Co-Development of DELFIA Technology. - Assessment of DELFIA technology in drug biodistribution in small animals

Transcription

1 Co-Development of DELFIA Technology PerkinElmer - Wallac Inc & - Assessment of DELFIA technology in drug biodistribution in small animals Project Report Aki Koivistoinen, 1

2 BACKGROUND Dissociation of Europium from DTPA & DOTA chelates - Comparative study Early findings 2

3 BACKGROUND DISSOCIATION OF EUROPIUM FROM CHELATES DTPA[Eu] ITC chelate vs. peptide-dota[eu] (comparison done to Eu-control) Comparison of the signal of Eu-DTPA ITC chelate to three different DOTAcoupled peptides: 1nM solution of Eu-control counts - 1nM content of measured compounds - Compounds diluted to Delfia Enhancement solution - Total volume 200ul (in Delfia Enhancement solution) Two identical sets (six parallel wells) 1) direct measurement 2) 40 min pre-incubation at 37C 3

4 BACKGROUND DISSOCIATION OF EUROPIUM FROM CHELATES Results Eu-control Eu-DTPA ITC RGD-DOTA[Eu] TCTP-DOTA[Eu] AETP-DOTA[Eu] Counts % of Eu-control 65 3,25 0,01 0,12 Counts % of Eu-control 64 3,22 0,04 0,17 Pep-DOTA / DTPA (%) 5,00 0,02 0,18 Pep-DOTA / DTPA (%) 5,03 0,06 0,26 RT 37ºC DOTA-coupled peptides gave significantly lower signals compared to Eu-DTPA ITC chelate from 0,02 % to 5% DOTA chelate is not useful with DELFIA technology without a separate dissociation step (2 M HCl) 4

5 Counts (615nm) Counts (615nm) BACKGROUND TAKE HOME: In vivo injected Europium-labelled peptides can be detected from the lysates of mouse organs Biodistribution profiles: RGDfK-DOTA[Eu] vs. Eu(III)Cl hexahydrate Biodistribution of RGD-DOTA-[Eu] in HT1080 tumour bearing mice (2/3 mice) - DELFIA assay Biodistribution of Eu(III) Cl hexahydrate in HT1080 tumour bearing mice (2/5 mice) -DELFIA assay A A /3 mice Tissue type 2/5 mice Brain Liver Skin Kidney Ovarios Heart Lungs Spleen Intestine Muscle Tumour 1 Tumour 2 Brain Liver Skin (NA) Kidney Ovarios Heart Lungs Spleen Tissue type Intestine Muscle (NA) Tumour 1 Tumour 2 A247 A284 RGDfK-DOTA-[Eu] shows identical biodistribution profiles in mice A296 & A298 verifying the potential of DELFIA technology in biodistribution analysis. Eu(III)Cl hexahydrate shows identical biodistribution profiles in mice A247 & A284 verifying the potential of DELFIA technology in biodistribution analysis. 5

6 The determination of biodistribution profile of DTPA[Europium] labelled compounds using DELFIA technology Aims: 1) Compound specific biodistribution patterns. 2) Reliability comparison of results to ICP-MS results. Aki Koivistoinen, 6

7 In vivo biodistribution with DELFIA technology Repetition of the biodistribution study using DELFIA technology with higher number of mice, and comparison the result with those of Inductively coupled plasma mass spectrometry (ICP-MS) Wallac - contribution 1) Eu-DTPA-iodoacetamido chelate and consultation for chemistry. 2) VICTOR fluorometer and reagents Karyon - contribution 1) Test compound (Thx1-targeting peptide) and a control compound DTPA[Eu]. 2) Cell lines and animal models (20 mice) 3) Development of process for biodistribution assesment 7

8 MATERIALS AND METHODS CHEMISTRY Tested Compounds 1) Thx-DTPA[Eu] Coupling of Thx-peptide* having sulphydryl group at its carboxy terminus to Eu-DTPA-iodoacetamido *Karyon TM Targeting Unit (KTU) specific for Lung cancer 2) DTPA[Eu] In vivo biodistribution with DELFIA technology Europium (III) Cl-hexahydrate was chelated to DTPA 8

