COMPARATIVE APPROACHES BETWEEN ......MSI of m/z 1320.1894 ( H-Chloroquine) Figure 2: MSI vs WBA in...
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COMPARATIVE APPROACHES BETWEEN QUANTITATIVE-MSI AND QUANTITATIVE-WBA: APPLICATION TO CHLOROQUINE ADMINISTRATION IN A LONG-EVANS MALE RAT MODEL Introduction G. Hochart 1 , J. Marini 2 , A. Tomezyk 1 , P. Manteufel 2 , B. Jung 2 , D. Bonnel 1 , S. Linehan 3 , D. McKenzie 2 and J. Stauber 1 1 ImaBiotech, Lille, FRANCE; 2 Covance Laboratories Inc., Wisconsin, USA; 3 ImaBiotech, Massachusetts, USA. Mass Spectrometry Imaging (MSI) and Quantitative Whole Body Autoradiography (QWBA) are related techniques to assess the distribution of drug related material. While QWBA is the gold standard for quantification in biological tissues the technique does not allow individual components to be identified. MSI has long been used to provide molecular distribution information in a specific mass range and has recently shown capabilities in quantifying material in biological tissues (QMSI). This study demonstrates the QMSI capabilities as a complementary approach with QWBA to follow chloroquine and one of its metabolites (desethyl-chloroquine) at different time points following a single oral administration of 3 H-Chloroquine to Long-Evans rats. Experimental Long-Evans rats were administered a single dose of 30 mg/kg of 3 H-Choloroquine (300µCi/kg). They were euthanized under deep anesthesia (isoflurane) at 4, 24, 72, 168, and 336 hours post-dose and carcasses were immediately frozen in a hexane/dry ice bath for approximately 8 minutes, embedded in chilled carboxymethylcellulose and frozen into blocks for analysis. Sections were collected onto adhesive tape for QWBA and QMSI analysis in the sagittal plane at 20 µm thickness. QWBA images were obtained following exposure to phosphorimaging screens and scanned using a GE Typhoon scanner. For QMSI, MALDI matrix spiked with a known concentration of stable labeled chloroquine-d4 and its metabolite desethyl-chloroquine-d4 were sprayed onto the slide to normalize the data by evaluating the extinction of the compounds into the different organs. Analyses were performed by a 7T MALDI-FTICR in positive mode in the head region of the animal targeting the eye and in the mid whole-body region for other organs of interest. Distribution and quantification were performed with Quantinetix TM software. Results Conclusions High sensitivity and linearity of QBWA methodology was combined with high resolution mass spectrometry and specificity of the MALDI to discriminate the chloroquine from its metabolite and enable the quantifications of each compound. High correlation was found between the two methods in the distributions and the normalized quantifications. Limits of the QMSI were illustrated in the low and high limits of detection compared to the QWBA because of the restricted range of linearity. Quantification results A calibration range was prepared on a liver tissue section by spotting a mixture of the parent and its metabolite before analysis by MSI (Figure 3). This calibration curve allowed the limit of detection (LOD) and lower limit of quantification (LLOQ) of the two compounds to be determined. Tissue Extinction Coefficients were calculated with the deuterated forms sprayed with the matrix to evaluate the suppression effects of the different organs (other than liver) of the dosed tissue. The quantification was processed with Quantinetix TM software for both 1 H-choloroquine and 1 H-desethyl-chloroquine taking into account the direct absolute quantification of the liver and the different TEC values for other organs. Results of the absolute quantification are summarized in the Table 1. A correlation between QMSI and QWBA was observed by totaling the parent + metabolite concentrations with the exception of the uveal tract and Harderian gland for which a ratio 10:1 QWBA:QMSI was observed. This difference appears to be due to the limited region of interest scanned by MSI as compared to the broader region of interest observed in the QWBA image (Figure 1). The low levels observable in the uveal tract is most likely related to melanin binding of chloroquine and insufficient extraction by the matrix solvent. The high