampliamente en la medicina tradicional para tratar diversas enfermedades&#44; incluido el c&#225;ncer&#46; El objetivo de este estudio fue evaluar el lapachol marcado con tecnecio 99m como una sonda de im&#225;genes para la identificaci&#243;n del c&#225;ncer de mama&#46;</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">M&#233;todos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Para lograr este prop&#243;sito&#44; el lapachol se marc&#243; con tecnecio-99m&#44; se determin&#243; la pureza radioqu&#237;mica y la estabilidad in vitro&#46; Tambi&#233;n se evalu&#243; el aclaramiento en sangre en ratones sanos y la biodistribuci&#243;n en ratones con tumor 4T1&#46;</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Lapachol fue exitosamente marcado con tecnecio-99m&#44; con altos valores de rendimiento radioqu&#237;mico &#40;95&#46;9<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;4&#37;&#41;&#46; La estabilidad in vitro mostr&#243; que el complejo radiomarcado permaneci&#243; estable durante hasta 24<span class="elsevierStyleHsp" style=""></span>h&#44; con valores superiores al 90&#37; tanto para soluci&#243;n salina como para plasma &#40;95&#44;6<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#44;6&#37; y 96&#44;4<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#44;7&#37;&#44; respectivamente&#41;&#46; El complejo radiomarcado decae de forma bif&#225;sica&#44; con una vida media de distribuci&#243;n y eliminaci&#243;n igual a 3&#46;3 y 50&#46;0<span class="elsevierStyleHsp" style=""></span>min&#44; respectivamente&#46; La biodistribuci&#243;n y las im&#225;genes gammagr&#225;ficas mostraron una alta captaci&#243;n en los &#243;rganos de excreci&#243;n &#40;ri&#241;ones&#44; h&#237;gado e intestino&#41;&#46; Tambi&#233;n se puede observar que la captaci&#243;n tumoral fue mayor que en el m&#250;sculo en todos los puntos temporales&#46; La relaci&#243;n de tumor &#47; m&#250;sculo alcanza &#8764; 4&#46;5 a las 24<span class="elsevierStyleHsp" style=""></span>h despu&#233;s de la administraci&#243;n&#46;</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclusi&#243;n</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Estos hallazgos sugieren que <span class="elsevierStyleSup">99m</span>Tc-Lapachol puede ser un potencial agente de diagn&#243;stico para los tumores de mama&#46;</p></span>"
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        "nota" => "<p class="elsevierStyleNotepara" id="npar0005">Please cite this article as&#58; Miranda SE&#44; Lemos JA&#44; Fernandes RS&#44; Ottoni FM&#44; Alves RJ&#44; Ferretti A&#44; et al&#46; Lapachol marcado con tecnecio 99m como sonda de im&#225;genes para la identificaci&#243;n de tumores de mama&#46; Rev Esp Med Nucl Imagen Mol&#46; 2019&#59;38&#58;167&#8211;172&#46;</p>"
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        "nota" => "<p class="elsevierStyleNotepara" id="npar0010">The work was carried out in accordance with our institutes guidelines and&#44; as appropriate&#44; in accordance with the EU directive 2010&#47;63&#47;EU&#46;</p>"
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Article information
ISSN: 22538089
Original language: English
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