Name: preparation and evaluation of 99mtc-labeled tridentate chelates for pre-targeting using bioorthogonal chemistry
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Description: Watch this Scientific Journal Video about Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry at JoVE.com

This work supported by research grant funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Ontario Institute for Cancer Research (OICR, #P.SI.015.8), and the Canadian Cancer Society (CCS, #703857). The authors acknowledge the contributions of Dr. Denis Snider who provided assistance in preparing the manuscript.

Djurstudier har godkänts av Animal forskningsetik styrelsen vid McMaster University i enlighet med kanadensiska rådet om Animal Care (CCAC) riktlinjer. 1. Radiomärkning av Tz-tretandade ligander med 99m Tc VARNING: Följande procedurer kräver användning av radioaktiva föreningar. Arbetet bör endast ske i en licensierad laboratorium med anslutning till säkerhet och bortskaffande regler. Mikrovågsugn reaktioner bör utföras i en mikrovågs...

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En samling av tetrazin bunden tretandade kelat av varierande polariteter framställdes och nyttan av sina 99m Tc-komplex i IEDDA reaktion med en TCO-derivat in vivo bedömdes. En effektiv och reproducerbar 99m Tc märkningsmetod har utvecklats för fem tetrazin-kelat, där ligandkoncentrationen var 10 -3 M. steg märkning följdes av avlägsnande av T- butylgrupper (för föreningarna 2-5). Den höga koncentrationen av ligand användes för att förbät...

99m Tc fortfarande den dominerande radioisotoper som används inom diagnostisk nukleärmedicin, med över 50 miljoner avbildningsförfaranden som sker per år i hela världen en, två, tre. Majoriteten av 99m Tc medel som används kliniskt är perfusion typ radioaktiva läkemedel. Det finns ett begränsat antal aktivt målinriktade föreningar i vilka 99mTc är riktad för att binda en specifik biomarkör genom ligering till en målkonstruktion. Skapandet av riktade 99mTc radiofarmaka ofta hindras av inverkan av 99m Tc-ligandkomplex på förmågan hos målmolekylen att binda biomarkör av intresse, eller isotop halveringstid är inte tillräckligt lång för att användas med högre molekylvikt biomolekyler såsom antikroppar. Det senare kräver vanligen flera dagar innan bilder förvärvas för att biomolekylen att rensa från icke-mål tiss<html> deringar. Pre-målinriktning erbjuder en alternativ metod för att övervinna dessa utmaningar. Pre-inriktning i kombination med bioorthogonal kemi har visat sig vara ett effektivt sätt att utveckla nya molekylära avbildningssonder för både fluorescens och radio avbildning 4, 5, 6, 7, 8. Den inversa efterfrågan elektron Diels-Alder (IEDDA) reaktion mellan 1,2,4,5-tetrazin (Tz) och trans -cyclooctene (TCO) derivat, såsom visas i figur 1, har visat sig vara särskilt effektivt 6. Den IEDDA reaktion med dessa komponenter kan uppvisa snabb kinetik i PBS (k 2 ≈ 6000 M -1 s -1) och hög selektivitet, vilket gör den idealisk för in vivo pre-inriktade program 9, 10. e_content &quot;&gt; Den vanligaste metod som används innebär att administrera en TCO-härledda målsökningsvektom och efter en tillräcklig fördröjningsperiod, är en radiomärkt tetrazin administreras. Radiomärkta tetrazines baserat på 11 C, 18 F, 64 Cu, 89 Zr, och 111 i har varit rapporterade 11, 12, 13, 14, 15. däremot finns det endast en rapport av en 99m Tc-märkta Tz, som framställdes med hjälp av en HYNIC ligand kräver användning av co-ligander för att förhindra proteinbindning och nedbrytning in vivo 16. som ett alternativ, rapporterar vi här syntesen av 99m Tc (i) märkt tetrazines med användning av en familj av ligander som bildar stabila tretandade komplex med en [99m Tc (CO) 3] ^ kärna. Figur 1: bioorthogonal IEDDA reaktion mellan tetrazin och trans -cyclooctene. <a href="http://ecsource.jove.com/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">Klicka här för att se en större version av denna siffra.</a> Familjen av ligander framställda innehålla tretandade kelater som varierar i polaritet och arten av länkgruppen mellan metallbindande regionen och den Tz (Figur 2). Målet var att identifiera en 99m Tc-tetrazin konstruera som effektivt kan lokalisera och reagera med TCO-märkta ställen in vivo och snabbt klart inte är förenade i syfte att ge hög mål till icke-mål-förhållanden. För att testa ligander, var en TCO-derivat av en bisfosfonat (TCO-BP) används 17. Vi har visat tidigare att TCO-BP lokaliserar till områden med aktiv benmetabolism och kan reagera medradiomärkta tetrazines in vivo 18. Det är en bekväm reagens för att testa nya tetrazines, eftersom det kan framställas i ett enda steg och experiment kan utföras i normala möss, där lokaliseringen inträffar primärt i lederna (knän och axlar).

