biodisponibilidade de edtmp-153 samÁrio tratados com...

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

CENTRO DE CIÊNCIAS DA SAÚDE

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

BIODISPONIBILIDADE DE EDTMP- 153SAMÁRIO EM RATOS

TRATADOS COM DOCETAXEL

ARTHUR VILLARIM NETO

Natal – RN 2009

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Tese apresentada ao Programa de Pós-

Graduação em Ciências da Saúde da

Universidade Federal do Rio Grande do Norte

para a obtenção do título de Doutor

Orientador: Prof. Dr. Aldo da Cunha Medeiros

Co-orientador: Prof. Dr. Mário Bernardo Filho

Natal – RN 2009

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Presidente da Banca Examinadora:

Prof. Dr. Aldo da Cunha Medeiros - UFRN

Banca Examinadora:

Prof. Dr. Adenilson de Souza da Fonseca (UERJ)

Prof. Dr. Jael Soares Batista (UFERSA)

Prof. Dr. Artur da Silva Carriço (UFRN)

Prof. Dr. Irami Araújo Filho (UFRN)

Aprovada em 02/07/2009

CATALOGAÇÃO NA FONTE

V722b Villarim Neto, Arthur.

Biodisponibilidade de EDTMP-153Samário em ratos tratados com docetaxel / Arthur Villarim Neto. – Natal, 2009.

58p. Orientador: Prof. Dr. Aldo José da Cunha Medeiros. Co-orientador: Prof. Dr. Mário Bernardo Filho.

Tese (Doutorado) – Programa de Pós-Graduação em Ciências da Saúde. Centro de Ciências da Saúde. Universidade Federal do Rio Grande do Norte.

1. Quimioterapia – Tese. 2. Metástase óssea – Tese. 3. Radiofármacos – Tese. 4. EDTMP-153Samário – Tese. I. Bernardo Filho, Mário. II. Bernardo Filho, Mário. III. Título.

RN-UF/BS-CCS CDU: 615.28(043.2)

iii

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

CENTRO DE CIÊNCIAS DA SAÚDE

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

COORDENADORA DO PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS

DA SAÚDE:

Profa. Dra. Tércia Maria de Oliveira Maranhão

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DEDICATÓRIA

A José Martins, meu querido pai, maior incentivador dos meus estudos,

exemplo de superação pessoal, e a minha mãe, Rejane, pela dedicação à

família, minha eterna gratidão.

A Rachel, esposa e Sol da minha vida.

A Camila e Pedro, frutos do nosso amor.

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AGRADECIMENTOS

A Deus, Ao Professor Dr. Aldo da Cunha Medeiros, meu orientador, pela amizade, apoio, inteligência e integridade exemplares. Ao Professor Dr. Artur da Silva Carriço, pelo incentivo e pelas boas idéias. A Rodrigo e Lídia, irmãos de sangue e companheiros de caminhada nesta vida. A Gerôncio e Suely, amigos de todas as horas. Ao Professor Dr. Irami Araújo Filho, pelo apoio prático e decisivo na execução deste projeto. A Amália Rego, pela presença constante e atenciosa no laboratório. A Ítalo Medeiros de Azevedo, pela boa vontade e pela inteligência na análise estatística. A Dra. Marla Medeiros, médica nuclear e amiga, por ter trabalhado em dobro nas horas em que estive ausente. A Kércia e Kadja, companheiras nesta jornada e colegas de trabalho. Ao Dr. Maciel Matias, pelo estímulo e apoio institucional através da Liga Norteriograndense Contra o Câncer.

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SUMÁRIO

RESUMO............................................................................................................ix

1 INTRODUÇÃO ........................................................................................ 1

2 REVISÃO DE LITERATURA ................................................................... 4

2.1. Mecanismo de formação das metástases ósseas ................................... 4

2.2. Tratamento da dor causada por metástases ósseas............................... 6

2.2.1. Medicações antiinflamatórias e opióides ................................................. 6

2.2.2. Bifosfonatos e bloqueadores hormonais.................................................. 7

2.2.3. Tratamento cirúrgico................................................................................ 7

2.2.4. Radioterapia externa ............................................................................... 8

2.2.5. Radiofámacos utilizados no alívio da dor óssea...................................... 8

2.2.6. Quimioterapia ........................................................................................ 14

3 ANEXAÇÃO DE ARTIGOS PUBLICADOS .......................................... 17

3.1. Biodistribution of samarium-153-EDTMP in rats treated with docetaxel.18

3.2. Biodistribution of the Radiopharmaceutical Sodium Pertechnetate after

Biliopancreatic Bypass with a Duodenal Switch. ................................... 27

3.3. Biodistribution of the radiophamarceutical sodium pertechnetate

(Na99mTcO4) after massive small bowel resection in rats.................... 38

3.4. Influence of Splenectomy on the Biodistribition of Technetium-99m

Dimercaptosuccinic Acid (99mTc-DMSA).............................................. 51

3.5. Biodistribution of the Radiopharmaceutical Technetium-99m-Sodium

Phytate in Rats After Splenectomy ........................................................ 58

4 COMENTÁRIOS, CRÍTICAS E SUGESTÕES ...................................... 65

5 REFERÊNCIAS ..................................................................................... 68

ABSTRACT

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LISTA DE ABREVIAÇÕES, SIGLAS E SÍMBOLOS

MBq megabequerel

MBq/Kg megabequerel por kilograma

% ATI percentual de atividade injetada

% ATI/g percentual de atividade injetada por grama

UFRN Universidade Federal do Rio Grande do Norte

µCi micro Curie 153Sm 153Samário

EDTMP etilenodiamina-tetrametileno fosfonato

EDTMP-153Sm 153Samário complexado a etilenodiamina-

tetrametileno fosfonato

Emax energia máxima de emissão

MeV Mega elétron-volt

keV quilo electron-volt

Gy Gray (unidade de dose absorvida)

Bcl-2 B-cell lymphoma 2 (linfoma 2 de célula B)

MDP-Tc 99m

99m

Tecnécio complexado a metileno-disfofonato

IPEN Instituto de Pesquisas Energéticas e Nucleares

USP Universidade de São Paulo

CNEN Comissão Nacional de Energia Nuclear

PSA Prostate-specific antigen (antígeno específico da

próstata)

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RESUMO

A dor óssea decorrente das metástases é um sintoma comum nos tumores

avançados de mama e próstata. Nenhuma opção terapêutica isolada é

completamente eficaz, e uma série de modalidades costuma ser empregada,

entre eles a terapia com radiofármacos, como o samário153-etilenodiamina-

tetrametileno fosfonato (EDTMP-153Sm). O docetaxel, um taxano com ação

sobre tumores avançados de mama e próstata, tem-se apresentado como uma

nova opção de tratamento quimioterápico. Muitos pacientes fazem uso

simultâneo de EDTMP-153Sm e docetaxel. Este estudo procurou avaliar a

influência do docetaxel na biodisponibilidade de EDTMP-153Sm em ratos

Wistar, aleatoriamente alocados em 2 grupos de 6 animais cada. O grupo DS

(docetaxel/samário) recebeu docetaxel (15 mg/kg) intraperitoneal em dois

ciclos com 11 dias de intervalo. Os ratos do grupo S (samário/controle) não

foram tratados com docetaxel. Nove dias após a quimioterapia, todos os

animais receberam 0.1ml de EDTMP-153Sm via plexo orbital (25µCi). Após 2

horas, os animais foram mortos, e realizaram-se análises de amostras de

cérebro, tireóide, pulmão, coração, estômago, cólon, fígado, rim e fêmures. O

percentual de radioatividade por grama (%ATI/g) de tecido de cada biópsia foi

determinado em contador gama automático (Wizard-1470, Perkin-Elmer,

Finland). No 9º dia após 2º ciclo de docetaxel, os ratos tiveram perda de peso

significante, passando de 353.66± 22.8g (controle/pré-tratamento) para

314,50±22,09g (p<0.5). Os %ATI/g nos órgãos dos ratos tratados com EDTMP-153Sm e docetaxel tiveram redução significante nos fêmures direito e esquerdo,

rim, fígado e pulmão, quando comparados aos animais não tratados com

docetaxel. Em conclusão, a combinação de docetaxel com EDTMP-153Sm foi

associada à menor concentração do radiofármaco em órgãos alvo. Futuras

investigações sobre o impacto do docetaxel na biodisponibilidade do EDTMP-153Sm poderão complementar estes achados. Deve-se ressaltar o caráter

multidisciplinar deste estudo, que contou com a participação ativa e com a

troca constante de conhecimentos entre profissionais das áreas de Medicina

Nuclear, Cirurgia, Oncologia, Biologia e Estatística.

1 INTRODUÇÃO

Metástases ósseas são complicações frequentes do câncer, ocorrendo em até

70 % dos pacientes acometidos por tumores avançados da mama e da próstata(1).

A dor óssea decorrente das metástases é um sintoma comum nestes casos e

freqüentemente determina a qualidade de vida nos últimos estágios da doença(2). O

principal mecanismo de dor óssea nas metástases é a distensão e o aumento da

pressão mecânica sobre as terminações nervosas locais(3). Tal sintoma interfere na

qualidade de vida do paciente e requer tratamento efetivo. Infelizmente, várias

modalidades terapêuticas, como analgésicos, terapia hormonal, orquiectomia,

quimioterápicos, bifosfonatos e tratamento cirúrgico não são eficazes em todos os

casos, especialmente no estágio final da doença(1). Radioterapia pode ser realizada

apenas em lesões ósseas bem definidas. A extensão do campo irradiado pode ser útil,

mas geralmente é acompanhada por graves efeitos colaterais(4).

Desta forma, a terapia sistêmica com radioisótopos, utilizando radiofármacos

com afinidade pelo tecido ósseo, torna-se uma opção valiosa e eficaz no tratamento da

dor óssea em pacientes com metástases difusas, especialmente no câncer de mama e

de próstata.

Os principais radiofármacos com eficácia estabelecida no tratamento da dor

óssea metastática são: 32Fósforo, 89Estrôncio, 186Rênio, 188Rênio e 153Samário. No

Brasil, a produção nacional restringe-se ao 153Samário (153Sm). O 153Samário possui

meia vida física de 46,3h e decai por emissão de fótons gama e de partícula beta

(Emax 0,81 Mev). Este isótopo forma um complexo estável com

etilinodiaminotetrametiletilenodifosfonato (EDTMP)(5). EDTMP-153Sm fixa-se à

hidroxiapatita nos locais de atividade osteoblástica aumentada, na proporção de 4:1 a

2

7:1 entre a metástase óssea e o osso normal(6). Após a administração de EDTMP-

153Sm, 65 a 80% dos pacientes apresentam alívio da dor óssea(7). A resposta é

tipicamente rápida, ocorrendo dentro de uma semana, com duração média de 8

semanas (variação de 4 a 35 semanas)(8).

Estudos randomizados, duplos cegos e controlados demonstraram alta eficácia

clínica e estabeleceram a atividade ótima administrada de 37 MBq/Kg (8-10). A

toxicidade para esta atividade é limitada, com discreta mielossupressão com um nadir

de quatro semanas. O tratamento pode ser repetido com segurança(8). Portanto, com

eficácia já estabelecida e com disponibilidade regular em nosso meio, o EDTMP-153Sm

torna-se uma ferramenta importante no manejo dos pacientes com dor óssea

metastática.

Novas opções terapêuticas têm surgido no tratamento do carcinoma de próstata

avançado. Entre os novos quimioterápicos disponíveis, destaca-se o docetaxel, um

taxano que inativa a proteína Bcl-2, presente em células metastáticas, causando

inativação e morte das células tumorais(9).

Alguns protocolos têm empregado docetaxel nos pacientes com carcinoma

prostático avançado que se tornam refratários ao bloqueio hormonal, com resultados

superiores àqueles obtidos com os esquemas quimioterápicos tradicionais

(mitoxantrona associada a prednisona ou hidrocortisona)(12-14).

O crescente emprego de docetaxel, neste grupo de pacientes, que normalmente

apresenta dor óssea significativa e é elegível para terapia com EDTMP-153Sm, conduz

a situações clínicas em que ocorre o emprego simultâneo das duas terapêuticas. Faz-

se necessário avaliar se a biodisponibilidade do EDTMP-153Sm, especialmente nos

ossos, é alterada pela administração de docetaxel, uma vez que, nesta situação, pode

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ser necessária uma correção da atividade radioativa administrada aos pacientes,

visando à otimização terapêutica.

Uma eventual alteração da concentração óssea de EDTMP-153Sm, em pacientes

em tratamento com docetaxel, poderia levar a uma redução da eficácia terapêutica do

radiofármaco. Adicionalmente, é necessário esclarecer se ocorre alguma modificação

na sua biodistribuição, pois uma eventual redistribuição do EDTMP-153Sm poderia gerar

um aumento indesejável da sua concentração em outros tecidos além do tecido ósseo,

com aumento indesejável dos níveis dosimétricos. Considerando que o efeito

terapêutico deste radiofármaco baseia-se em sua ação no tecido ósseo, sua fixação

extra-óssea é contraproducente e pode estar associada a um aumento de reações

adversas e da toxicidade do tratamento. A busca de respostas para essas questões

constitui a base de partida deste experimento.

A pesquisa foi realizada no Núcleo de Cirurgia Experimental do Centro de

Ciências da Saúde da Universidade Federal do Rio Grande do Norte. Os experimentos

foram possíveis através de convênio firmado entre a Universidade Federal do Rio

Grande do Norte e a Liga Norteriograndense Contra o Câncer, Serviço de Medicina

Nuclear.

Deve-se ressaltar o seu caráter interdisciplinar, uma vez que contou com a

participação ativa e com a troca constante de conhecimentos entre profissionais das

áreas de Medicina Nuclear, Cirurgia, Oncologia, Biologia e Estatística.

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2 REVISÃO DE LITERATURA

2.1. Mecanismo de formação das metástases ósseas

Aproximadamente 70% dos pacientes com câncer avançado apresentam dor

decorrente de metástases ósseas especialmente nos tumores de mama, próstata e

pulmão (1;3;3;10;11). As células tumorais podem atingir o tecido ósseo por extensão

direta ou, mais freqüentemente, por via hematogênica, atingindo, em primeira instância,

a medula óssea(3).

Entre os pacientes com doença óssea metastática, 70% apresentam

envolvimento da coluna vertebral e arcos costais, 40% da pelve, 25% dos fêmures e

15% da calota craniana. Fraturas patológicas ocorrem em 10% das lesões

metastáticas, especialmente no câncer de mama(3). Outras complicações da doença

óssea metastática são: hipercalcemia e déficit neurológico ocasionado por compressão

de raízes nervosas(12).

A metástase de células tumorais para o tecido ósseo altera a sua arquitetura e a

homeostase mineral, possibilitando um meio propício para a multiplicação das células

tumorais(13). As lesões ósseas são classificadas através da radiografia, como

osteolíticas ou osteoblásticas. Nas lesões osteolíticas, a destruição ocorre pela ação

dos osteoclastos, como visto em pacientes portadoras de câncer de mama. As lesões

osteoblásticas predominam no câncer de próstata e caracterizam-se pelo aspecto de

esclerose na análise radiográfica(14). Uma complexa interação entre as células

tumorais e o tecido ósseo leva à formação de lesões osteolíticas, com reabsorção

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óssea, ou lesões osteoblásticas, onde ocorre formação óssea(12;13). Contudo, um

padrão misto é comum em muitas lesões, com reabsorção e formação óssea ocorrendo

simultaneamente(14).

A matriz óssea é um rico depósito de fatores de crescimento, que são liberados

durante a reabsorção óssea. Análise histológica das metástases ósseas osteolíticas

indica que a destruição óssea é mediada pelos osteoclastos e não diretamente pelas

células tumorais(12;15;16). Essas observações sugerem um ciclo vicioso, levando à

formação de metástases osteolíticas: células tumorais secretam fatores de estímulo

aos osteoclastos dentro da medula óssea, causando reabsorção de osso e liberação de

fatores de crescimento a partir da matriz óssea. Esses fatores de crescimento são

essenciais para a colonização e subseqüente invasão do microambiente ósseo pelas

células tumorais(12;13;15;17).

No câncer de próstata, o comportamento osteolítico é, em parte, determinado

pela ativação dos osteoclastos, com elevação de marcadores séricos associados à sua

formação, entre eles o fator de diferenciação de osteoclastos (18). No câncer de

pulmão, estudo in vitro da colonização tumoral do tecido ósseo identificou moléculas

sinalizadoras e proteínas capazes de modular o fator de crescimento beta a estimular a

formação de novos osteoclastos (19). No câncer de mama, o fator de diferenciação de

osteoclastos foi identificado como um pré-requisito para a formação e manutenção de

osteoclastos a partir de precursores hematopoéticos(20).

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2.2. Tratamento da dor causada por metástases óssea s

O mecanismo exato da dor óssea metastática não é conhecido(14). A distensão

e o aumento da pressão mecânica sobre as terminações nervosas locais estão entre os

principais desencadeadores da dor(3).

As metástases ósseas costumam levar a um quadro de dor progressiva,

exigindo o emprego de diversas modalidades terapêuticas, incluindo medicamentos

(ex: antiinflamatórios, opióides, bloqueadores hormonais, bifosfonatos e

quimioterápicos), intervenções cirúrgicas e uso de radiações ionizantes, através de

radioterapia externa ou do tratamento com radiofármacos(3;21;22). O seu manejo

apresenta, portanto, um caráter interdisciplinar, visando ao controle da dor e à melhora

na qualidade de vida do paciente.

2.2.1. Medicações antiinflamatórias e opióides

Analgesia com medicamentos antiinflamatórios não esteróides é a primeira

opção na maioria dos pacientes, com progressão para opióides mais potentes à

medida que ocorre piora no quadro doloroso(14).