concentration of the drug in the stomach was limited in the QMSI evaluation due to the potential of saturation of the signal, crystallization differences, or suppression effects impacting the linearity of the method. Another representation to correlate the quantification data of WBA (µg equivalents/g of tissue) with MSI is presented in the Table 2 and Figure 4 where the liver was normalized to 1 (100%) and all the organs were compared to this level. Figure 4 highlights the closeness of the results between the two methods for relative quantification purposes. MSI of m/z 320.1894 ( 1 H-Chloroquine) MSI of m/z 292.1581 ( 1 H-desethyl chloroquine) Choroid Retina Ciliary Body Lens Harderian gland Cornea Skin Vitreous humor QWBA image of LE rat at 24h dosed with 3 H-Chloroquine Figure 1: Distribution of 1 H-Chloroquine and 1H-desethyl chloroquine at T24h *1-Desethyl Chloroquine not detected or under the LOD Table 2 Figure 4: Relative quantification in whole-body region by QMSI and QWBA (Reference organ: Liver) Organ Relative QWBA (µg Equivalents [3H]Chloroquine/g of tissue) Relative QMSI (µg [1H]Chloroquine/g of tissue) Relative QMSI (µg [1H]Desethyl Chloroquine/g of tissue) Liver 1.00 1.00 1.00 Kidney, Cortex 0.17 0.16 0.57 Kidney, Medulla 0.29 0.26 0.50 Kidney 0.23 0.20 0.53 Stomach* 1.69 2.91 BLOD Lung 0.40 0.28 0.60 Uveal tract* 0.25 0.02 BLOD Lens* 0.01 0.00 BLOD Harderian gland* 0.09 0.02 BLOD Skin* 0.03 0.01 BLOD Whole-body mid region In the whole-body mid region, both drug parent and metabolite were detected in various tissues from the prepared T4h sections (Figure 2). The two molecules were mainly co- localized within the different tissues analyzed and the quantitative distribution data obtained by QMSI matched QWBA parent-equivalent concentration data. The parent drug data from QMSI within the stomach contents was compared directly to the QWBA data since the bulk of the contents would be parent drug at T4h; whereas the liver can provide a good understanding of drug metabolite distribution. MSI of m/z 320.1894 ( 1 H-Chloroquine) Figure 2: MSI vs WBA in the whole-body region at T4h 9.1mm QWBA image of LE rat at 4h dosed with 3 H-Chloroquine MSI of m/z 292.1581 ( 1 H-Desethyl Chloroquine) Optical image T4h whole-body Combining the strengths of QWBA and MALDI QMSI allowed for the sensitive detection of drug related material (QWBA) and the quantitative differentiation of parent and metabolite (QMSI). Discriminate images were generated for chloroquine and desethyl-chloroquine so that each compound could be directly quantified to generate tissue concentrations. Distribution results Eye region 1 H-Chloroquine was clearly detected and identified by MALDI-FTICR at the first time-point T4h in the different regions of the eye, especially the uveal tract. 1 H-desethyl-Chloroquine was also detected and identified in ocular tissues. Figure 1 below demonstrates the tissue distribution at the time-point T24h to allow for a better visualization of the drug related metabolite. Table 1: QMSI vs QWBA QMSI (µg/g of tissue) Organ 1 H-Chloroquine 1 H-Desethyl Chloroquine TOTAL QMSI TOTAL QWBA (µg eq/g of tissue) Liver 111.4 3.1 114.5 103.0 Stomach 293.8* BLOQ 293.8 174.0 Kidney, Cortex 13.5 BLOQ 13.5 17.6 Kidney, Medulla 25.2 BLOQ 25.2 29.6 Kidney 17.7 BLOQ 17.7 23.3 Lung 26.3 0.7 27.0 41.7 Uveal tract 0.6 BLOD 0.6 26.0 Harderian gland 0.6 BLOD 0.6 9.6 Lens BLOD BLOD BLOD 0.9 Skin BLOQ BLOD BLOQ 3.0 Y = 3468716.833x + 726297.7096 R²= 0.9955 LOD (1H-Chloroquine) =0.056 µg/g of tissue LLOQ (1H-Chloroquine) = 0.406 µg/g of tissue Optical image T24h eye Choroid Retina Ciliary Body Lens Cornea Skin Vitreous humor Harderian gland Choroid Retina Ciliary Body Lens Harderian gland Cornea Skin Vitreous humor Figure 3: Calibration curve on a Liver section of 1 H-Chloroquine 0 0.5 1 1.5 2 2.5 3 3.5 Ratio QWBA eq 1H Chloroquine QMSI 1H Chloroquine QMSI 1H-Desethyl Chloroquine Spleen Stomach content 1 Kidney Lung Liver Heart Stomach content 2 Intestines Spleen Stomach content Kidney Lung Liver Heart Stomach content Intestines Spleen Stomach content 1 Kidney Lung Liver Heart Stomach content 2 Intestines
Transcript of COMPARATIVE APPROACHES BETWEEN ......MSI of m/z 1320.1894 ( H-Chloroquine) Figure 2: MSI vs WBA in...