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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Argon gas&#160;</td>
			<td style="width:255px;">Alphagaz</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2CO3</td>
			<td style="width:255px;">EMD Millipore</td>
			<td style="width:199px;">106395</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2B4O7.10H2O</td>
			<td style="width:255px;">Anachemia</td>
			<td style="width:199px;">S9640</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KNaC4H4O6.4H2O</td>
			<td style="width:255px;">Anachemia</td>
			<td style="width:199px;">217255</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Technelite 99mTc generator</td>
			<td style="width:255px;">Lantheus medical imaging</td>
			<td style="width:199px;">---</td>
			<td>Source of 99mTcO4-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">0.9% Saline</td>
			<td style="width:255px;">Lantheus medical imaging</td>
			<td style="width:199px;">---</td>
			<td>To elute generator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M HCl</td>
			<td style="width:255px;">Lab Chem</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MeOH</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACN</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td>HPLC grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Millipore H2O</td>
			<td style="width:255px;">Thermo Fisher Scientific&#160;&#160;</td>
			<td style="width:199px;">Barnstead Nanopure</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">DCM</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TFA</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">PBS</td>
			<td style="width:255px;">Thermo Fisher Scientific</td>
			<td style="width:199px;">10010023</td>
			<td>pH 7.4 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">BSA</td>
			<td style="width:255px;">Sigma Aldrich</td>
			<td style="width:199px;">A7906</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tween80</td>
			<td style="width:255px;">Sigma Aldrich</td>
			<td style="width:199px;">P8047</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isoflurane&#160;</td>
			<td style="width:255px;">CDMV</td>
			<td style="width:199px;">108737</td>
			<td>Supplier: Fresenius Kabi Animal Health&#160;</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:367px;">HPLC&#160;</td>
			<td style="width:255px;">Waters&#160;</td>
			<td style="width:199px;">1525 Binary Pump, 2998 Photodiodde Array Detector, E-SAT/IN, Bioscan Flowcount PMT detector (item # 15590)</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">HPLC column for analysis and purification of compounds 2-4</td>
			<td style="width:255px;">Phenomenex</td>
			<td style="width:199px;">00G-4435-E0</td>
			<td>Gemini&#174; 5 &#181;m C18 110 &#197;, LC Column 250 x 4.6 mm,</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">HPLC column for analysis and purification of compounds 1 and 5</td>
			<td style="width:255px;">Waters&#160;</td>
			<td style="width:199px;">186003115</td>
			<td>XBridge BEH C18 Column, 130 &#197;, 5 &#181;m, 4.6 mm X 100 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microwave Reactor&#160;</td>
			<td style="width:255px;">Biotage&#160;</td>
			<td style="width:199px;">Initiator 8</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Biotage V10 Evaporator</td>
			<td style="width:255px;">Biotage&#160;</td>
			<td style="width:199px;">Serial # V1041</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Dose calibrator</td>
			<td style="width:255px;">Capintec, Inc.&#160;</td>
			<td style="width:199px;">CRC-25R</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">Gamma counter</td>
			<td style="width:255px;">Perkin Elmer</td>
			<td style="width:199px;">Wizard 1470 Automatic Gamma Counter</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Animal room scale&#160;</td>
			<td style="width:255px;">Mettler Toledo</td>
			<td style="width:199px;">XP105 Delta Range</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microwave vials&#160;</td>
			<td style="width:255px;">Biotage&#160;</td>
			<td style="width:199px;">355629</td>
			<td>0.5-2 mL&#160;</td>
		</tr>
	</tbody>
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preparation and evaluation of 99mtc-labeled tridentate chelates for pre-targeting using bioorthogonal chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Ligandema syntetiserades med olika linkers och kelatorer via en enkel reducerande aminering strategi (Figur 2), följt av koppling av produkten till en kommersiellt tillgänglig tetrazin 22, 23. Radiomärkning utfördes med användning av samma metod för alla föreningar och var mycket reproducerbar. Processen optimerades genom att variera pH, mängden ligand, reaktionstid och temperatur, varefter den 99m</...

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Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Beredning och utvärdering av<sup&gt; 99m</sup&gt; Tc-märkt tridentat Chelates för Pre-inriktning Använda Bioorthogonal kemi

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

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Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

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