Aproximadamente dois terços dos pacientes com doença óssea metastática

necessitam de medicação analgésica, em geral um opióide, e podem ser afetados

pelos efeitos colaterais desses medicamentos, como letargia e constipação(23;24).

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2.2.2. Bifosfonatos e bloqueadores hormonais

Os bifosfonatos são análogos do pirofosfato, um componente normal da matriz

óssea. Estes agentes ligam-se aos cristais de hidroxiapatita, tornando-os menos

suscetíveis à reabsorção pelos osteoclastos. Adicionalmente, os bifosfonatos inibem o

recrutamento de precursores dos osteoclastos, previnem a sua migração para o tecido

ósseo e inibem a produção de prostaglandina-E2, interleucina 1 e enzimas

proteolíticas(25).

Estudos clínicos controlados e randomizados comprovaram sua eficácia no

tratamento de pacientes com metástases ósseas decorrentes do câncer de próstata,

reduzindo as complicações esqueléticas relacionadas a esta patologia(25-27).

Os bloqueadores hormonais são utilizados no câncer de próstata e no câncer de

mama, tumores cujo crescimento é estimulado pela presença dos hormônios sexuais,

respectivamente testosterona e estrógeno. O bloqueio desses hormônios é realizado

com o emprego de análogos do hormônio liberador do hormônio luteinizante(25;28-32).

Entretanto, durante a evolução natural da doença, as células tumorais

freqüentemente desenvolvem mecanismos que permitem a sua sobrevivência e

proliferação em um ambiente com mínima quantidade de estrógeno e, portanto,

tornam-se refratárias ao bloqueio hormonal(28;33-36).

2.2.3. Tratamento cirúrgico

Pouco utilizado, restringe-se à correção de fraturas patológicas e a casos onde

há compressão da medula espinhal pelo tumor(37).

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2.2.4. Radioterapia externa

A radioterapia local apresenta resposta em cerca de 80% dos casos, com

resposta completa em 30 a 60% das situações. Seu efeito analgésico tem duração

média de quatro meses, restringindo-se, entretanto, ao sítio irradiado.(21;22;38). A

radioterapia de hemicorpo isolado ou seqüencial, com doses de 6 a 8 Gy, pode ser

empregada em pacientes com metástases disseminadas, apresentando resposta

completa em aproximadamente 20% e parcial em 50-60% dos casos.

A irradiação de grandes áreas é limitada pela toxicidade hematopoética, pulmonar e

gastrointestinal, decorrentes da alta dose de radiação absorvida, com mielossupressão

em 10 a 30% dos pacientes(21;38;39).

2.2.5. Radiofámacos utilizados no alívio da dor óss ea

Em pacientes com múltiplas metástases ósseas, onde o emprego da

radioterapia externa torna-se proibitivo, em virtude dos seus efeitos colaterais,

radiofármacos de uso sistêmico podem ser utilizados no alívio da dor óssea com bons

resultados(1;3;10;11;14;22;25;25;40;41). Esses agentes terapêuticos são emissores de

partículas beta negativas e possuem afinidade química por sítios de neoformação

óssea. Desta forma, sua captação é maior nas áreas onde há formação de osso novo,

9

devido ao aumento do fluxo sanguíneo nessas áreas e a maior presença de superfície

de contato com os cristais de hidroxiapatita recém formados(3).

Os principais radiofármacos utilizados são compostos fosfonados marcados com

153Samário, 188Rênio, 186Rênio, 89Estrôncio e 32Fósforo. Os dois mais empregados

mundialmente são 89Estrôncio e 153Samário(42;43).

Embora apresentem eficácia analgésica similar(44), esses radiofármacos

apresentam diferenças significativas na meia-vida física e na emissão de partículas, o

que resulta em diferentes efeitos colaterais, principalmente na supressão medular(25).

O 89Estrôncio possui longa meia-vida física no osso (quase 60 dias), e pode levar

à queda substancial no número de leucócitos e especialmente de plaquetas. Isto torna

difícil a sua indicação em um grupo de pacientes que, muitas vezes, possui indicação

de realizar quimioterapia simultaneamente(25).

O EDTMP-153Sm, em contraste, pode ser administrado com baixa toxicidade

medular(45;46). A dose 37 MBq/Kg demonstrou ser mais efetiva que placebo no

controle da dor(47). No Brasil, a disponibilidade e o custo do EDTMP-153Sm o tornaram

o agente de escolha nesta modalidade de tratamento(22;48-51).

2.2.5.1. EDTMP-153Sm

O 153Samário é ligado à molécula de etilenodiamina-tetrametileno fosfonato,

formando o complexo EDTMP-153

Sm, que é captado na matriz mineral óssea por

10

adsorção aos cristais de hidroxiapatita(22). O complexo EDTMP-153

Sm demonstra

captação altamente seletiva pelo tecido ósseo, de forma similar aos radiofármacos

utilizados na cintilografia óssea(10). Seu efeito terapêutico deve-se à radiação beta

negativa de curto alcance emitida pelo radionuclídeo. Sua produção nacional é

realizada pelo Instituto de Pesquisas Energéticas e Nucleares (IPEN), autarquia

estadual associada à Universidade de São Paulo (USP) e gerenciada pela Comissão

Nacional de Energia Nuclear (CNEN).

153Samário é um metal terra rara, produzido em reator. Possui um esquema de

decaimento complexo, incluindo raios-x e raios gama, além do decaimento principal por

partículas beta. Sua meia-vida física é de 46,3h. A energia das emissões beta é

relativamente baixa, resultando em penetração média de 0,83 mm na água e máxima

de 1,7 mm no osso e 3,1 mm em tecido mole. A emissão gama média de 103,2 keV é

satisfatória para aquisição de imagens em gama-câmara(10).

Do ponto de vista prático, essas características físicas significam que o

153Samário tem seu efeito terapêutico limitado ao tecido alvo, evitando a irradiação

desnecessária das áreas circunjacentes. Além disso, o fato de ser também um emissor

gama possibilita a realização de uma cintilografia óssea após a sua aplicação, servindo

de controle e eventual comparação com tratamentos posteriores. O 153Samário é

facilmente complexado ao EDTMP. A preparação é quimicamente estável e não

apresenta decomposição apreciável por mais de 48h(11). A dose sugerida para

tratamento da dor óssea é de 37 MBq/Kg, administrada lentamente em um acesso

endovenoso anteriormente preparado, evitando extravasamento.

11

2.2.5.2 EDTMP-153

Sm : biodistribuição e dosimetria

EDTMP-153

Sm liga-se ao tecido ósseo normal e ao patológico, a uma razão

lesão-osso normal de 4,04±2,62 e lesão-tecido mole de 6,87±3,18(5). Esses valores

são bastante similares aos encontrados nos radiofármacos utilizados para realização

da cintilografia óssea, como o metileno-disfofonato marcado com 99m

Tecnécio (MDP-

Tc 99m

)(52). EDTMP-153

Sm compartilha com MDP-Tc 99m

a mesma qualidade de

imagem e sensibilidade na identificação de lesões ósseas(52).

Em estudo realizado em ratos normais, sem doença óssea, EDTMP-153

Sm

demonstrou elevada captação óssea (>55% da dose injetada), com baixa concentração

no sangue 2h após a injeção(11). A porcentagem total que se liga ao osso é

geralmente 50% ou mais da atividade injetada, dependendo da extensão da doença

óssea metastática. O restante da dose é rapidamente excretada por filtração

glomerular. A excreção urinária é virtualmente completada após 6 horas(52), o que é

de interesse prático, pois possibilita o tratamento ambulatorial, com liberação do

paciente para casa após este período.

Após a administração, EDTMP-153

Sm é rapidamente clareado do espaço

intravascular, fixando-se ao tecido ósseo. A excreção urinária e a captação óssea

identificam a bexiga e a medula óssea como os órgãos críticos em relação à radiação.

A real exposição da bexiga à radiação é inversamente proporcional à extensão da

12

doença metastática e depende da freqüência miccional do paciente, que deve ser

orientado a ingerir líquidos em abundância e urinar regularmente. A taxa de exposição

da medula óssea à radiação é um ponto crítico, uma vez que a toxicidade da medula

óssea é o fator dose-limitante na prática clínica(10). Para o propósito clínico, é

necessário manter a dose administrada em torno de 37MBq/Kg, com avaliação prévia

da contagem de plaquetas e leucócitos. Admitindo-se uma queda de até 50% nessas

contagens, é contra-indicada a administração de EDTMP-153

Sm em pacientes com

contagens de plaquetas inferiores a 100 x 109/litro e número de leucócitos inferiores a

3.5 x 109/litro. Quimioterapia e radioterapia externa realizadas recentemente também

devem ser consideradas. Tomadas essas precauções, a supressão medular costuma

ser transitória e de grau leve a moderado e constitui o único efeito adverso da

administração de EDTMP-153

Sm. A queda no número de plaquetas e leucócitos

apresenta nadir (ponto mais baixo, onde ocorre a maior depressão) entre a 3ª e 4ª

semanas após a administração, com recuperação aos níveis anteriores em 8

semanas(25;47;53). A repetição do tratamento pode ser feita após esse período, com

resultados semelhantes(54). Estes dados atestam a segurança do emprego de

EDTMP-153

Sm no tratamento da dor óssea metastática e sugerem que o seu uso pode

ser realizado em conjunto com a quimioterapia(25).

13

2.2.5.3 EDTMP-153

Sm : eficácia clínica

Após administração de EDTMP-153

Sm, 65 a 80% dos pacientes apresentam

alívio da dor óssea(7;8;47;55-63). A resposta é tipicamente rápida, ocorrendo dentro de

5 a 10 dias(14). A duração média da resposta é de 8 semanas (variação de 4 a 35

semanas)(41). Apesar da resposta clínica eficaz no alívio da dor, não há melhora da

sobrevida destes pacientes(14;47).

Homens com câncer de pulmão, lesões nas vértebras ou na pelve e metástases

adicionais nos ossos dos membros inferiores apresentam pior resposta ao EDTMP-

153Sm(14;64).

Em estudo que comparou a eficácia de duas doses de EDTMP-153

Sm no alívio

da dor óssea metastática, empregando-se uma dose menor (18,5 MBq/Kg) e uma dose

maior (37MBq/kg), verificou-se um redução global no nível de dor em todos os

pacientes, mas aqueles que receberam a dose maior apresentavam melhora mais

significativa na 4ª semana após a avaliação (p=0,04). A proporção de pacientes que

conseguia dormir à noite aumentou de 33% para 45% naqueles que receberam a dose

mais baixa e para 59% para os que receberam a dose mais alta(14;65). Achados

semelhantes foram encontrados em outro estudo duplo cego controlado, com melhor

eficácia ao se utilizar a dose maior (37MBq/kg): melhora da dor em 67% dos pacientes,

em relação àqueles que utilizaram dose menor (18,5 MBq/Kg), com alívio da dor em

42% dos casos. Neste trabalho, verificou-se aumento progressivo no consumo de

analgésicos pelo grupo placebo em relação aos níveis anteriores ao tratamento,

14

enquanto nos grupos tratados com EDTMP-153Sm houve redução no uso de

analgésicos(8;14).

Em outro estudo clínico, randomizado e controlado, a eficácia de EDTMP-153Sm

foi avaliada em 152 homens com câncer de próstata e diversas lesões ósseas

dolorosas(14;47). Os pacientes foram selecionados para receber EDTMP-153Sm na

dose de 37MBq/kg ou o mesmo produto, na sua forma não radioativa. Os diários dos

pacientes foram utilizados para registrar o uso de analgésicos e o nível de dor, que foi

avaliado através de uma escala visual e de uma escala de palavras. Entre 3 e 4

semanas, o consumo de analgésico decresceu significativamente no grupo tratado em

relação ao grupo placebo (p<0,05). Além disso, tanto a escala visual de dor quanto a

escala de palavras demonstrou significativa melhora no grupo tratado entre 2 e 4

semanas (p=0,0004).Os grupos não apresentaram diferença em relação à

sobrevida(14;47).

2.2.6. Quimioterapia

A quimioterapia apresenta um papel preponderante no tratamento de pacientes

com dor óssea provocada por metástases ósseas. É sabido que, durante a sua

evolução clínica, os tumores de próstata e mama tornam-se refratários ao bloqueio

hormonal. O tratamento, a partir deste momento, inclui o emprego de diversos

quimioterápicos. Além do alívio da dor e melhora da qualidade de vida dos pacientes,

alguns esquemas terapêuticos têm demonstrado melhora na sobrevida no câncer de

mama e de próstata refratários ao tratamento hormonal(25;40;66-70). A combinação de

15

radiofármacos com quimioterápicos pode levar a um aumento da eficácia no alívio da

dor em relação ao emprego dos dois agentes separadamente(71-74).

Entre os quimioterápicos que vêm sendo empregados nesta situação, destaca-

se a classe dos taxanos, incluindo viblastina, paclitaxel e docetaxel(25;32;75-81). Entre

estes, o docetaxel demonstrou ser o mais ativo, isoladamente ou associado à

etamustrina(33;82-84). O docetaxel é um taxano semissintético com vários

mecanismos de ação anti-neoplásica. O mais aceito desses mecanismos é a

estabilização de microtúbulos, Na divisão celular normal, os microtúbulos agem como o

citoesqueleto da célula em mitose. O docetaxel liga-se aos microtúbulos, impedindo o

seu processo de polimerização e, desta forma, interrompendo o processo de divisão

celular, levando à apoptose da célula tumoral(25). Um segundo mecanismo proposto

para a ação citotóxica do docetaxel é seu efeito sobre a proteína BCL-2. Foi

demonstrado que a expressão deste oncogene protege a célula tumoral, evitando sua

apoptose. Docetaxel atua na fosforilação da BCL-2, levando à perda de sua função

anti-apoptótica(25;85-87).

Estudos com utilização de docetaxel em pacientes com câncer de próstata

refratário ao tratamento hormonal demonstraram vantagem em relação aos esquemas

quimioterápicos anteriores, com aumento na sobrevida e melhor resposta na

diminuição da dor, redução dos níveis de PSA (prostate-specific antigen – antígeno

específico da próstata) e melhora na qualidade de vida(40;69;70). Após estes achados,

o docetaxel passou a ser considerado o quimioterápico de primeira linha nesta situação

clínica(25;40).

16

O crescente emprego de docetaxel neste grupo de pacientes já vem sendo

observado na prática médica. Isto conduz a situações em que ocorre o seu emprego

simultâneo com EDTMP-153Sm. A influência do docetaxel na biodisponibilidade do

EDTMP-153Sm, especialmente nos ossos, precisa ser avaliada, uma vez que, nesta

situação, pode ser necessária uma correção da atividade radioativa administrada aos

pacientes.

17

3 ANEXAÇÃO DE ARTIGOS PUBLICADOS

Artigo I: Biodistribution of samarium-153-EDTMP in rats treated with docetaxel.

Publicado no periódico Acta Cirúrgica Brasileira, 2009 Jan-Feb;24(1):62-6, Qualis

Internacional C

Artigo II: Biodistribution of the Radiopharmaceutic al Sodium Pertechnetate after

Biliopancreatic Bypass with a Duodenal Switch.