9 In vivo biodistribution with DELFIA technology MATERIALS AND METHODS Biology - Thx testing in Non-small cell lung cancer mouse models. Cell lines: A549: human adenocarcinoma cell line NCI-H520: Human epidermoid carcinoma cell line Tumour model: Mouse bearing both A549 and NCI-H520 tumours on their flanks (n=20). 9

10 In vivo biodistribution with DELFIA technology MATERIALS AND METHODS Biology - Thx testing in Non-small cell lung cancer mouse models. COMPOUND AMOUNT INJECTED MICE (n) Thx DTPA[Eu] 0,275 umol 7 DTPA[Eu] 0,275 umol 5 DTPA[Eu] 0,550 umol 5 DTPA[Eu] 2,20 umol 5 Tested compounds were injected i.v. into tail vein of mice circulation time being 15 min before perfusion. 10

11 In vivo biodistribution with DELFIA technology MATERIALS AND METHODS Preparing of samples with minimized lysate proportion Lysate Enhancement WELL SAMPLES (0,275umol, 0,55umol, 2,2umol) 5 µl of 1:5 to 1:50 diluted tissue lysate (dilution to lysis buffer) 195 µl of DELFIA Inducer solution TISSUE SPECIFIC STANDARDS FOR COMPOUDS 5 µl of Tissue Lysate (Eu-free lysate from blank mouse) 195 µl of DELFIA Inducer solution containing appropriate amount of Eu-labelled peptide BLANK 5 µl of tissue lysate µl of DELFIA Inducer 11

12 counts (614nm) A615nm In vivo biodistribution with DELFIA technology Process Development Tissue specific standards Tissue specific standards were analysed to determine differences of fluorescence signal from lysates of different tissue types Tissue specific standards of RGDfK-DOTA[Eu] for in vivo biodistribution brains Tissue-specific standards study of Thx-DTPA[Eu] liver 0,00E+00 5,00E-12 1,00E-11 1,50E-11 2,00E-11 2,50E-11 3,00E-11 mol of Thx-DTPA[Eu] RGDfK-DOTA[Eu] (ng/well) skin brains kidney liver ovarios/womb skin kidney heart ovarios + womb lungs heart spleen lungs muscle spleen small intestine small intestine muscle HTB-182 tumour A549 blood Plasma Minimised lysate proportion in DELFIA assay no need for tissue-specific standards 12

13 The determination of biodistribution profile of Thx-DTPA[Eu] using DELFIA technology Results 13

14 Amount of Europium (ng/g) Amount of Europium (ng/g of tissue) Biodistribution of Thx-DTPA[Eu] with DELFIA ICP-MS confirms the reliability of DELFIA technology Biodistribution of Thx-DTPA[Eu] in NSCLC xenografts with DELFIA (0,275umol of Thx-DTPA[Eu] injected) Brain Liver Kidney Ovarios Heart Lungs Spleen Muscle Intestine HTB-182 A549 A372 A373 A375 A378 A379 Biodistribution of Thx-DTPA[Eu] in NSCLC xenografts with ICP-MS (0,275 umol of Thx-DTPA[Eu] injected) Brain Liver Kidney Ovarios Heart Lungs Spleen Muscle Intestine HTB-182 A549 A372 A373 A375 A378 A379 14

15 Amount of Europium (ng/g of tissue) Amount of Europium (ng/g of tissue) Biodistribution of DTPA[Eu] with DELFIA ICP-MS confirms the reliability of DELFIA technology Biodistribution of DTPA[Eu] in NSCLC xenografts with DELFIA (2,2umol of DTPA[Eu] injected) Brain Liver Kidney Ovarios Heart Lungs Spleen Muscle Intestine HTB-182 A549 A367 A368 A369 A370 A371 Biodistribution of DTPA[Eu] in NSCLC xenografts with ICP-MS (2,2umol of DTPA[Eu] injected) Brain Liver Kidney Ovarios Heart Lungs Spleen Muscle Intestine HTB-182 A549 A367 A368 A369 A370 A371 15

16 Conclusions DELFIA appears to be reliable method for biodistribution tests Detection sensitivity as good as for radionuclides Safe to use and stable (excellent alternative for radionuclides) DELFIA is a good alternative for biodistribution studies 16