Publicado no periódico Brazilian Archives of Biology and Technology, 2007; 50:189-

197,Qualis Internacional B

Artigo III: Biodistribution of the radiophamarceuti cal sodium pertechnetate

(Na99mTcO4) after massive small bowel resection in rats

Publicado no periódico Acta Cirúrgica Brasileira, 2007 Nov-Dez;22(6):430-435, Qualis

Internacional C

Artigo IV: Influence of Splenectomy on the Biodistr ibition of Technetium-99m

Dimercaptosuccinic Acid (99mTc-DMSA)



Artigo V: Biodistribution of the Radiopharmaceutica l Technetium-99m-Sodium

Phytate in Rats After Splenectomy



18

3.1. Biodistribution of samarium-153-EDTMP in rats treated with docetaxel.

Publicado no periódico Acta Cirúrgica Brasileira, 2009 Jan-Feb;24(1):62-6,

Qualis Internacional C

19

ORIGINAL ARTICLE Effects of Drugs

Biodistribution of samarium-153-EDTMP in rats treated with docetaxel

Biodistribuição de EDTMP-153-samário em ratos tratados com docetaxel

Arthur Villarim Neto I, Maria Kadja Meneses Torres AçucenaI, Kércia Regina Santos Gomes PereiraI, Amália Cínthia Meneses RêgoI, Ítalo Medeiros AzevedoII , Mário Bernardo-FilhoIII , Aldo Cunha MedeirosIV I Fellow PhD degree, Postgraduate Program in Health Sciences, UFRN, Brazil. II Statistician, Department of Surgery, UFRN, Brazil. III PhD, Full Professor, Department of Biophysics and Biometry, State University of Rio de Janeiro, Brazil. IV PhD, Full Professor, Department of Surgery, UFRN, Brazil. ABSTRACT Purpose: Many patients with metastatic bone disease have to use radiopharmaceuticals associated with chemotherapy to relieve bone pain. The aim of this study was to assess the influence of docetaxel on the biodistribution of samarium-153-EDTMP in bones and other organs of rats. Methods: Wistar male rats were randomly allocated into 2 groups of 6 rats each. The DS (docetaxel/samarium) group received docetaxel (15 mg/kg) intraperitoneally in two cycles 11 days apart. The S (samarium/control) group rats were not treated with docetaxel. Nine days after chemotherapy, all the rats were injected with 0.1ml of samarium-153-EDTMP via orbital plexus (25µCi). After 2 hours, the animals were killed and samples of the brain, thyroid, lung, heart, stomach, colon, liver, kidney and both femurs were removed. The percentage radioactivity of each sample (% ATI/g) was determined in an automatic gamma-counter (Wizard-1470, Perkin-Elmer, Finland). Results: On the 9th day after the administration of the 2nd chemotherapy cycle, the rats had a significant weight loss (314.50±22.09g) compared (p<0.5) to pre-treatment weight (353.66± 22.8). The % ATI/g in the samples of rats treated with samarium-153-EDTMP had a significant reduction in the right femur, left femur, kidney, liver and lungs of animals treated with docetaxel, compared to the control rats. Conclusion: The combination of docetaxel and samarium-153-EDTMP was associated with a lower response rate in the biodistribution of the radiopharmaceutical to targeted tissues. Further investigation into the impact of docetaxel on biodistribution of samarium-153-EDTMP would complement the findings of this study. Key words: Taxoids. Samarium. Biological Availability. Drug Therapy. Rats. RESUMO Objetivo: Muitos pacientes com metastases ósseas são tratados com radiofármacos associados com quimioterapia para alívio da dor óssea. O objetivo do trabalho foi estudar a influência do docetaxel na biodistribuição do EDTMP-153-samário nos ossos e outros órgãos de ratos. Métodos: Ratos Wistar foram aleatoriamente alocados em 2 grupos de 6 animais cada. O grupo DS (docetaxel/samário) recebeu docetaxel (15 mg/kg) intraperitoneal em dois ciclos com 11 dias de intervalo. Os ratos do grupo S (samário/controle) não foram tratados com docetaxel. Nove dias após a quimioterapia, todos os animais receberam 0.1ml de EDTMP-153-samário via plexo

20

orbital (25µCi). Após 2 horas, os animais foram mortos e feitas biópsias de cérebro, tireóide, pulmão, coração, estômago, cólon, fígado, rim e fêmures. O percentual de radioatividade por grama (%ATI/g) de tecido de cada biópsia foi determinado em contador gama automático (Wizard-1470, Perkin-Elmer, Finland). Resultados: No 9º após 2º ciclo de docetaxel os ratos tiveram perda de peso significante, passando de 353.66± 22.8g (controle/pré-tratamento) para 314,50±22,09g (p<0.5). Os % ATI/g nos órgãos dos ratos tratados com EDTMP-153–samário e docataxel tiveram redução significante nos fêmures direito e esquerdo, rim, fígado e pulmão, quando comparados com os não tratados com docetaxel. Conclusão: A combinação de docetaxel com EDTMP-153-samário foi associada com resposta mais baixa na biodistribuição do radiofármaco em órgãos alvo. Futuras investigações sobre o impacto do docetaxel na biodistribuição do EDTMP-153-samário poderão complementar os achados teste estudo. Key words: Taxóides. Samário. Disponibilidade Biológica. Quimioterapia. Ratos. __________________________________________________________________ 1 Research performed at the Postgraduate Program in Health Sciences, Federal University of Rio Grande do Norte (UFRN), Brazil.

Introduction Bone metastases are a frequent complication of cancer, occurring in up 70% of patients

suffering from advanced breast or prostate cancer1. Bone metastases patients often present with severe bone pain, especially in the advanced stage of disease1,2. The major pain mechanism of small metastases appears to be the stimulation of nerve endings in the endosteum by a variety of chemical mediators. Larger bone metastases produce stretching of the periosteum, which leads to pain2. The resulting bone pain interferes with the patient’s quality of life and requires effective treatment. Unfortunately, various non-radiotherapeutic modalities such as analgesics, hormone therapy, orchidectomy, cytostatic and cytotoxic drugs, bisphosphonates, and surgery are not effective in all cases, especially in the late stage of the disease1,2. Systemic radionuclide therapy using boneseeking radiopharmaceuticals is considered a valuable and effective method for treating patients with widespread skeletal metastases and increased bone turnover, especially in patients with bone metastases from prostate and breast cancer3,4,5.

In the 1980s the radiopharmaceutical samarium-153 ethylenediamine tetramethylene phosphonate (Sm-153-EDTMP) with short physical half-life was developed, and has been considered effective in the treatment of bone pain4,5. Docetaxel is one of the taxane class chemotherapies that is being increasingly used to treat various solid tumors, including those of prostate and breast cancer. It acts by Bcl-2 protein inactivation in the metastatic cells, causing the death of tumor cells by apoptosis6. In patients with prostate cancer, docetaxel has been suggested in various studies as the first-line choice for treating metastatic hormone-refractory disease. This treatment increases survival, compared to previously accepted chemotherapy schemes6.

A significant number of patients with metastatic bone disease are often subjected to a series of concomitant treatments, including the use of radioisotopes to relieve bone pain, associated with chemotherapy. Accordingly, the aim of this study was to assess the influence of docetaxel on the biodistribution of Sm-153-EDTMP, especially in the bones of rats, enabling a more thorough knowledge of its applicability.

21

Methods

We used male rats provided by the Health Science Center vivarium, Federal University of Rio Grande do Norte, Brazil. The animals were randomly allocated into 2 groups of 6 rats each. They were weighed and placed in individual cages with water and food (Labina Purina ®) "ad libitum" and acclimatized in the lab for 7 days. The rats were kept under temperature (21ºC), humidity (60 - 70%) and lighting (12 / 12 hour cycle light / dark) control and they were handled in accordance with International Standards of Care and Use of Laboratory Animals, following the guidelines of the Brazilian College of Animal Experimentation. The animals of the DS group (docetaxel/samarium) were administered docetaxel (Trixotene ®, Novartis, São Paulo, Brazil), intraperitoneally, using a 15 mg/kg dose in two administration cycles 11 days apart. The S group (samarium/control) rats were not treated with docetaxel. The animals remained under observation for 9 days after the 2nd cycle and were then injected with 0.1 mL of Sm-153-EDTMP via the orbital plexus, corresponding to radioactivity of 925 Bq (25µCi). Two hours after radiopharmaceutical administration, the animals were killed with an anesthesia overdose (thiopental sodium, via the intracardiac cannula) and underwent surgery for removal of samples from the brain, thyroid, lung, heart, stomach, colon, liver, kidney and both femurs. The tissue samples were washed in 0.9% saline, weighed on a precision scale (Mark 160®, Bel equipment, Italy) and their radioactivity was determined in an automatic gamma counter (Wizard 1470, Perkin-Elmer, Finland). The results are shown in counts per minute (CPM), corrected by disintegrations per minute (DPM). The efficiency of the gamma counter was 86%, as specified by the manufacturer. The specific activity of each sample was calculated by dividing its absolute count DPM by its weight (DPM/g). The percentage of radioactivity of each sample (% ATI/g) was calculated by dividing its specific activity (DPM/g) by the total radioactivity injected in each animal. The total activity administered to each animal was calculated from the average of three patterns with the same volume injected.

The data were expressed as mean ± standard deviation. Statistical analysis for comparison between groups was performed by the Student and Tukey tests, using a significance level of 0.05.

Results No animal died during the experiment. On the eleventh day after the 1st chemotherapy cycle with docetaxel, the animals showed weight loss (mean 329.16±29.2 g), but not significantly different from pre-treatment weight (mean 353.66± 22.8). Meanwhile, on the 9 th day after administration of the 2nd chemotherapy cycle, there was significant weight loss (314.50±22.09g) compared to pre-treatment weight (Figure 1). We observed no changes in animal behavior or side effects, such as diarrhea and hair loss. During the laparotomy to remove organ samples, few peritoneal adhesions were observed in DS group rats. No macroscopic changes were detected in the abdominal organs.

The analysis of the percentage of total activity of Sm-153-EDTMP injected per gram of tissue (% ATI/g) in the samples analyzed showed greater radiopharmaceutical activity in the liver, kidney, right femur, left femur and thyroid, when compared to other organs, in both the DS group (treated with docetaxel) and control group S (Table 1). In the rats treated with docetaxel, the Sm-153-EDTMP activity was smaller in all the organs studied, when compared to the control group, except in the brain. Using the Student t-test, we showed that this reduction was statistically significant in the right femur, left femur, kidney, liver and lungs of animals treated with docetaxel, compared to the control group rats (p<0.05). Consequently, there was no

22

significant reduction in radioactivity biodistribution in brain, thyroid, heart, stomach and colon samples (Table 1).

Pre 1st 2nd

Cycle

280

300

320

340

360

380

400

Wei

ght (

g)

*

*

FIGURE 1 – The evolution of rats weight comparing pretreatment and after the first (1st) and second (2nd) post-docetaxel treatment * Significant difference, p<0.05, Tukey test.

23

TABLE 1 - Comparison of percentage of radioactivity/g (% ATI/g) of samples from each organ of rats treated with Sm-153-EDTMP (group S) and with docetaxel + Sm-153-EDTMP (group DS)

% ATI/g Organs

Group S Group DS p

Brain 0.01 ± 0.002 0.01 ± 0.005 0.753022

Thyroid 0.40 ± 0.079 0.30 ± 0.066 0.07301

Lung 0.17 ± 0.033 0.13 ± 0.025 0.043126

Heart 0.12 ± 0.019 0.11 ± 0.019 0.300239

Stomach 0.18 ± 0.042 0.14 ± 0.032 0.091458

Colon 0.13 ± 0.041 0.11 ± 0.038 0.484202

Liver 1.48 ± 0.187 0.96 ± 0.267 0.007438

Kidney 1.21 ± 0.160 0.86 ± 0.144 0.007411

Femur (right) 0.73 ± 0.105 0.46 ± 0.093 0.002484

Femur (left) 0.72 ± 0.004 0.50 ± 0.086 0.003599

Mean ± SD, Student t test. S, samarium; DS, docetaxel+samarium.

Discussion

Bone pain is a common symptom of metastatic cancer and may be difficult to control with analgesic medication alone. Systemic agents such as chemotherapy, bisphosphonates and hormonal therapy, as well as external beam radiation in the form of hemibody or focal bone irradiation are also used as palliative measures to control metastatic bone pain7. Radiation in the form of radiopharmaceuticals has been investigated for the relief of bone metastasis pain and several systemically administered beta-emitters have been evaluated for the treatment of painful bone metastases, two of which (32-P and 89-Sr) have been studied extensively7. Both agents are effective in relieving pain from osteoblastic metastases, but they have properties that limit their usefulness. The very high beta energy of 32-P coupled with a low lesion-to-normal bone ratio has inhibited widespread use of this radiopharmaceutical as a palliative agent for bone pain7. The long physical half-life of 89-Sr (50.5 days) results in a slow delivery of the radiation dose. Consequently, the onset of pain relief may not occur for several weeks after administration8. The long half-life can also produce a prolonged and variable suppression of hematopoietic elements8, limiting the availability or efficacy of concurrent myelosuppressive therapies (e.g. external radiation, surgery or chemotherapy) that these patients frequently require. Therefore, we used 153-samarium in this study because of its physical and biological proprieties, ease of use in laboratory, as well as its efficacy in treating bone pain5. The physical half-life of 153-Sm is 46.3 h, and its beta particle has a maximum range of 1.7 mm in bone and 3.1 mm in soft tissue. The gamma emission at 103 keV (29% abundance) is suitable energy for imaging using standard scintigraphic cameras, and is low enough result in minimal external exposure levels when administered at therapeutic levels9. Although many radiopharmaceuticals have been examined for bone pain palliation, samarium-153 has been the most extensively examined in clinical trials.

24

The advantages of radiopharmaceutical use include the ease of administration, the ability to treat multiple sites of metastatic disease, the improved potential therapeutic ratio due to bone location

and the potential of combining with chemotherapy agents and external beam radiation for an enhanced therapeutic effect4,5 . A subset of trials has suggested improved pain palliation by combining a radiopharmaceutical agent with chemotherapy4,5,12. Taxanes (docetaxel and paclitaxel) are currently one of the most extensively studied new chemotherapeutic agents for metastatic breast cancer. Single-agent docetaxel has demonstrated significant survival advantages over other recognized regimens in two trials involving patients with anthracycline-pretreated metastatic breast cancer10. Some data suggest that docetaxel is one of the most active agents available for treating these patients11. The primary objective of this study was to evaluate if combined chemotherapy interferes with the biodistribution of Sm-153-EDTMP to bone and other organs. It was based on the possibility of using Sm-153-EDTMP combined with docetaxel for the treatment of bone metastasis pain in patients with prostate or breast cancer. Recently, Ricci et al.12 reported that Sm-153-EDTMP is effective in bone pain relief, with minimal toxicity. When administered in combination with chemotherapy (estramustine phosphate or mitoxantrone plus prednisone), it prolonged survival. Many factors, such as drug therapy, radiation therapy, surgery, in addition to the pathological process, could affect the biodistribution of different radiopharmaceuticals13-15. In this work, when Sm-153-EDTMP was administered to rats previously treated with docetaxel, the biodistribution of the radiopharmaceutical to bone, kidney, liver and lung was significantly lower than that of controls. Based on these data, we can infer that an additional dose of Sm-153-EDTMP may be necessary to get the desired pain relief effect when docetaxel is used in combination with this radiopharmaceutical. It was reported that the average duration of bone pain palliation in patients treated with Sm-153-EDTMP alone was about 3–4 months, while it was about 9–10 months in patients who started receiving estramustine phosphate or mitoxantrone plus prednisone after treatment with the bone-seeking radionuclide12. However, information about the clinical effect of docetaxel associated to Sm-153-EDTMP is scarce. The results obtained in the present work provide a rationale for further studies aimed at assessing combined chemotherapy and bone-targeted radiotherapy (docetaxel plus Sm-153-EDTMP) as a therapeutic strategy in patients with advanced hormone resistant prostate cancer. In conclusion, the combination of docetaxel and Sm-153-EDTMP was associated with a lower response rate in the biodistribution of the radiopharmaceutical to targeted tissues. Further investigation into the impact of docetaxel on the biodistribution of Sm-153-EDTMP would complement the findings of this study. References

1. Liepe K, Runge R, Kotzerke J. The benefit of bone-seeking radiopharmaceuticals in the treatment of metastatic bone pain. J Cancer Res Clin Oncol. 2005;131:60-6.

2. Nielsen OS, Munro AJ, Tannock IF. Bone metastases: pathophysiology and management policy. J Clin Oncol. 1991;9:509–24.

3. Sapienza MT, Ono CR, Guimaraes MI, Watanabe T, Costa PA, Buchpiguel CA. Retrospective evaluation of bone pain palliation after samarium-153-EDTMP therapy. Rev Hosp Clin Fac Med São Paulo. 2004;59:321-8.

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4. Ubieto MA, Abos MD, Tardin AL, Razola P, Prats E, Garcia F, Polo E, Yubero A, Banzo J. Treatment of bone metastatic pain with Sm-153-EDTMP. Evaluation of the analgesic response and the existence of differences according to the primary tumor and the metastatic pattern. Rev Esp Med Nucl. 2005;24:297-304.

5. Serafini AN. Systemic metabolic radiotherapy with samarium-153 EDTMP for the treatment of painful bone metastasis. Q J Nucl Med. 2001;45:91-9.

6. Petrilak DP, Pangen CM, Hussain MH, Lara PN Jr, Jones JA, Taplin MA, Burch PA, Berry D, Moinpour C, Kohli M, Benson MC, Small EJ, Raghavan D, Crawford ED. Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med. 2004;351:1513-20.

7. Serafini AN. Current status of systemic intravenous radiopharmaceuticals for the treatment of painful metastatic bone disease. Int J Radiat Oncol Biol Phys. 1994;30:1187-94.

8. Laing AH, Ackery DM, Bayly RJ, Buchanan RB, Lewington VJ, McEwan AJ, Macleod

PM, Zivanovic MA. Strontium-89 chloride for pain palliation in prostatic skeletal malignancy. Br J Radiol. 1991;64:816-22.

9. Eary JF, Collins C, Stabin M, Vernon C, Petersdorf S, Bakre M, Hartnett S, Ferency S,

Addison SJ, Appelbaum F. Samarium-153-EDTMP biodistribution and dosimetry estimation. J Nucl Med. 1993;34:1031-6.

10. Nabholtz JM, Senn HJ, Bezwoda WR, Melnychuk D, Deschenes L, Douma J, Vandenberg

TA, Rapoport B, Rosso R, Trillet-Lenoir V, Drbal J, Molino A, Nortier JW, Richel DJ, Nagykalnai T, Siedlecki P, Wilking N, Genot JY, Hupperets PS, Pannuti F, Skarlos D, Tomiak EM, Murawsky M, Alakl M, Aapro M. 304 Study Group: Prospective randomized trial of docetaxel versus mitomycin plus vinblastine in patients with metastatic breast cancer progressing despite previous anthracycline-containing chemotherapy. J Clin Oncol. 1999;17:1413-24.

11. Crown J: A review of the efficacy and safety of docetaxel as monotherapy in metastatic

breast cancer. Semin Oncol. 1999;26(1 Suppl 3):5-9.

12. Ricci S, Boni G, Pastina I, Genovesi D, Cianci C, Chiacchio S, Orlandini C, Grosso M, Alsharif A, Chioni A, Di Donato S, Francesca F, Selli C, Rubello D, Mariani G. Clinical benefit of bone-targeted radiometabolic therapy with 153Sm-EDTMP combined with chemotherapy in patients with metastatic hormone-refractory prostate cancer. Eur J Nucl Med Mol Imaging. 2007;34:1023-30.

13. Brito DMM, Gomes ML, Rodrigues PC, Paula EF, Gutfilen B, Bernardo-Filho M. Effect of the chemotherapeutic drugs on the biodistribution of 99mTc-DTPA in Balb/c mice. J Exp Clin Cancer Res. 1998;17:313-6.

14. Holanda CMCX, Leite RCH, Catanho MTJ, Souza GM, Bernardo-Filho M. The effect of glucantime™ on the labeling of blood constituents with technetium-99m. Acta Cir Bras. 2005;20:81-5.

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15. Chacon DA, Araújo-Filho I, Villarim-Neto A, Rêgo AC, Azevedo IM, Bernardo-Filho M, Brandão-Neto J, Medeiros AC. Biodistribution of the radiophamarceutical sodium pertechnetate (Na99mTcO4) after massive small bowel resection in rats. Acta Cir Bras. 2007;22:430-5.

Conflict of interest: none Financial source: CNPq

Correspondence: Aldo Cunha Medeiros Av. Miguel Alcides Araújo, 1889 59078-270 Natal – RN Brazil [email protected]

Received: August 26, 2008 Review: October 28, 2008

Accepted: November 27, 2008 How to cite this article Villarim Neto A, Açucena MKMT, Pereira KRSG, Rêgo ACM, Azevedo IM, Bernardo-Filho M, Medeiros AC. Biodistribution of samarium-153-EDTMP in rats treated with docetaxel. Acta Cir Bras. [serial on the Internet] 2009 Jan-Feb;24(1). Available from URL: http://www.scielo.br/acb

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3.2. Biodistribution of the Radiopharmaceutical Sod ium Pertechnetate after

Biliopancreatic Bypass with a Duodenal Switch.

Publicado no periódico Brazilian Archives of Biology and Technology, 2007;

50:189-197,Qualis Internacional B

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Biodistribution of Sodium Pertechnetate after Biliopancreatic Bypass with a Duodenal Switch.

Irami Araújo-Filho, Amália Cínthia Meneses Rêgo, José Brandão-Neto, Arthur Villarim-Neto, Eryvaldo Sócrates Tabosa Egito, Ítalo Medeiros Azevedo and Aldo Cunha Medeiros* Programa de Pós-Graduação em Ciências da Saúde; Universidade Federal do Rio Grande do Norte; [email protected]; Rua Cordeiro de Faria S/N; 59010-180; Natal - RN - Brasil

ABSTRACT

Study with the purpose to examine the effects of duodenal switch (DS), regularly performed in morbidly obese patients, on biodistribution of sodium pertechnetate in several organs of rats. There was no early or late mortality in either rats groups. The values of percent radioactivity per gram of tissue (%ATI/g), showed no significant difference in liver, stomach, small bowel, duodenum, kidney, heart, bladder, bone and brain, when compared the DS rats with sham and controls rats. A postoperative significant increase in mean %ATI/g levels was observed in spleen, pancreas and muscle in group DS rats, as compared to group S and C rats (p<0.05). In the lung there was an increase and in thyroid a decrease in mean %ATI/g of DS rats, when compared to sham rats (p>0.05). In conclusion, the biliopancreatic diversion with duodenal switch in rats modified the biodistribution of sodium pertechnetate in tireóide, lung, pancreas, spleen and muscle. Key words: Bariatric surgery, duodenal switch, sodium pertechnetate, biodistribution * Author for correspondence. INTRODUCTION Worldwide, it is estimated that more than 300 million people are obese (Haslam & James., 2005). Obesity, particularly abdominal obesity, is associated with increased risks of hypertension, diabetes, hyperlipidemia, sleep apnea, coronary heart disease, and stroke (Li et al., 2005). The accumulating evidence identifying obesity-related mortality and comorbidities is an important factor that has led to increased numbers of patients seeking treatment through bariatric surgery. This is a surgical procedure that reduces caloric intake by modifying the anatomy of the gastrointestinal tract and provides effective treatment for many patients with morbid obesity. Bariatric operations are classified as either restrictive or malabsorptive. Restrictive procedures limit intake by creating a small gastric reservoir with a narrow outlet to delay emptying. Malabsorptive procedures bypass varying portions of the small intestine where nutrient absorption occurs (DeMaria & Jamal.,

2005). The biliopancreatic diversion with duodenal switch (DS) is a hybrid operation involving both components of weight loss surgery. In the DS, a lateral gastrectomy provides a restricted gastric volume, while excess fat absorption is limited by shortening the functioning length of the intestine. This involves diversion of the biliopancreatic secretions by partitioning the bowel into two limbs – an alimentary channel, and the biliopancreatic (afferent) limb. These two limbs of small bowel are reconnected to form the common channel (Hess., 1998; Marceau et al., 1998). DS produces a sustained weight loss, with low side effects and without any increase in the perioperative morbidity and mortality rate, comparing to other bariatric operations (Biron et al., 2004; Rabkin., 2004; Anthone et al., 2003). There are numerous complications that may arise following any of the bariatric surgical procedures that require understanding and delineation of the specific anatomy of the operation performed. These complications

29

may include nutrient deficiencies or gastrointestinal pathology. Anastomotic leak and stricture commonly occurs (Schauer et al., 2000; DeMaria et al., 2002). The most frequently reported complication of gastric band placement is prolapse of stomach superiorly through the band producing obstruction at the band (O’Brien et al., 1999). Radiographs and scintigraphs may show an air fluid level in the gastric pouch, malposition, angulation of the band bands, problems with gastric emptying, and gastric obstruction (O’Brien et al., 2005). The radiopharmaceuticals are frequently used in diagnostic procedures. Examinations of gastric emptying, patency of anastomoses, enterogastric reflux, hepatic and thyroid diseases, osteoporosis, metastasis, etc, frequently are carried after bariatric surgery (Kitabaiashi et al., 2002; Fonseca et al., 2000; Badiali et al., 2001; Obradovic et al., 2000). Several works have studied the relationship between chemotherapy, phytotherapy and other drugs with the biodistribution of sodium pertechnetate (Braga et al., 2000; Oliveira et al., 2002; Gomes et al., 1998; Ripoll-Hamer et al, 1995; Simões et al., 1997; Feliciano et al., 2002., Abreu et al., 2006., Santos et al., 1995). With regard to potential consequences of bariatric surgery in the biodistribution of radiopharmaceuticals, little or no study was published until now. As DS is a restrictive and malabsorptive operation, of raised anatomical and metabolic repercussion, postoperative evaluation of patients through scintigraphy can be necessary. If the biodistribution of sodium pertechnetate to organs and tissues is modified as a result of bariatric surgery, scintigraphic examinations can be false-positive or false-negative, resulting in repetition of examinations with unnecessary exposition of patient to ionizing radiations. The purpose of this study was to examine the effects of DS, similar to that performed in morbidly obese patients, on biodistribution of sodium pertechnetate in several organs and tissues of rats.

MATERIAL AND METHODS

Male Wistar rats who were 12 weeks of age and weighing 328g ± 33g were obtained from Center of Experimental Surgery-UFRN, Brazil. Animals were housed in polypropilene cages for 1 week to acclimatize them to the study laboratory: 12-h light/dark cycle, room temperature of 25°C, and 50% relative humidity. Rats were allowed free access to water and standard rat chow (Labina, Purina®). The study was approved by the Institutional Animal Care Committee of the University Hospital-UFRN, Brazil and the international guidelines for the care and use of laboratory animals were followed throughout the study. Rats were randomly divided into three groups: duodenal switch group (DS), control group (C), and sham-operated group (S). After 12 h of food deprivation, rats were anesthetized with a ketamine and xylazine mixture (200 mg: 5 mg, 0.1 ml/100 g, IM), the abdomen was shaved and prepared, and the operations were performed with aseptic technique. All the surgical procedures were performed by the same investigator, a well trained and experienced surgeon in animal surgery and three previous series of experiments were performed in sequence to develop the DS model. A single intramuscular dose of 75 mg/kg of ceftriaxone sodium (Roche, SP,Brazil) was given as antimicrobial prophylaxis 30 minutes before the surgical procedures. In the DS rats (n=7) the surgery was performed via an upper 3cm midline incision. A sleeve 75% longitudinal gastrectomy was performed leaving a tabularized stomach. (Figure. 1). The duodenum was divided about 1 cm beyond the pylorus. The stump of duodenum was closed with running sutures. The small bowel was divided at its midpoint, and the distal end (alimentary limb) was anastomosed to the proximal duodenum. The proximal end of the divided small bowel, now the distal end of the iliopancreatic limb, was anastomosed to the ileum 5cm from the ileocecal valve to create a 10cm common channel. (Figure. 1). The anastomosis were hand sewn using interrupted polypropilene 6-0 sutures (Prolene® - Ethicon), using a surgical microscope (DFV- M900, São Paulo, Brazil). The hydration was done with normal saline (10 ml) injected subcutaneously into the back of the rats for the first 2 postoperative days. Postoperatively pain was treated with tenoxicam (Roche Pharm., Brazil); 0.5 mg/kg was given im to the rats once a day for 3 days.

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Figure 1 - The biliopancreatic diversion with duodenal switch operation.

Rats were allowed to drink and eat 24 h after surgery. Liquid diet (Nestogeno, Nestlé, SP-Brazil, 1 cal/g) was provided for the first 2 days, followed by ground Purina Labina chow. The sham rats (n=7) were submited to a laparotomy and soft malipulation of stomach, duodenum and small bowel. The control rats (n=7) were not operated. Body weight, using a digital scale (Filizola®, São Paulo, Brasil), and operative complications were evaluated daily for 10 days. On the 10th day all the animals were anaesthetized again, and injected with 0.lmL of Na99mTcO4 IV in the orbital plexus, corresponding to radioactive activity of 0.66MBq. After 30 minutes, the animals were killed by lethal dose of anesthetic.

Samples of the liver, spleen, stomach, small intestine, duodenum, pancreas, kidney, heart, lung, thyroid, bladder, muscle, bone (femur) and brain were harvested. The samples were washed in 0.9% saline , weighed on a high-precision digital scale (Bel-Mark 160-II Itália) and subjected to radioactivity detection using a 1470 WizardTM Gamma Counter- Perkin-Elmer, with automatic correction of radiation decline. The percentage of radioactive activity/g (%ATI/g) of each organ was calculated by dividing the activity/g of the tissue by the total activity administered to each animal. Data are expressed as mean ±SD. Statistical analysis was performed using one-way analysis of variance and the Tukey test as appropriate. P values <0.05 were considered to be statistically significant.

RESULTS There was no early or late mortality in either rats groups. The values of all sodium pertechnetate biodistribution, expressed as percent radioactivity per gram of tissue (%ATI/g), are shown in Table 1. There was no significant difference in %ATI/g in liver, stomach, small bowel, duodenum, kidney, heart, bladder, bone and brain, when compared the DS rats with S and C rats. A postoperative significant increase in mean %ATI/g levels was observed in spleen, pancreas and muscle in group DS rats, as compared to group S and C rats (p<0.05). Looking at each group separately, in the lung there was an increase and in thyroid a decrease in mean %ATI/g of DS rats, when compared to sham rats (p>0.05).

75% subtotal gastrectomy

Biliopancreatic Limb

Common channel

Cecum

Alimentary Limb

31

Table 1 - Results of the percentage of radioactivity/g of organs and tissues(%ATI/g).

Organs %ATI/g

Switch C Sham P(1)

Liver 0.42 ± 0.103 0.35 ± 0.069 0.35 ± 0.097 0.256

Spleen(2) 0.26 ± 0.079ab 0.18 ± 0.028a 0.17 ± 0.045b 0.016

Stomach 3.39 ± 1.321 2.98 ± 1.507 4.23 ± 0.941 0.205

Small bowel 0.19 ± 0.052 0.19 ± 0.052 0.26 ± 0.109 0.209

Duodenum 0.65 ± 0.541 0.44 ± 0.088 0.61 ± 0.484 0.633

Pancreas(2) 0.27 ± 0.089ab 0.14 ± 0.045a 0.15 ± 0.078b 0.006

Kidney 0.53 ± 0.157 0.42 ± 0.075 0.36 ± 0.174 0.115

Heart 0.28 ± 0.131 0.27 ± 0.061 0.16 ± 0.079 0.050

Lung(2) 0.44 ± 0.075a 0.39 ± 0.143 0.28 ± 0.075a 0.030

Thyroid(2) 1.48 ± 2.070a 3.60 ± 1.578 4.44 ± 1.143a 0.010

Bladder 0.30 ± 0.115 0.33 ± 0.094 0.32 ± 0.173 0.929

Muscle(2) 0.09 ± 0.028ab 0.06 ± 0.018a 0.05 ± 0.026b 0.008

Bone 0.14 ± 0.032 0.14 ± 0.032 0.14 ± 0.050 0.958

Brain 0.03 ± 0.033 0.01 ± 0.003 0.01 ± 0.007 0.244

Mean ± standard deviation 1. P-value. 2. Groups identified by the same letter differ significantly. Significance 0.05 using the

Tukey test. Preoperative and postoperative data of weight loss of DS rats are summarized in Figure 2. Before treatments, there were no significant differences between groups in terms of weight. The operative time for the sham operation was equivalent to that of DS. Postoperatively in group DS there was a significant decrease in body weight during the 10

days of observation attributed to the effects of operation (p<0.05). Body weight in the C and S groups gradually increased by day 10, when the rats were euthanized (Figure 2). So, the differences in the mean weight of DS rats at the end of the 10 days were significant, when compared to C and sham rats (p<0.05).

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Figure 2 - Effect of duodenal switch, sham and control on body weight in rats during the observation period.

1 2 3 4 5 6 7 8 9 10

Day

180

200

220

240

260

280

300

320

340

360

380

400

420

440

Wei

ght (

g)

Sham Switch Control

Body weight decreased significantly in the duodenal switch group vs the sham and control, P<0.05. No difference was observed between sham and control rats. In day 1 (operative day) no difference was observed among all the rats weights, meaning that the groups were uniform.

33

DISCUSSION Obesity has become an epidemic condition and in the United States, the percentage of adults who are obese increased from 15.3% in 1995 to 23.9% in 2005. Approximately 4.8% are considered to be extremely or morbidly obese. Worldwide, it is estimated that more than 300 million people are obese (Ogden et al., 2006; Haslam & James., 2005). Obesity results in a major risk for serious diseases, including diabetes mellitus, cardiovascular disease, hypertension, dyslipidemia, degenerative arthritis, certain forms of cancer and respiratory problems and may result in socioeconomic and psychosocial impairment (Ali et al., 2006). Therefore many weightlowering therapies, such as dietary and pharmaceutical regimens completed with physical exercise, have been proposed. However, almost 95% of morbidly obese (BMI ≥40 kg/m2) patients fail to achieve acceptable long-term weight loss with any form of non-operative treatment Ali et al., 2006). Hence, great efforts have been made to achieve better results using surgery. The bariatric operations have proved very successful and cost effective in achieving marked and maintained weight loss. In spite of good results, bariatric surgery may cause anatomic and metabolic complications (DeMaria et al., 2002). The diagnostic of these side effects may require image exams such as radiography and scintigraphy. To understand and explore the relationship between bariatric surgery and biodistribution of radiopharmaceuticals to organs and tissues, a well-characterized and reproducible animal model was used. To achieve this purpose, this is a report of the experience with a biliopancreatic diversion with duodenal switch rat model. Technetium- m99 (m99Tc) is the most used radioisotope in nuclear medicine as well as in basic research. It has a low mean life (6 h), low radiation and a low doses is needed for diagnostic procedures (Braga et al., 2006). It has been used in vivo and in vitro under the form of sodium pertechnetate, in the study of diseases, drugs, chemotherapics and phytoterpics that interfere in its biodistribution (Oliveira et al, 2002; Amorim et al., 2003). Pertechnetate labelled red blood cells and leukocytes have been used in the study of drugs, and in the evaluation of the mononuclear system (Palestro et al., 2006). Postoperative scintigraphic exams are accomplished to diagnose digestive bleeding (Bingener-Casey et al., 2002), gastroesofageal reflux (Adachi et al., 1999), as well as anastomoses patency (Blachar., 2004). Diagnosis of cancer metastasis (Leen., 1999), and postoperative chances in kidney, liver, lung, heart and other organs are done using scintigraphy with radiotracers and pertechnetate (Aktas et al., 2005; Chalela., 1999). In the present work the DS did not affect the biodistribution of sodium pertechnetate in liver, stomach, small bowel, duodenum, kidney, heart, bladder, bone and brain. The stomach is commonly examined in the postoperative of bariatric surgery to diagnose mainly leaks, because leakage from the reservoir or the connecting tube is a late complication of bariatric surgery. In small leaks the escape rate of a radio-contrast agent may be low, and hence these leaks may be overlooked on radiography. By contrast, using scintigraphy, the slowly diffusing 99mTc-pertechnetate is re-absorbed by peritoneal blood vessels and subsequently absorbed into the gastric mucosa, because of high gastric affinity for pertechnetate. This fact may explain because, in this study, biodistribution of pertechnetate was not affected in stomach. It has been suggested a higher accuracy of scintigraphy as compared with radiography in the assessment of leakage in bariatric surgery. (Van DenBossche et al., 2002). The liver, small bowel, duodenum, kidney, heart, bladder, bone and brain have a few affinity for pertechnetate and we did not find alteration in biodistribution of this radiopharmaceutical in them. So, if a scintigraphy is to be done to study anyone after bariatric surgery, false results certainly are not expected. The biodistribution of pertechnetate showed elevated in spleen and this organ may be the target of future scintigraphs. Splenectomy during exploratory laparotomy after bariatric surgery significantly increases morbidity and mortality. Peters et al, (1990) related that six of 200 patients having primary or revisional vertical banded gastroplasty for morbid obesity or failure of previous bariatric surgery had splenic injury. Eventual scintigraphy in these patients should be interpreted with caution. Obese patients often complain of dyspnea despite not having demonstrable lung disease It has been hypothesized that increased chest wall mass along with increased abdominal size imparts a restrictive ventilatory defect, which then imposes an increased work of breathing (Sahebjani, 1996). The bariatric

34

procedures are performed in morbidly obese patients who tend to have reduced chest wall compliance, reduced lung volume, less functional residual capacity, and increased physiologic intrapulmonary shunt during mechanical ventilation (Damia et al., 1988). Therefore, morbidly obese patients may be at risk for intraoperative and postoperative complications, which may be diagnosed by scintigraphy. Together, pulmonary emboli, anastomotic leaks, and respiratory failure account for 80 percent of all deaths in the first 30 days following bariatric surgery (Virji & Murr, 2006). As in this study the biodistribution of pertechnetate was higher in lungs of operated rats then in controls, the interpretation of eventual lung exams (Giordano et al., 1997) in patients has to be with caution. In the past five years, several confirmed cases of pancreatic disorders occurred in persons who had undergone bariatric surgery (Service et al., 2005). Some patients presented with repeated episodes of symptoms of profound postprandial neuroglycopenia associated with endogenous hyperinsulinemic hypoglycemia and demonstration of diffuse beta-cell hypertrophy and hyperplasia in resected pancreatic tissue. A plausible explanation, with broader implications, is that bariatric surgery results in long-term stimulation of beta-cell growth and activity by gut hormones (glucagon-like peptide 1) that are perturbed as a result of the altered gastrointestinal transit. These metabolic disorders may explain the high radioactivity observed in pancreas of DS operated rats (D’Alessio & Vahl, 2004). Moreover, at least in rodents, GLP-1 triggers beta-cell neogenesis and proliferation while inhibiting apoptosis (Brubaker & Drucker., 2004). In this study the %ATI/g in thyroid was decreased in DS rats compared to sham rats. Paradoxically, a great percentage of obese patients have hypothyroidism, that is improved after bariatric surgery (Raftopoulos et al., 2004). The reduction in the %ATI/g in thyroid can be related to the postoperative energy deficiency, knowing that half of the small bowel is defunccionalyzed and the stomach is highly reduced. Steps et al had attributed this phenomenon to a reduction of the transport of sodium pertechnetate to the thyroid gland, as happen with iodine in malnourished patients (Passos et al., 2000; Passos et al., 2002). Further studies are necessary to explain these findings. Bariatric surgery has become the treatment of choice for morbid obesity and it greatly changes the body composition for years following surgery. The lean body mass (muscle) is significantly reduced in the postoperative period (Tanner et al., 2002). In this study we found a paradoxical relationship between weight loss, muscle loss, and increased muscle %ATI/g. So that, questions remain with regard to the physiological mechanisms and pathophysiological consequences of duodenal switch, and biodistribution of radiopharmaceuticals is one of them. It has been related that the polyneuropathy and miopathy after bariatric surgery are resultant of the deficiency of B12 vitamin, tiamin and vitamin D respectively (Koffman et al., 2006). The highly significant muscular captation of sodium pertechnetate in DS rats may be a consequence of muscle inflammation for vitamin D deficit (Plotnikoff & Quigley., 2003). In conclusion, the data of this study permits the conclusion that the biliopancreatic diversion with duodenal switch in rats modified the biodistribution of sodium pertechnetate in tireóide, lung, pancreas, spleen and muscle. ACKNOWLEDGEMENTS The authors are grateful to Mr. Michael Germain for English language manuscript review. RESUMO Estudo objetivando de examinar efeitos da operação switch duodenal (SD) na biodistribuição do pertecnetato de sódio em vários órgãos de ratos. Não ocorreu mortalidade precoce e tardia nos animais operados. Os valores do percentual de radioatividade/grama de tecido (%ATI/g) mostraram nenhuma diferença significante no fígado, estômago, intestino, duodeno, rim, coração, bexiga, osso e cérebro, comparando-se o grupo SD com sham e controle. Aumento significante na média de %ATI/g foi observado no baço, pâncreas e músculo (grupo SD), comparado com os grupos sham e controle (p<0,05). No pulmão houve aumento e na tireóide diminuição no %ATI/g nos ratos SD, comparados com os sham e controles (p>0,05). Em conclusão, a operação denominada derivação biliopancreática

35

com switch duodenal em ratos modificou a biodistribuição de pertecnetato de sódio na tireóide, pulmão, pâncreas, baço e músculo, podendo significar que a interpretação de exames cintilográficos nesses órgãos deve ser feita com cautela . REFERENCES Abreu, P.R., Almeida, M.C., Bernardo, R.M., Bernardo, L.C., Brito, L.C., Garcia, E.A., Fonseca, A.S., Bernardo-

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22:947-954. Obradovic, V.B., Artiko, V., Chebib, H.Y., Petrovic, M.N., Vlajkovic, M., Petrovic, N.M. (2000), Estimation of the

enterogastric reflux by modified scintigraphic method. Hepatogastroenterology., 47,738-741. O’Brien, P.E., Brown, W.A., Smith, A., McMurrick, P.J., Stephens, M. (1999), Prospective study of a

laparoscopically placed, adjustable gastric band in the treatment of morbid obesity. Br. J. Surg., 85,113–118. O’Brien, P.E., Dixon, J.B., Laurie, C., Anderson, M. (2005), A prospective randomized trial of placement of the

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Oliveira, J.F., Avila, A.S., Braga, A.C., Oliveira, M.B., Boasquevisque, E.M., Jales, R.L., Cardoso, V.N., Bernardo-Filho, M. (2002), Effect of extract of medicinal plants on the labeling of blood elements with Technetium-99m and on the morphology of red blood cells: I--a study with Paullinia cupana. Fitoterapia., 73,305-312.

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Passos, M.C., Ramos, C.F., Bernardo-Filho, M., Mattos, D.M., Moura, E.G. (2000), The effect of protein or energy restriction on the biodistribution of Na99TcmO4 in Wistar rats. Nucl. Med. Commun., 21,1059-62.

Passos, M.C., Ramos, C.F., Dutra, S.C., Bernardo-Filho, M., Moura, E.G. (2002), Biodistribution of 99mTc-O4Na changes in adult rats whose mothers were malnourished during lactation. J. Nucl. Med., 43,89-91.

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Van DenBossche, B., Goethals, I., Dierckx, R.A., Villeirs, G., Pattyn, P., Van de Wiele, C. (2002), Leakage assessment in adjustable laparoscopic gastric banding: radiography versus 99mTc-pertechnetate scintigraphy. Eur. J. Nucl. Med., 29,1128–1131.

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3.3. Biodistribution of the radiophamarceutical sod ium pertechnetate

(Na99mTcO4) after massive small bowel resection in rats

Publicado no periódico Acta Cirúrgica Brasileira, 2007 Nov-Dez;22(6):430-435,

Qualis Internacional C

39

ORIGINAL ARTICLE

Biodistribution of the radiophamarceutical sodium pertechnetate (Na99mTcO4) after massive small bowel resection in rats1

Biodistribuição do radiofármaco pertecnetato de sódio (Na99mTcO4) em ratos submetidos a ressecção extensa de intestino delgado

Dâmaso de Araújo Chacon2, Irami Araújo-Filho 2, Arthur Villarim-Neto 2, Amália Cínthia Meneses Rêgo3, Ítalo Medeiros Azevedo4, Mário Bernardo-Filho 5, José Brandão-Neto6, Aldo Cunha Medeiros6

1. Research performed at Nucleus for Experimental Surgery, Federal University of Rio Grande do Norte (UFRN), Brazil.

2. Fellow PhD degree, Postgraduate Program in Health Sciences, UFRN, Brazil. 3. Graduate Student, Scientific Initiation Program, UFRN, Brazil. 4. Statistician, Department of Surgery, UFRN, Brazil. 5. PhD, Chairman, Biophysics and Biometry Department, UERJ, Brazil 6. PhD, Full Professor, Postgraduate Program in Health Sciences, UFRN, Brazil. ABSTRACT Purpose: To evaluate the biodistribution of sodium pertecnetate (Na99mTcO4) in organs and tissues, the morphometry of remnant intestinal mucosa and ponderal evolution in rats subjected to massive resection of the small intestine. Methods: Twenty-one Wistar rats were randomly divided into three groups of 7 animals each. The short bowel (SB) group was subjected to massive resection of the small intestine; the control group (C) rats were not operated on, and soft intestinal handling was performed in sham rats. The animals were weighed weekly. On the 30th postoperative day, 0.l mL of Na99mTcO4, with mean activity of 0.66 MBq was injected intravenously into the orbital plexus. After 30 minutes, the rats were killed with an overdose of anesthetic, and fragments of the liver, spleen, pancreas, stomach, duodenum, small intestine, thyroid, lung, heart, kidney, bladder, muscle, femur and brain were harvested. The biopsies were washed with 0.9% NaCl.,The radioactivity was counted using Gama Counter WizardTM 1470, PerkinElmer. The percentage of radioactivity per gram of tissue (%ATI/g) was calculated. Biopsies of the remaining jejunum were analysed by HE staining to obtain mucosal thickness. Analysis of variance (ANOVA) and the Tukey test for multiple comparisons were used, considering p<0.05 as significant. Results: There were no significant differences in %ATI/g of the Na99mTcO4 in the organs of the groups studied (p>0.05). An increase in the weight of the SB rats was observed after the second postoperative week. The jejunal mucosal thickness of the SB rats was significantly greater than that of C and sham rats (p<0.05). Conclusion: In rats with experimentally-produced short bowel syndrome, an adaptive response by the intestinal mucosa reduced weight loss. The biodistribution of Na99mTcO4 was not affected by massive intestinal resection, suggesting that short bowel syndrome is not the cause of misleading interpretation, if an examination using this radiopharmaceutical is indicated.

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Key words: Short Bowel Syndrome. Sodium Pertechnetate Tc 99m. Pharmacokinetics. Rats. RESUMO Objetivo: Avaliar em modelo animal com ressecção extensa do intestino delgado a biodistribuição de pertecnetato de sódio (Na99mTcO4) em órgãos e tecidos, a evolução ponderal e a morfometria da mucosa do intestino delgado remanescente. Métodos: Vinte e um ratos Wistar foram aleatoriamente divididos em três grupos de sete animais cada. O grupo intestino curto (IC) foi submetido a ressecção extensa do intestino delgado, o grupo controle (C) não foi operado e o grupo sham foi submetido a leve manipulação cirúrgica das alças intestinais.Todos foram pesados semanalmente. No 30º dia pós-operatório foi administrado 0,l mL de Na99mTcO4 aos animais dos três grupos, IV no plexo orbital, com atividade radioativa média de 0,66MBq. Após 30 minutos os ratos foram mortos e retirados fragmentos do fígado, baço, pâncreas, estomago, duodeno, intestino delgado, tireóide, pulmão, coração, rim, bexiga, músculo, fêmur, e cérebro. As amostras foram lavadas com solução de NaCl 0,9%.A radioatividade foi contada peloContador Gama 1470, WizardTM Perkin-Elmer e calculado o percentual de atividade radioativa por grama (%ATI/g) de cada órgão. Biópsias do jejuno foram submetidas a análise da espessura da mucosa (coloração HE). Utilizou-se avaliação estatística paramétrica (ANOVA) e teste de Tukey, considerando p<0,05 como significante. Resultados: Não houve diferenças significantes da %ATI/g nos órgãos dos grupos estudados (p>0,05). Verificou-se acentuada redução inicial de peso, em seguida um aumento do peso dos animais tratados a partir da segunda semana de observação e aumento da espessura da mucosa jejunal do grupo IC, comparado com os demais. Conclusão: Em ratos com síndrome do intestino curto, uma adaptação na espessura da mucosa contribuiu para reversão na perda de peso inicial e para que a biodistribuição do Na99mTcO4 não fosse afetada pela ressecção extensa do intestino, sugerindo que o intestino curto não é causa de interpretações duvidosas, quando exame cintilográfico com este radiofármaco estiver indicado. Descritores: Síndrome do Intestino Curto. Pertecnetato Tc99m de Sódio. Farmacocinética.

Ratos.

Introduction

Radioisotopes are used in nuclear medicine for diagnostic and therapeutic purposes. The labelling capacity of these isotopes for the plasmatic proteins is well known, and their bioavailability and pharmacokinetics can be modified by drugs and diseases1,2. Among the most useful artificial radioisotopes, technetium-99m, in the chemical form of sodium pertechnetate (Na99mTc04), is the most important. Its use in nuclear medicine is due to the emission of gamma energy of 140 KeV, enabling it to take advantage of the scintillation detectors and obtain enhanced image quality; this is known as scintigraphy3. It has a short life, is low cost, and is obtained from a molybdenum/technetium generator (99Mo/99mTc).3 It is easily distributed through the vascular fluid, intersticial space with uptake by the salivary glands, thyroid, stomach, intestines and other organs3. It is rapidly eliminated by the urine and when incorporated to specific substances, produces organ images of different densities and functions4. Experimental studies carried out with the labelling of red blood cells with 99mTc identified important biological effects, in addtion to alterations in the labelling process5,7. Gomes et al carried out a study with mitomicin-C that described alterations in the labelling of red blood cells6. The literature reports that several natural drugs reduce the efficiency of labelling red blood

41

cells with 99mTc7. An experimental study with Vincristin, used in oncology protocols, showed an interaction of this drug with 99mTc in several organs8. However, there are no reports of research studying the biodistribution of radiopharmaceuticals after surgical procedures. In the present study, we used an experimental model of massive resection of the small intestine, characterizing the short bowel syndrome, which results in unsuitable water and nutrient absorption, causing malnutrition9,10. In spite of the short bowel syndrome, the intestine can be adapted through physiologic, cellular and moleculares mechanisms9. In some patients, dilation and lengthening of the remnant small intestine occur as a phenomenon of functional adaptation. Surgical techniques have been reported that attempt to lengthen this intestinal segment. Such procedures are complex and frequently ineffective, and call for assessments of their efficacy11. Recently, new therapeutic methods, such as isolated small intestine transplantation or combined with liver transplantation, have been an alternative for cases of hepatic failure due to total parenteral nutrition in the treatment of short bowel syndrome12.

Given the antiabsorptive effect of the operation, with great repercussions on the metabolism, radioisotope images may be necessary in the postoperative, in order to control the series of pathological conditions resulting from short bowel syndrome. Scintigraphy can be used in the postoperative of intestinal resections to assess the morphology and metabolism of several organs. Under these conditions, it becomes relevant to study the biodistritubion of Na99mTc04 in specific organs and tissues.

Therefore, the present paper aims to study, in an animal model of massive resection of the small intestine, the biodistribution of Na99mTcO4 in several organs by means of radiation counting in organs and tissues in the postoperative period. We also evaluated the ponderal evolution of the animals after the operation, as well as the mucosal morphometry of the remnant small intestine. Methods

Twenty-one Wistar rats, age around 180 days, weighing 265g±31g were used. They were supplied by the vivarium of the Nucleus for Experimental Surgery of the Federal University of the Rio Grande do Norte, Brazil. All the animals were weighed and observed in individual cages with water and food (Labina ® Purina) ad libitum and acclimated in the laboratory for 7 days. They were maintained under temperature control (21ºC), air humidity (60 – 70 %) and lighting (12/12 hours light/dark cycle) and handled in accordance with the Animal Experimentation Code of Ethics (Council for International Organizations of Medical Sciences ) and the rules of the Brazilian College of Animal Experimentation. They were randomly divided into three groups: the experimental group rats, denominated short bowel, (SB, n=7) were subjected to massive resection of the small intestine (90%); control group rats were not operated on (C, n=7) and the third group underwent a simulated operation, called sham (n=7). Rats were fasted overnight before surgery, and anesthetized with sodium pentobarbital (20mg/kg intraperitoneal) and ketamine (20mg/kg intramuscular); they were operated on under sterile conditions.

A 3 cm midline laparotomy was performed and intestinal transections were done 5 cm above the ileocecal junction and 5 cm from the duodenojejunal transition. With the aid of a surgical microscope (DF Vasconcelos, São Paulo, Brazil), interrupted sutures of 6-0 prolene (Ethicon®, Brazil) were used for bowel anastomosis. The animals typically have a small bowel length of 100 cm, and accordingly, residual length was 5 cm of jejunum and 5 cm of ileum (10 cm), corresponding to 90% of resection. After surgery, the abdomen was closed with interrupted sutures of 4-0 nylon suture (Ethicon®). The animals were allowed water immediately after

42

surgery and food on the second postoperative day. The sham rats were subjected to a 3 cm medium laparotomy and mild manipulation of the small bowel. The rats were weighed weekly with a digital scale (Filizola® São Paulo, Brazil) and observed for 30 days. On the 30th day all the animals were anaesthetized again, and injected with 0.lmL of Na99mTcO4 in the venous orbital plexus, corresponding to radioactive activity of 0.66MBq. After 30 minutes, the animals were killed by lethal dose of anesthetic. Samples of the liver, spleen, pancreas, stomach, duodenum, small intestine, thyroid, lung, heart, kidney, bladder, muscle, femur and brain were harvested. The samples were washed in 0.9% NaCl , weighed on a high-precision digital scale (Bel-Mark 160-II Itália) and subjected to radioactivity detection using a 1470 WizardTM Gamma Counter- Perkin-Elmer, with automatic correction of radiation decline. The percentage of radioactive activity/g (%ATI/g) of each organ was calculated by dividing the activity/g of the tissue by the total activity administered to each animal. Samples with 2cm of jejunum were harvested 2 cm below the anastomosis. After washed in 0.9% saline, the excised tissues were fixed in 10 % buffered formalin for 48 h, dehydrated and embedded in paraffin. Sections cut at 5µm thickness were stained with hematoxilyn and eosin and morphology was assessed by an observer, who was unaware of the tissue origin. For the morphometric study of intestinal mucosa, Media Cybernetics – LP, USA, Image Pro-Plus software was used with an Olimpus BX-50 microscope fitted with a digital (Samsung®) video camera. The video camera transferred the image from the microscope to the computer screen. The measurement of the mucosal thickness was delimited with a computer mouse from the apex of the villus to the muscularis mucosae and was expressed in microns (µm). The analysis was made under 40x magnification using specimens in which the villi and the crypts were perpendicular to the muscularis mucosae.

For the analysis of the different data related to postsurgical weight loss, to the measurements of total mucosal thickness, and to the biodistribution of sodium pertechnetate of the different groups, parametric variance (ANOVA) was used. For the multiple comparisons, the Tukey test was used. A significance level of 5% (p<0.05) was established. Results

All the animals survived the surgical procedures. Table 1 shows the results of the

differences in %ATI/g among groups SB, C and sham. We observed an increase in the biodistribution of Na99mTcO4 in the thyroid and duodenum of SB rats, when compared to control rats. However, since the standard deviation was high, there was no significant difference (p>0.05). In the stomach, an apparent tendency for reduced biodistribution of the %ATI/g occurred in the SB rats, compared to sham rats, but without statistical significance (p>0.05). In several organs the percentages of radioactive activity (%ATI/g) had very similar values among the groups, without significant differences (Table 1).

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TABLE 1 – Biodistribution of Na99mTcO4 in the organs of the respective groups

%ATI/g

Organs

SB C Sham

ANOVA (1)

Liver 0.35 ± 0.089 0.36 ± 0.079 0.39 ± 0.113 0.794400

Spleen 0.22 ± 0.090 0.18 ± 0.031 0.19 ± 0.044 0.565470

Estomach 2.58 ± 0.730 2.72 ± 0.614 4.11 ± 1.793 0.116180

Small bowel 0.28 ± 0.107 0.20 ± 0.052 0.28 ± 0.130 0.690700

Duodenum 1.73 ± 1.814 0.41 ± 0.062 1.13 ± 1.719 0.378723

Pancreas 0.16 ± 0.063 0.14 ± 0.055 0.18 ± 0.125 0.811183

Kidney 0.41 ± 0.086 0.42 ± 0.082 0.36 ± 0.187 0.738872

Heart 0.17 ± 0.075 0.27 ± 0.057 0.17 ± 0.084 0.076831

Lung 0.35 ± 0.105 0.38 ± 0.125 0.31 ± 0.058 0.581337

Thyroid 5.35 ± 1.979 3.71 ± 1.256 3.80 ± 1.058 0.187603

Bladder 0.39 ± 0.114 0.33 ± 0.109 0.27 ± 0.139 0.309546

Muscle 0.07 ± 0.028 0.06 ± 0.019 0.05 ± 0.035 0.570391

Femur 0.16 ± 0.055 0.14 ± 0.036 0.15 ± 0.050 0.760950

Brain 0.02 ± 0.013 0.01 ± 0.003 0.03 ± 0.027 0.482193

Mean ± Standard deviation 3. P- from analysis of variance (ANOVA). The results of the test did not show statistically significant differences (p>0.05), for all the variables. %ATI/g, percent radioactivity per gram of tissue.

The weight of SB rats decreased during the first and second weeks of survival and, after that, their weight gradually increased until the 30th postoperative day, when it was nearly the same as the C and SHAM rats, which continually increased their weight over time (Figure 1). The differences in the mean weight of SB rats at the end of the second week were significant, when compared to C and SHAM rats (p<0.05).

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Preoperative 1st week 2nd week 3rd week 4th week180

200

220

240

260

280

300

320

340

360

380W

eigh

t (g)

SB Sham Control

FIGURE 1 – Mean weight of rats in each group and postoperative period; SB, short bowel.

The presence of an increase in intestinal mucosa thickness was detected in all IC rats, when compared to C and SHAM rats until the end of the observation period, as seen in Table 2 (p<0.05) and Figure 2.

TABLE 2 – Jejunal mucosa thickness of rats and their respective groups.

Groups Variable

SB C Sham

ANOVA (1)

Mucosal

thickness in

µm(2)

334.34 ± 25.9ab 194.40 ± 39.0a 194.22 ± 33.2b 0.0000

Values expressed in Mean ± Standard deviation (1) p-value of analysis of variance (ANOVA). (2) Groups identified with the same letter differ significantly at a level of 5% (Tukey test). SB = Short bowel; C = control.

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A

B

46

FIGURE 2 - Small intestine morphology and mucosal cell proliferation on day 30 in short bowel (A) control (B) and sham (C) animals. Massive intestinal resection (A) induced significant increases in total mucosal thickness (arrows), when compared to control and sham animals (B, C) (see Table 2; p<0.05). HE, 40x Discussion

The alterations in the biodistribution of Na99mTcO4 in organs and tissues are very well identified in several studies that used no experimental surgical models 1,,6,,8,13,14. However, in the postoperative of major surgical procedures, there are no reports concerning the biodistribution of radiopharmaceuticals. The present experimental model of the short bowel syndrome in rats submitted to massive resection of the small intestine was used to determine the biodistribution profile of Na99mTcO4 in several organs and tissues. Intestinal failure is characterized by malnutrition and/or dehydration as a result of the inadequate digestion and absorption of nutrients. The most common cause of intestinal failure is short bowel syndrome, which occurs when the functional small bowel mass is reduced below the level necessary for adequate nutrient and water absorption. This condition frequently results from a massive resection of the small bowel. Following resection, the intestine is capable of adaptating in response to enteral nutrients as well as other trophic stimulation. Rodents are commonly used in well-characterized models to assess the process of intestinal adaptation16. Following small bowel resection in the rat, the remnant intestinal mucosa undergoes compensatory alterations in an attempt to restore normal absorptive capacity. Morphologic and functional changes include increases in mucosal length, enterocyte proliferation, as well as increased electrolyte, glucose and amino acid uptake16,17.

In humans, the alterations of intestinal absorption due to massive resection of the small intestine usually cause significant weight loss15. However, in rodents, there is a rapid adaptation of the intestinal mucous membrane, which minimizes weight loss16. These mechanisms of intestinal adaptation take place at physiologic, cellular and molecular levels and they do not correspond to what occurs in the human intestine 17. Nutrients, electrolytes, hormones, cytokines and other elements take part in the process, which involves mainly the intestinal mucous membrane. The process begins with apoptosis and continues with an increase in epithelial cells,

C

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vilosities and mucosal crypts, and a consequent remodeling of their architecture. Functionally, this allows for increased substance transport through the intestinal mucosa17.

In the present study a significant decrease was observed in the weights of rats submitted to massive intestinal resection, in the immediate postoperative period, and weight recovery beginning at the end of the second week. These data coincide with a classic study on the subject, where morphological and functional adaptations of the jejunum were observed between the first and second postoperative weeks18. This phenomenon was also shown in the morphometry of the jejunal mucous membrane of the animals subjected to massive resection of the present study. Therefore, the mucous membrane hyperplasia observed in the jejunal mucosa of the SB rats of the present experiment, likely contributed to the rapid weight recovery of the animals, starting from the second postoperative week. Welters et al (2002) verified that intestinal function recovery begins with the hyperplasia of the intestinal mucosa and that absorptive function depends on the maturity of the enterocytes, a fundamental factor for nutrient metabolism19.

The precocious postoperative recovery of the animals, represented by weight and morphology of the intestinal mucosa recovery, and the healthy behavior of the SB rats, comparable to the controls, certainly contributed to the absence of significant clinical alterations and malnutrition. Consequently, there was no negative effect on the biodistribution of Na99mTcO4 in the vital organs. In an experimental study using malnutrition-inducing diets, Passos et al20 (2000) showed that malnutrition affected the biodistribution of Na99mTcO4 in different organs such as the thyroid, brain, stomach and heart. In their study, the intestine was not surgically manipulated.

Studies in animals have been investigating substances that regulate the absorptive function of the intestine21. These mechanisms are mediated by multiple factors, including enteral or parenteral nutrition, hormones and growth factors22. Recently, studies on the use of the human growth hormone (GH), the epidermal growth factor (EGF) and the glucagons-like peptide-2 (GLP-2), produced in the L-cells of the small intestine, have confirmed them as agents that increase intestinal adaptation after massive resection23. The study suggests that, whereas GLP-2 is important in controlling adaptation, there are spatial or regional systems in place that use varying pathways. The significant increase in nutrient-stimulated GLP-2 secretion suggests that GLP-2 is involved not only in the initiation, but also in maintaining the ongoing adaptive process. The increases in mucosal proliferation that are temporally associated with a maintained GLP-2 release, suggest that GLP-2 is important in initiating and maintaining the small intestine’s adaptive response to resection24.

Curtis et al (1984) studied rats submitted to massive resection of the small intestine using marker 51mCrl3mC and protein, and observed the animals for one week. They concluded that the rats had no alteration in absorption and digestion time when compared to the treated group and the control; this demonstrated the fast physiological adaptation of the animals25. A growing number of tissue factors are being investigated for having great potential in promoting intestinal adaptation in animals and humans with short bowel syndrome, in the hope of obtaining effective therapies for the syndrome in the future23,26.

In summary, massive intestinal resection in the current study did not interfere significantly with the biodistribution of the radiopharmaceutical Na99mTcO4 in the organs studied. Certainly the mucosal hyperplasia of the remnant intestine was a preponderant factor for the quick weight loss reversal of the animals, and consequent preservation of their healthy metabolism. The present study does not allow us to comment on the mechanisms by which intestinal resection results in the stimulation of trophic effects and mucosal adaptation, allowing normal biodistribution of Na99mTcO4 in rats. Identifying factors that may enhance the process of

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intestinal adaptation is an exciting area of research with important potential clinical applications. This area will require further studies. Conclusion

In rats with experimentally-induced short bowel syndrome, an adaptive response by the intestinal mucosa reduced weight loss. The biodistribution of Na99mTcO4 was not affected by massive intestinal resection, suggesting that short bowel syndrome could not be the cause of misleading image interpretation when an examination with this radiopharmaceutical is indicated. References

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6. Gomes ML, Mattos MMD, Freitas RS, Diré GF, Lima EAC, Souza SMS, Bernardo-Filho M. Evaluation of the effect of mitomicin-C on the bioavailability of technetium-99m-labelled sodium pyrophosphate in mice. Cell Mol Biol. 2002; 48:757-9.

7. Oliveira JF, Avila AS, Braga ACS, de Oliveira MB, Boasquevisque EM, Jales RL, Cardoso VN, Bernardo-Filho. Effect of extract of medicinal plants on the labeling of blood elements with Technetium-99m and the morphology of red blood cells: a study with Paullinia cupana. Fitot. 2002; 73:305-12.

8. Mattos D M M, Gomes M L, Freitas R S, Boasquevisque EM, Cardoso VN, Paula EF, Bernardo-Filho M. The effect of vincristine on the biodistribution of technetium-99m-DTPA, GHA and DMSA in balb/c female mice. J Nucl Med Technol. 2000; 28:271-4.

9. Zhou X, Li YX, Li N,Li JS. Effect of bowel rehabilitative therapy on structural adaptation of remnant small intestine: animal experiment. World J Gastroenterol. 2001;7:66-73.

10. Tannuri U. Síndrome do intestino curto na criança - tratamento com nutrição parenteral domiciliar. Rev Assoc Med Bras. 2004;50:330-7.

11. Bianchi A. Intestinal loop lengthening: a technique for increasing small intestinal length. J Pediatr Surg. 1980; 15:145-51.

12. Iyer K, Kaufman S, Sudan D, Horslen S, Shaw B, Fox I, Langnas A. Long-term results of intestinal transplantation for pseudo-obstruction in children. J Pediatr Surg. 2001; 36:174-7.

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13. Santos-Filho,SD, Bernardo-Filho,M. Efeito de um extrato de Hipérico (Hypericum perforatum) na marcação in vitro de elementos sangüíneos com tecnécio-99m e na biodisponibilidade do radiofármaco pertecnetato de sódio em ratos Wistar. Acta Cir Bras. 2005;20 (suppl.1):121-5.

14. Holanda CMCX, Leite CH, Nunes RAS, Oliveira HA, Catanho MTJ, Souza GM, Bernardo-Filho M. Effect of antimalarial drugs on the bioavailability of the methylenediphosphonic acid labeled with technetium99m (99mTc-MDP) in Wistar rats. Braz Arch Biol Technol. 2006; 49:207-14.

15. Goulet O, Baglin-Gobet S, Talbotec C, Fourcade L, Colomb V, Sauvat F, Jais JP, Michel JL, Jan D, Ricour C. Outcome and long-term growth after extensive small bowel resection in the neonatal period: a survey of 87 children. Eur J Pediatr Surg. 2005; 15:95-101.

16. O'Connor TP, Lam MM, Diamond J. Magnitude of functional adaptation after intestinal resection. Am J Physiol Regul Integr Compar Physiol. 1999; 276: 1265-75.

17. Drozdowski L, Thomson ABR. Intestinal mucosal adaptation. World J Gastroenterol. 2006; 12: 4614-27.

18. Hanson WR, Osborne JW, Sharp JG. Compensation by the residual intestine after intestinal resection in the rat. II. Influence of postoperative time interval. Gastroenterology. 1977; 72:701-5.

19. Welters CFM, Dejong CHC, Deutz NEP, Heineman E. Intestinal adaptation in short bowel syndrome. ANZ J Surg. 2002; 72: 229–36.

20. Passos MC, Ramos CF, Bernardo-Filho M, Mattos DMM, Moura EG. The effect of protein and energy restriction on the biodistribution of Na99TcmO4 in Wistar rats. Nucl Med Commun. 2000;21:1069-62.

21. DiBaise JK, Young RJ, Vanderhoof JA. Intestinal rehabilitation and the short bowel syndrome: Part 2. Am J Gastroenterol. 2004;99:1823–32.

22. Spadoni JM, Aguilar-Nascimento JE, Silva MHGG, Spadoni-Neto B, Costa PATFB, Aléssio DMT. Effects of the combined use of glutamine and growth hormone in the intestinal adaptation after massive resection of the small bowel in rats. Acta Cir Bras. 2005; 20:382-9.

23. Cisler JJ, Buchman AL. Intestinal adaptation in short bowel syndrome. J Invest Med. 2005; 53:402-13.

24. Martin GR, Wallace LE, Hartmann B, Holst JJ, Demchyshyn L, Toney K, Sigalet DL. Nutrient-stimulated GLP-2 release and crypt cell proliferation in experimental short bowel syndrome. Am J Physiol Gastrointest Liver Physiol. 2005;288:431–8.

25. Curtis KJ, Sleisenger MH, Kim YS. Protein digestion and absorption after massive small bowel resection. Dig Dis Sci. 1984; 29:834-40.

26. Drucker D, Ehrlich P, Asa SL, Brubaker P. Induction of intestinal epithelial proliferation by glucagon-like peptide 2. Proc Natl Acad Sci USA. 1996; 93:7911–6.

Conflict of interest: none Financial source: CNPq

Correspondence: Dâmaso de Araújo Chacon Rua Almte. Nelson Fernandes, 797/1200 59022-600 Natal - RN Brazil [email protected]

Received: April 10, 2007 Review: June 12, 2007

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Accepted: July 18, 2007

How to cite this article Chacon DA, Araújo-Filho I, Villarim-Neto A, Rego ACM, Azevedo IM, Bernardo-Filho M, Brandão-Neto J, Medeiros AC. Biodistribution of the radiopharmaceutical sodium pertechnetate (Na99mTcO4) after massive small bowel resection in rats. Acta Cir Bras. [serial on the Internet] 2007 Nov-Dec;22(6). Available from URL: http://www.scielo.br/acb *Color figures available from www.scielo.br/acb

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3.4. Influence of Splenectomy on the Biodistribitio n of Technetium-99m

Dimercaptosuccinic Acid (99mTc-DMSA)



52

Influence of Splenectomy on the Biodistribuition of Technetium-99m–Dirmercaptosuccinic Acid (Tc-99mDMSA) in Rats. Maria Kadja Meneses Torres Açucena, Kércia Regina Santos Gomes Pereira, Arthur Villarim Neto, Amália Cínthia Meneses Rêgo, Ítalo Medeiros Azevedo, Irami Araújo Filho, Aldo Cunha Medeiros. Programa de Pós-Graduação em Ciências da Saúde; Universidade Federal do Rio Grande do Norte; Rua Cordeiro de Faria, s/n; 59010-180; Natal – RN – Brasil.

ABSTRACT The present work had as objective to evaluate, in animal model, if the splenectomy modifies the biodistribuition of the radiopharmaceutical DMSA-Tc99m and if there is correlation with possible alterations of the renal function. Twelve divided adult wistar rats had been used andomly in two groups (splenectomy and have controlled). The values of the percentage of radioactivity por grama de tecido.(ATI%) they had shown that it had significant reduction in the kidney and blood (p<0,05) of the animals submitted to splenectomy.

Key words: Kidney, splenectomy, radiopharmaceutical, biodistribuition. INTRODUÇÃO: Dentre os procedimentos cirúrgicos que afetam a biodistribuição dos radiofármacos, suspeita-se que a esplenectomia seja um deles, visto que já foi demonstrado que a mesma provoca alterações metabólicas a nível hepático (um dos órgãos relacionados com o metabolismo de drogas), que podem repercutir sistemicamente, quando o fígado é submetido ao dano isquemia/reperfusão (Ito K et al, 2002). No entanto, casos graves como: pancitopenia persistente em níveis perigosos; doenças hematológicas com risco de complicações maiores, como hemorragias sistêmicas ou intracranianas incontroláveis; hiperesplenismo grave; ou traumas que comprometam irreversivelmente o baço ainda constituem indicações para a esplenectomia (Petroianu, A., 2001). Vários aspectos teóricos e práticos de interação de drogas terapêuticas com radiofármacos têm sido estudados por muitos pesquisadores utilizando modelos animais para avaliar as possíveis alterações na biodisponibilidade destes radiofármacos, induzidas por drogas

sintéticas (Hessleewood S et al, 1994; Gomes ML et al, 2002; Gomes ML et al, 2002a) ou naturais ( Diré G et al, 2001; Capriles PVSZ et al 2002; Oliveira JF et al 2003). A distribuição dos radiofármacos é baseada nos mesmos princípios farmacocinéticos já descritos para os agentes terapêuticos. Assim, quando um radiofármaco é administrado concomitantemente ao uso de diferentes drogas, ou após procedimentos operatórios mutilantes, pode sofrer alterações em sua biodistribuição, desviando-se do seu órgão alvo (Braga ACS et al, 2000).

Através de administração intravenosa, o ácido demercaptossuccínico (DMSA) marcado com Na99mTcO4 é distribuído no compartimento vascular. Cerca de 75% dos íons pertecnetato ligam-se inicialmente às proteínas plasmáticas e esta ligação é reversível (Nickel, 1995). DMSA scintigraphy is now the standard method to evaluate permanent renal damage in children with UTI. (Stokland ET AL, 2007; Piepsz, et al, 1999). The use of technetium99m -dimercaptosuccinic acid (Tc99m-DMSA) for renal cortical scintigraphy, introduced by Davies et al. in 1972, has been shown to be sensitive and specific for the diagnosis of pyelonephritis in children ( Majd et

53

al, 1992), but has the disadvantages of radiation dose, invasiveness and relative expensive ( Jakobsson et al, 1992). Quando um radiofármaco é administrado a um paciente, ocorre o processo de biodistribuição. Este processo consiste de absorção, distribuição, metabolismo e excreção da substância. A biodistribuição através da farmacocinética dos radiofármacos pode ser alterada por uma variedade de procedimentos cirúrgicos (Siegel et al, 1999), medicamentos (Gomes et al., 2002a) naturais e alopáticos administrados aos pacientes (Britto et al, 1998; Mattos et al., 2000a; Diré Aguiar et al, 2002; Diré et al., 2003ª; Santos-Filho et al, 2004; Diré et al, 2004), ou por doenças. Em Medicina Nuclear o 99m Tc é amplamente utilizado em estudos estáticos e dinâmicos por meio de cintilografia (Kowalsky RG et al, 1997). O 99m Tc tem tendência a se acumular onde existe uma excessiva neovascularização, sendo exemplo disso os tumores cerebrais. Após administração sérica seu tempo de permanência permite estudos de hemodinâmica, lesões orgânicas e estudos vasculares (Sampson CB et al, 1996). É largamente utilizado no diagnóstico de doenças dos rins, bexiga, fígado, pulmões, intestinos, ossos, tireóide, fluxo sanguíneo, coração, etc.. Logo, os radiofármacos em Medicina são utilizados para obtenção de imagens com seletividade por um órgão e/ou sistema e a radiação emitida é captada externamente, transformada ou registrada em papel, filme ou monitor de vídeo. Saha GB, 1997).

O trabalho teve como objetivo avaliar se a retirada do baço altera a biodistribuição do radiofármaco DMSA-Tc-99m em ratos Wistar, correlacionando possíveis alterações renais com a esplenectomia . MATERIAL AND METHOD O estudo é do tipo experimental, no qual foram utilizados 12 ratos Wistar adultos, pesando 328g ± 33g e fornecidos pelo Biotério do Núcleo de Cirurgia Experimental Prof. Travassos Sarinho. O estudo foi aprovado pelo Comitê de Ética do Hospital Universitário-

UFRN, Brasil. Os animais foram divididos aleatoriamente em 2 grupos, A (esplenectomizados) e B (controle) de 6 animais. Os mesmos foram observados em gaiolas individuais com água e alimento “ad libitum”, anestesiados com ketamina intramuscular na dose de 50 mg/Kg associado a tiopental sódico intraperitoneal na dose de 20 mg/Kg, e operados sob condições assépticas.

Os animais do grupo A foram submetidos a laparotomia mediana, na qual foi realizado uma esplenectomia total, procedendo-se a ligadura do pedículo do baço com fio poliglactina 5-0. A laparotomia foi fechada por planos com fios de mononylon 4-0.

No grupo B, os animas foram apenas submetidos a laparotomia mediana nas mesmas condições de anestesia e antissepsia, sem esplenectomia. Ambos os grupos permaneceram sob observação pós-operatória por 15 dias, durante os quais, foram observados os parâmetros variação de peso através da pesagem semanal em balança de precisão com sensibilidade para variação a partir de 1 grama. Após esse período, os animais dos grupos A e B foram submetidos à administração de uma alíquota de 0,1 ml de DMSA marcado com Na 99m Tc04 (pertecnetato de sódio) via plexo orbital dos animais, produzindo uma atividade radioativa de 0,41 MBq. Após 30 minutos da administração do radiofármaco, os animais foram mortos com dose letal de aestésico e em seguida foi retirado amostas de sangue, esôfago, estômago, fígado, pâncreas, intestinos delgado, pulmões, tireóide, rins, fêmur, cérebro. As amostras foram isoladas, pesadas em balança de precisão e determinada a detecção da radioatividade em cada uma delas com o emprego de um contador gama, Wizard Gama Counter Perkin-Elmer . O percentual de radioatividade (% ATI) foi calculado dividindo a atividade radioativa de cada órgão pela atividade total administrada a cada animal. Os dados obtidos foram expressos em média ± desvio padrão. A comparação entre os grupos foi feita pela análise de variância ANOVA e o teste de t de Student, usando-se significância de 0,05. RESULTADOS

A tabela 1 mostra descritivamente os resultados encontrados da biodistribuição do Tc99m-DMSA

54

em cada órgão por grupo, como também os testes para investigar a existência de diferenças

estatisticamente significantes entre os grupos esplenectomia e controle.

Tabela 1 - Estatísticas descritivas e testes estatísticos referentes ao %ATI/g.

%ATI/g

Órgãos Esplenectomia Controle

p-valor(1)

Fígado 0,11 ± 0,028 0,16 ± 0,041 0,02323

Rim 2,10 ± 0,308 3,27 ± 0,810 0,04181

Coração 0,13 ± 0,041 0,12 ± 0,021 0,06622

Pulmão 0,23 ± 0,051 0,12 ± 0,064 0,00826

Tireóide 0,07 ± 0,029 0,06 ± 0,026 0,35709

Estômago 0,08 ± 0,022 0,06 ± 0,023 0,22557

Bexiga 0,34 ± 0,437 0,42 ± 0,351 0,73614

Fêmur 2,04 ± 0,014 1,93 ± 0,019 0,92429

Sangue 0,30 ± 0,092 0,53 ± 0,165 0,01247

Média ± Desvio Padrão 4. P-valor da análise do teste t para amostras independentes.

Podemos verificar através dos dados apresentados na tabela 1, que no fígado, rim e sangue houve uma menor captação de radioatividade nos animais submetidos a esplenectomia em relação ao grupo controle, caracterizando uma diferença estatisticamente significante (p<0.05), Predominou a captação

radioativa no rim e osso (fêmur). No pulmão foi observada maior captação radioativa nos animais esplenectomizados do que nos controles (p<0.05) e nos demais órgãos (coração, tireóide, estômago, bexiga e fêmur) não foram detectadas diferenças significantes na captação radioativa, comparando-se com os controle.

Tabela 2 - Estatísticas descritivas e testes estatísticos referentes a análise bioquímica.

Variáveis Esplenectomia Controle p-valor(1)

Uréia (mg/dL) 88,83 ± 18,65 51,67 ± 1,63 0,001

Creatinina (mg/dL) 0,57 ± 0,08 0,37 ± 0,03 <0,001

Média ± Desvio Padrão 1. P-valor da análise do teste t para amostras independentes.

Na tabela 2 podemos verificar que houve alteração a nível renal pois os níveis de uréia e creatinina dosados no sangue nos mostram que a média dos animais do grupo que foi submetido a esplenectomia foi significativamente maior que os animais do grupo controle verificando-se que existe

diferença estatisticamente significante (p<0,05) entre os grupos em ambas dosagens.

55

DISCUSSÃO

Some physiological and/or pharmacological properties related with the radiobiocomplex Na99mTcO4 have been reported. After intravenous injection, it is weakly bound to serum proteins (70-80%). Unbound pertechnetate ion (99mTcO4

-) diffuses slowly through the capillary membranes to the interstitial fluids, from where it is cleared by various organs such as stomach wall, intestines, salivary glands, thyroid, choroid plexus, sweat glands, kidneys and mucous membrane. The thyroid uptake of 99mTcO4

- is by active transport, as a sodium-iodide symporter, and its uptake is 2-4%. In the kidneys, 99mTcO4

- is filtered in the glomeruli, but 86% are reabsorved in the proximal tubes. Only 30% of the injected dose is excreted in urine in 24 h. [Owunwanne, A.; Patel, M.; Sadek, S. Preparation of radiopharmaceuticals. The handbook of radiopharmaceuticals. Chapman & Hall: London, 1995.//// Zuckier, L.S.; Dohan, O.; Li, Y.; Chang, C.J.; Carrasco, N.; Dadachova, E. Kinetics of perrhenate uptake and comparative biodistribution of perrhenate, pertechnetate, and iodide by NaI symporter-expressing tissues in vivo. J. Nucl. Med. 2004, 45, 500-507]. In spite of the discrepancy in the published results [xx], most investigators encourage using DMSA scintigraphy for the early diagnosis of many diseases, because of its cost-effectiveness and safety. A focal reduction or absence of uptake in one or more areas of the kidney is considered abnormal, indicating renal damage. A kidney with a relative function of <45% is also classified as abnormal. [Piepsz A, Blaufox MD, Gordon I et al (1999) Consensus on renal cortical scintigraphy in children with urinary tract infection. Semin Nucl Med 29:160–174.] In positive cases, DMSA scintigraphy was found to be highly sensitive in detecting multiple (two or more) lesions [Stogianni A, Nikolopoulos P, Oikonomou I, Gatzola M, Balaris V, Farmakiotis D, Dimitriadis A. Childhood acute pyelonephritis: comparison of power Doppler sonography and Tc-DMSA scintigraphy. Pediatr Radiol (2007) 37:685–690].

Nas condições do presente estudo, os resultados mostraram que ocorre alteração na biodistribuição do DMSA-Tc99m em modelo animal e que a retirada do baço provoca alguma alteração à nível dos rins, constatados com as dosagens de uréia e creatinina. RESUMO O estudo permitiu avaliar se a esplenectomia altera a biodistribuição do radiofármaco DMSA-Tc-99m em ratos wistar e se há correlação com possíveis alterações da função renal. Os valores do percentual de radioatividade/grama de tecido (% ATI/g) mostraram que houve redução significante no rim e sangue (p<0,05) dos animais do grupo esplenectomizados comparado com o grupo controle e correlacionando-se com aumento significante (p<0,05) da ureia e creatinina, quando comparado com o grupo controle. Em conclusão, a esplenectomia altera a captação de DMSA-Tc-99m pelo rim de ratos, correlacionando-se com comprometimento da função renal. REFERENCES Ito K, Ozasa H, Yoniya R, Horikawa S.(2002), Splenectomy ameliorates hepatic ischemia and reperfusion injury mediated by heme oxygenase-1 induction in the rat. Liver International. 22,467-473.

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3.5. Biodistribution of the Radiopharmaceutical Tec hnetium-99m-Sodium

Phytate in Rats After Splenectomy



59

Biodistribution of the radiopharmaceutical Technetium-99m-sodium phytate

in rats after splenectomy.

Kércia Regina Santos Gomes Pereira1, Maria Kadja Meneses Torres Açucena1, Arthur Villarim Neto1, Amália Cínthia Meneses Rêgo1, Ítalo Medeiros Azevedo2, Irami Araújo Filho2, Aldo Cunha Medeiros2*

1Universidade Federal do Rio Grande do Norte, Programa de Pós-Graduação em Ciências da Saúde, Centro de Ciências da Saúde, Av. Nilo Peçanha, s/n – 59012-300 Natal-RN, Brasil; 2Universidade Federal do Rio Grande do Norte, Departamento de Cirurgia, Av. Nilo Peçanha, s/n – 59012-300, Natal-RN, Brasil; [email protected].

ABSTRACT

Some drugs and surgery can interfere with the biodistribution of radiopharmaceuticals and data about the effect of splenectomy in the metabolism of phytate-Tc-99m are scarce. This study aimed to evaluate the interference of splenectomy on biodistribution of phytate-Tc-99m and liver function in rats. The SP group rats (n=6) underwent splenectomy. In group C(control) the animals were not operated. After 15 days, all rats were injected with 0.1mL of Tc-99m-phytate via orbital plexus (0.48MBq). After 30 minutes liver samples were harvested, weighed and percentage of radioactivity per gram (%ATI/g) was determined by gama counter Wizard Perkin-Elmer. The ATI%/g in splenectomized rats (0.99±0.02) was significantly higher than in controls (0.4±0.02), (p=0.034). ALT, AST and HDL were significantly lower in SP rats (p= 0.001) and leukocytosis was observed in SP rats. In conclusion, splenectomy in rats changed the hepatic biodistribution of Tc-99m-phytate and liver enzimatic activity. Key words: Splenectomy, Sodium phytate, Biodistribution, Liver

INTRODUCTION

The spleen is well known to be the largest lymphoid organ in the body. The spleen’s functions are many, but they are generally in 1 of 4 categories: filtration, immunologic, reservoir, and hematopoietic functions. Many of the spleen’s immunologic functions, therefore, are in common with other immunologic organs. For example, the spleen is efficient at removing non-opsonized bacteria, mostly encapsulated organisms (Müftüoğlu et al., 2000). Splenectomy is frequently performed for a multitude of reasons, including trauma and various pathologic processes. Blunt abdominal trauma remains the most common indication for splenectomy, but patients with a variety of hematologic disorders also benefit from this procedure. Loss of the spleen, however, leads to a lifelong higher risk for sepsis and severe infection (Bader-Meunier et al., 2001, Waghorn et al., 1997) and may be associated with an increased rate of thromboembolic complications, enhanced arteriosclerosis, and late coronary heart disease (Schilling, 1997). It has been related that splenectomy might somewhat promote hepatic regeneration, prevent liver fibrosis (Akahoshi et al., 2002, Murata et al., 2001), reduce serum bilirubin concentration and improve liver function (Shimada et al., 2000, Lin et al., 1999). However, considering the relationship of spleen with the physiology of liver, the nuclear medicine may have an important role in studying the diagnose and physiology of liver after splenectomy.

Since its introduction some time ago, Tc-99m-phytate colloid has been used as an imaging agent for the liver and spleen (Huet et al., 1980). Subjective assessment of parameters such as the dimensions of

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the liver, colloid shift, and uptake of radiopharmaceutical in the bone marrow have been used for both diagnosis of liver diseases and evaluation of its progression. Subjective assessment of radiocolloid distribution has been shown to be unreliable, and quantitative techniques have been used to evaluate liver function (Jago et al., 1987). The distribution of radiocolloid uptake in the liver and bone marrow has been shown to correlate well with the severity of chronic liver disease, the severity of histologic fibrosis, prognosis, and hepatic function (Hoefs et al., 1997). Thus, quantitation of liver uptake of 99mTc-phytate colloid provides a practical index of hepatic function, by using planar and single photon emission computed tomography (SPECT) techniques (Strauss et al., 1984). The ability to quantitate individual organ volumes and radiopharmaceutical concentrations with SPECT (Front et al., 1987, Iosilevsky et al, 1989) stimulates the use of Tc-99m-phytate colloid scintigraphy of the liver as a quantitative test of hepatic function. Biodistribution of radiopharceuticals may give important information about the uptake of them to some target organs but, after surgery are scarce (Chacon et al., 2007, Araújo-Filho et al., 2007).

This study aimed do evaluate if the splenectomy interferes with liver function and on the biodistribution of Tc-99m-fytate in the liver of Wistar rats.

MATERIALS AND METODS

Animals: Wistar male rats weighing 274±21g were obtained from Vivarium of Center for Health Sciences, UFRN, Brazil. The animals were housed in popypropilene cages and received standard rat chow and water ad libitum. Prior to surgery, rats were fasted overnight in separate cages. At the start of the study, after 7 days of acclimatization, the rats were allocated into two groups (each group, 6 animals). The groups were named splenectomy(SP) and control (C). The protocol for this study was approved by the Institutional Animal Care Committee, and the research was performed in accordance with the guidelines from Brazilian College of Animal Experimentation. Surgical procedure for splenectomy: Rats were anesthetized with ketamine 50 mg/kg IM and thiopental 20 mg/kg IP, shaved and placed on an operative board and fixed with tapes. Midline laparotomy (3cm) was performed after skin sterilization with ethanol 70% and covering with a sterile small drape with a circle 6 cm aperture. In group SP (n=6) the spleen was identified and ressected after the ligature of the spleen vessels with vicryl 5-0 (Ethicon, São Paulo, Brazil). The peritoneal cavity was irrigated with warm (37°C) normal saline. The laparotomy wound was closed with 4-0 nylon in layers and individual rats were placed in separate cages. They were allowed water ad libitum and rat chow 24 h postoperatively. The C rats (n=6) were not anesthetized neither operated. The hydration was done with normal saline (10 mL/100g weight) injected subcutaneously in the back of the rats for the first two postoperative days. Postoperative pain was treated with tenoxicam (Roche Pharm., Brazil); 10 mg/kg was given i.m. to the rats once a day for three days. Body weight and clinical observation: Body weight was monitored weekly throughout the entire experimental period lasting 15 days. Activity, mucosa and skin color were observed daily. Radioactivity count: Sodium pertechnetate (Na99mTcO4) was obtained by elution of a 99Mo/99mTc generator (Instituto de Pesquisas Energéticas e Nucleares, Comissão Nacional de Energia Nuclear, Brazil), and 99mTc-phytate was prepared. On the 15th day all the animals were anaesthetized again, and injected with 0.lmL of 99mTc-phytate in the orbital plexus, corresponding to radioactivity of 0.66MBq. After 30 min, blood was collected by cardiac puncture for dosages and the animals were killed by lethal dose of anesthetic. Samples of the liver were harvested. The samples were washed in 0.9% saline, weighed on a high-precision digital scale (Bel-Mark 160-II Itália) and subjected to radioactivity detection using a 1470 WizardTM Gamma Counter - Perkin-Elmer, Finland, with automatic correction of radiation decline. The percentage of radioactivity/g (%ATI/g) of each organ was calculated by dividing the activity/g of the hepatic tissue by the total activity administered to each animal. Laboratory analysis: A blood sample was used for hematological analysis of white blood cells (Automatic Analyzer Abbot Cell Dyn 3500) and for the serum measure of alanine aminotransferase

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(ALT), aspartate aminotransferase (AST) and lactic desidrogenase activity, using the Spectrophotometer Konelab 60i, (assay kit from Weiner, São Paulo, Brazil). Statistics: The data were expressed as mean±standard deviation (SD). The comparison between groups were done using ANOVA and the post-hoc Student test, considering significant p<0.05. RESULTS

Postsplenectomy symptoms such as hematuria, pale mucosa and lethargy were observed in 3 animals and these were not seen in control rats. We did not observe a significant body weight loss in splenectomized rats, comparing with controls. Radioactivity per gram of liver tissue ((%ATI/g) was increased by prior splenectomy in the SP group rats (0.99±0.2), when compared to the control (C) group (0.4±0.2). Comparing the groups, the difference was significant (p=0.034; Table 1). To examine the effects of prior splenectomy on liver function of rats, we assessed the levels of ALT, AST and LDH activity, as marker of liver injury, at 15 days after splenectomy (Table 1). In splenectomized rats rats, ALT, AST and LDH values were markedly attenuated, compared with the control group rats (p=0.001; Table 1). In addition, splenectomy resulted in increasing on white blood cells count in the SP rats (13.8± 2.7 k/µL), when compared to the C rats (2.5±0.9 k/µL; Table 1).

Table 1 – Results of percentage of radioactivity per gram (%ATI/g)of liver, hepatic enzymes activity and .

Exams Splenectomy Control p(1)

Liver (%ATI/g) 0.99 ± 0.2 0.4 ± 0.2 0.034

ALT (U/L) 114.6 ± 10.9 157.5 ± 13.2 0.001

AST (U/L) 51.6 ± 7.8 62.0 ± 2.5 0.041

LDH (mg/dL) 8.2 ± 2.4 16.1 ± 1.1 0.001

WBC (k/µL) 13.8±2.7 2.5± 0.9 <0.001

Mean±standard deviation; ALT,alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactic dehidrogenase; p-value from t test for independent samples; WBC, white blood cells.

DISCUSSION

Based on current knowledge, there is no doubt that the spleen is a lymphoid organ. Filtration, immunology, reservoir, and hematopoiesis are such important functions, that partial splenectomy or the preservation of the organ by suture are preconized in cases of traumatic injuries (Resende and Petroianu., 2003). Complications of various types and beneficial effects of the operation are described in the post-operative splenectomy (Cadili and Gara., 2008). Splenectomy might somewhat promote hepatic regeneration and prevent liver fibrosis (Murata et al.,2001, Chen et al., 1998). In this study it was demonstrated that in splenectomized rats the biodistribution of 99mTc-phytate to the liver was higher than in controls, meaning that the operation favored the hepatic uptake of the radiopharmaceutic. This result coincided with the improvement in liver function, validated by the better alanine aminotransferase, aspartate aminotransferase and lactic dehidrogenase activities in splenectomized rats, compared with controls. In some studies splenectomy promoted hepatic regeneration (Akahoshi et al., 2002, Murata et al., 2001), prevented liver fibrosis (Chen et al., 1998), reduced serum bilirubin concentration and, consequently, reflected the effect of splenectomy on reducing the burden of hepatocyte bilirubin metabolism, improving liver function (Shimada et al, 2000, Lin et al., 1999). Tomikawa et al (1996) reported that splenectomy increased the hepatocyte growth factor (HGF)

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activities in the plasma, suggesting that the spleen played an inhibitory role in hepatic regeneration. HGF, first identified as the most potent mitogen for primary hepatocytes, not only stimulates hepatic regeneration but also accelerates liver function. Moreover, the fact that a large amount of splenic tissue connects to liver tissue through the portal vein system, suggests the existence of a humoral factor originating from the spleen, which thus inhibits hepatic regeneration and promotes liver fibrosis. In rats submitted do hepatic ischemia/reperfusion, prior splenectomy ameliorated acute multiple organ damage (Jiang et al., 2007). These findings, associated to the favorable serum activity of ALT, AST and LDH in our splenectomized rats, in part explain the high hepatic biodistribution of 99mTc-phytate in the operated animals. The postsplenectomy diagnosis of sepsis based on white blood cell (WBC) elevation is confounded by the fact that leukocytosis is considered a physiologic response to splenectomy (Horowitz et al., 1992). Some reports suggest that WBC count postsplenectomy in patients with sepsis is greater and more persistent than the WBC in patients without sepsis (Weng et al.,2005, Rutherford et al., 1994). In this study we were not able to diagnose sepsis in the splenectomized rats. Nevertheless, all the operated animals had leukocitosis (WBC ranging from 11 to 18 k/µL) in the 15th postoperative day. It has been related that by the fifth day following post-traumatic splenectomy, the WBC count may help the physician to confirm the development of sepsis and the need for treatment. If the WBC is greater than 15x103/µL on that day, the physician should seriously consider treatment by empiric antibiotics and further diagnostic work-up to prevent the untoward sequelae of postsplenectomy sepsis (Toutouzas et al., 2002). In conclusion, in rats the splenectomy ameliorated the liver uptake of Tc-99m-phyate, coinciding with changing in hepatic enzymatic activity.

RESUMO O radiofármaco fitato-Tc-99m. é usado no diagnóstico através de exames de imagem, na dependência de sua biodistribuição. O objetivo do trabalho foi avaliar efeito da esplenectomia na biodistribuição do fitato-Tc-99m e função hepática em ratos Wistar. Sob anestesia e técnica asséptica, os animais do grupo SP(n=6) foram esplenectomizados. Grupo C(controle; n=6) não operado. Após 15 dias, injeção de 0,1ml de fitato-Tc-99m via plexo orbital (0,48MBq). Após 30 minutos, retiradas biópsias hepáticas para determinação do percentual de radioatividade/grama (% ATI/g), usando-se contador gama WizardPerkin-Elmer. Realizada dosagem de ALT, AST e HDL, e leucometria. Estatística pelo teste t, significância 0,05. O %ATI/g nos ratos esplenectomizados foi 0,99 ± 0,2 e nos controles 0,40 ± 0,2 (p=0,034). ALT, AST e HDL tiveram dosagens significativamente menores nos esplenectomizados (p=0,01), com leucocitose, comparando com controles. Em conclusão, em ratos a esplenectomia provocou alteração na captação de fitato-Tc-99m pelo fígado, coincidindo com alteração da função hepática.

REFERENCES Akahoshi, T., Hashizume, M., Tanoue, K., et al. (2002). Role of the spleen in liver fibrosis in rats may be

mediated by transforming growth factor beta-1. J. Gastroenterol. Hepatol., 17, 59-65. Araújo-Filho, I., Rego, A.C.M., Brandao-Neto, J., et al. (2007). Biodistribution of the

radiopharmaceutical sodium pertechnetate after biliopancreatic bypass with a duodenal switch. Braz. Arch.Biol.Ttechnol., 50,189-197.

Bader-Meunier, B., Gauthier, F., Archambaud, F., et al. (2001). Long-term evaluation of the beneficial

effect of subtotal splenectomy for management of hereditary spherocytosis. Blood., 97,399–403. Cadili, A., Gara, C. (2008). Complications of splenectomy. Am. J. Med., 121, 371-375.Chacon, D.A., Araújo-Filho, I., Villarim-Neto, A., et al. (2007). Biodistribution of the

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radiophamarceutical sodium pertechnetate (Na99mTcO4) after massive small bowel resection in rats. Acta. Cir. Bras., 22, 430-435. Chen, D., Liu, W., Leng, E., Wu, B. (1998). Effect of splenectomy on CCl4-induced liver fibrosis in rats.

Chin. Med. J., 111, 779-783.

Front, D., Ioselevsky G, Frenkel A, et al. (1987). In vivo quantitation using SPECT of radiopharmaceutical uptake by human meningiomas. Radiology., 164,93–96.

Hoefs, J.C., Wang, F., Kanel, G. (1997). Functional measurement of nonfibrotic hepatic mass in cirrhotic patients. Am. J. Gastroenterol., 92,2054–2058.

Horowitz J, Leonard D, Smith J, Brotman S. Postsplenectomy leukocytosis: physiologic or an indicator of infection? Am Surg. 1992;58:387-390.

Huet, P-M., Chartrand, R., Marleau, D. (1980). Extrahepatic uptake of Tc-99m-phytate: its mechanism and significance in chronic liver disease. Gastroenterology.,78,76–80.

Iosilevsky, G., Israel, O., Frenkel, A., et al. (1989). A practical SPECT technique for quantitation of drug delivery to human tumors and organ absorbed radiation dose. Semin. Nucl. Med.,19,33–46.

Jago, J.R., Gibson, C.J., Diffey, B.L. (1987). Evaluation of subjective assessment of liver function from radionuclide investigations. Br. J. Radiol., 60,127–132.

Jiang H, Meng F, Li W, Tong L, Qiao, H Sun X. Splenectomy ameliorates acute multiple organ damage induced by liver warm ischemia reperfusion in rats. Surgery 2007;141:32-40.

Lin, M.C., Wu, C.C., Ho, W.L., Yeh, D.C., Liu, T.J., Peng, F.K. (1999). Concomitant splenectomy for hypersplenic thrombocytopenia in hepatic resection for hepatocellular carcinoma. Hepatogastroenterology., 46, 630-634.

Müftüoğlu, T,M.; Köksal, N and Ozkutlu, D. (2000). Evaluation of phagocytic function of macrophages in rats after partial splenectomy. J. Am. Coll. Surg., 191,668-671.

Murata, K., Shiraki, K., Sugimoto, K., et al. (2001). Splenectomy enhances liver regeneration through

tumor necrosis factor (TNF)-alpha following dimethylnitrosamine-induced cirrhotic rat model. Hepatogastroenterology.,48,1022-1027.

Resende, V., Petroianu, A. (2003). Functions of the splenic remnant after subtotal splenectomy for

treatment of severe splenic injuries. Am. J. Surg., 185,311-315. Rutherford EJ, Morris JA Jr, van Aalst J, Hall KS, Reed GW, Koestner JA. The white blood cell response

to splenectomy and bacteraemia. Injury. 1994;25:289-292. Schilling, R,F. (1997). Spherocytosis, splenectomy, strokes, and heart attacks. Lancet., 350,1677–1678 . Shimada, M., Hashizume, M., Shirabe, K., Takenaka, K., Sugimachi, K. (2000). A new surgical strategy

for cirrhotic patients with hepatocellular carcinoma and hypersplenism. Performing a hepatectomy after a laparoscopic splenectomy. Surg. Endosc., 14,127-130.

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Strauss, L.G., Clorius, J.H., Frank. T., Van Kaick, G. (1984). Single photon emission computerized tomography (SPECT) for estimates of liver and spleen volume. J. Nucl. Med., 25,81–85.

Tomikawa M, Hashizume M, Highashi H, et al. (1996). The role of the spleen, platelets, and plasma hepatocyte growth factor activity on hepatic regeneration in rats. J. Am. Coll. Surg., 182,12–6.

Toutouzas KG, Velmahos GC, Kaminsk A, Chan L, Demetriades D. Leukocytosis After Posttraumatic

Splenectomy. A Physiologic Event or Sign of Sepsis? Arch Surg. 2002;137:924-929. Waghorn, D.J., Mayon-White, R.T. (1997). A study of 42 episodes of overwhelming postsplenectomy

infection: is current guidance for asplenic individuals being followed? J. Infect., 35,289 –294. Weng J, Brown CV, Rhee P, Salim A, Chan L, Demetriades D, Velmahos GC. White blood cell and

platelet counts can be used to differentiate between infection and the normal response after splenectomy for trauma: prospective validation. J Trauma. 2005;59(5):1076-80.

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4 COMENTÁRIOS, CRÍTICAS E SUGESTÕES

Os objetivos traçados durante o anteprojeto foram satisfatoriamente

atingidos durante a execução deste trabalho experimental. O modelo

desenhado correspondeu às expectativas, sendo capaz de produzir dados

comparáveis aos estabelecidos na literatura(16). A análise estatística dos

dados obtidos, realizada de forma simples e elegante, mostrou-se adequada

como instrumento auxiliar na interpretação dos achados deste estudo.

Com a realização de um estudo piloto, foi possível detectar e corrigir

uma variação na atividade radioativa injetada nos animais. Esta variação

estava ocorrendo devido ao pequeno volume de líquido a ser injetado, com

uma quantidade não desprezível de atividade radioativa remanescente na

seringa após a injeção. A detecção dessa variação só foi possível devido à

realização do estudo piloto, reforçando a sua importância em projetos

experimentais dentro desta linha de pesquisa.

A solução encontrada foi o estabelecimento de um padrão, formado pela

atividade radioativa média em três tubos de ensaio, cada qual com o conteúdo

de uma seringa contendo EDTMP-153Sm, na mesma atividade radioativa

empregada durante o estudo, esvaziada diretamente dentro do tubo, simulando

a situação real encontrada, ao se injetar o radiofármaco no animal.

Como crítica à metodologia empregada, verificamos, após a análise dos

dados, que o trabalho poderia ter sido enriquecido pela aferição da atividade

radioativa no sangue, uma vez que a análise da proporção sangue/órgão alvo

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traria informações adicionais importantes, considerando-se a natureza do

experimento, que buscou verificar o efeito de um medicamento (docetaxel)

sobre a biodisponibilidade de outro (EDTMP-153Sm).

Essa limitação será corrigida em um projeto complementar, a ser

executado dentro da mesma linha de pesquisa, que buscará avaliar o impacto

desta associação terapêutica sobre a contagem de hemácias, leucócitos e

plaquetas, bem como a aferição da atividade radioativa de EDTMP-153Sm no

sangue.

Deve-se ressaltar o esforço empreendido neste trabalho e a publicação

gerada a partir dele, uma vez que o docetaxel é um quimioterápico de

aplicação recente na Oncologia Clínica, e, desta forma, também o são todos os

estudos que validaram a sua utilização. Adicionalmente, estudar este

medicamento em associação com EDTMP-153Sm restringe ainda mais as

fontes de consulta, tornando realmente inédita essa linha de pesquisa.

Até o momento, um único estudo relata a utilização de EDTMP-153Sm

em associação com docetaxel em humanos, demonstrando eficácia clínica

superior no controle da dor no tratamento concomitante, quando comparado ao

emprego de um dos tratamentos isoladamente(72). Esse trabalho foi publicado

no mesmo período que o nosso, atestando a originalidade da nossa linha de

pesquisa.

Isto demonstra a real importância da melhor compreensão das possíveis

interações entre estes tratamentos, tendo em vista que eles tendem a se tornar

cada dia mais freqüentes, em virtude do seu grande potencial de utilização.

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Apesar das características específicas do grupo de pacientes que dele se

beneficiam, fato que termina por restringir a sua indicação, quando se

considera o grande número de pessoas com diagnóstico de câncer de próstata,

o potencial impacto dos achados do nosso estudo não é desprezível.

Este pensamento, aliado ao indiscutível crescimento intelectual daqueles

que se acharam envolvidos em sua execução, serve de recompensa ao esforço

desprendido e atua como norte, ampliando nossos horizontes, para que seja

consolidada uma linha de pesquisa em quimioterápicos e radiofármacos

vinculada ao Núcleo de Cirurgia Experimental do CCS-UFRN e ao Programa

de Pós-graduação em Ciências da Saúde.

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ABSTRACT

Pain related to bone metastasis is a frequent symptom in advanced

prostate and breast carcinoma. Treatments with systemic radioisotopes like

153Samarium-EDTMP in association to chemotherapy have been shown to have

palliative benefits in this population. The aim of this study was to assess the

influence of docetaxel chemotherapy on the biological availability of

153Samarium-EDTMP in bones and other organ of rats. Wistar male rats were

randomly allocated into 2 groups of 6 rats each. DS group

(docetaxel/153Samarium-EDTMP) received docetaxel (15mg/Kg)

intraperitoneally in cycles 11 days apart. S group (153Samarium-EDTMP/control)

was not treated with docetaxel. Nine days after chemotherapy, all rats were

injected with 0,1ml (25 µCi) of 153Samarium-EDTMP via orbital plexus. After 2

hours, the animals were killed and samples of brain, thyroid, lung, heart,

stomach, colon, liver kidney and both femurs were removed. Calculated values

of the %dose/g of tissues (%ID/g) were determined in an automatic gamma-

counter (Wizard-470, Perkin-Elmer, Finland). Nine days after 2nd chemotherapy

cycle, the rats showed a significant weight loss (314.50±22.09g) compared

(p<0.5) to pre-treatment weight (353.66± 22.8). %ID/g in DS group showed a

significant reduction in right femur, left femur, kidney, liver and lungs compared

to S group. The use of 153Samarium-EDTMP plus docetaxel was associated to

lower radioisotope concentration in target tissues. Further investigations about

the impact of docetaxel on 153Samarium-EDTMP biological availability would

complement these findings. This study had a multidisciplinary feature, include

participation among various fields of knowledge, especially Nuclear Medicine,

Surgery, Oncology, Biology and Statistics.

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biodisponibilidade de edtmp-153 samÁrio tratados com...

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