COMPARATIVE APPROACHES BETWEEN QUANTITATIVE-MSI AND QUANTITATIVE-WBA: APPLICATION TO CHLOROQUINE ADMINISTRATION IN A LONG-EVANS MALE RAT MODEL
Introduction
G. Hochart1, J. Marini2, A. Tomezyk1, P. Manteufel2, B. Jung2, D. Bonnel1, S. Linehan3, D. McKenzie2 and J. Stauber1 1 ImaBiotech, Lille, FRANCE; 2Covance Laboratories Inc., Wisconsin, USA; 3 ImaBiotech, Massachusetts, USA.
Mass Spectrometry Imaging (MSI) and Quantitative Whole Body Autoradiography (QWBA) are related techniques to assess the distribution of drug related material. While QWBA is the gold standard for quantification in biological tissues the technique does not allow individual components to be identified. MSI has long been used to provide molecular distribution information in a specific mass range and has recently shown capabilities in quantifying material in biological tissues (QMSI). This study demonstrates the QMSI capabilities as a complementary approach with QWBA to follow chloroquine and one of its metabolites (desethyl-chloroquine) at different time points following a single oral administration of 3H-Chloroquine to Long-Evans rats.
Experimental Long-Evans rats were administered a single dose of 30 mg/kg of 3H-Choloroquine (300µCi/kg). They were euthanized under deep anesthesia (isoflurane) at 4, 24, 72, 168, and 336 hours post-dose and carcasses were immediately frozen in a hexane/dry ice bath for approximately 8 minutes, embedded in chilled carboxymethylcellulose and frozen into blocks for analysis. Sections were collected onto adhesive tape for QWBA and QMSI analysis in the sagittal plane at 20 µm thickness. QWBA images were obtained following exposure to phosphorimaging screens and scanned using a GE Typhoon scanner. For QMSI, MALDI matrix spiked with a known concentration of stable labeled chloroquine-d4 and its metabolite desethyl-chloroquine-d4 were sprayed onto the slide to normalize the data by evaluating the extinction of the compounds into the different organs. Analyses were performed by a 7T MALDI-FTICR in positive mode in the head region of the animal targeting the eye and in the mid whole-body region for other organs of interest. Distribution and quantification were performed with QuantinetixTM software.
Results
Conclusions High sensitivity and linearity of QBWA methodology was combined with high resolution mass spectrometry and specificity of the MALDI to discriminate the chloroquine from its metabolite
and enable the quantifications of each compound. High correlation was found between the two methods in the distributions and the normalized quantifications. Limits of the QMSI were illustrated in the low and high limits of detection compared to the QWBA because of the restricted range of linearity.
Quantification results A calibration range was prepared on a liver tissue section by spotting a mixture of the parent and its metabolite before analysis by MSI (Figure 3). This calibration curve allowed the limit of detection (LOD) and lower limit of quantification (LLOQ) of the two compounds to be determined. Tissue Extinction Coefficients were calculated with the deuterated forms sprayed with the matrix to evaluate the suppression effects of the different organs (other than liver) of the dosed tissue. The quantification was processed with QuantinetixTM software for both 1H-choloroquine and 1H-desethyl-chloroquine taking into account the direct absolute quantification of the liver and the different TEC values for other organs. Results of the absolute quantification are summarized in the Table 1. A correlation between QMSI and QWBA was observed by totaling the parent + metabolite concentrations with the exception of the uveal tract and Harderian gland for which a ratio 10:1 QWBA:QMSI was observed. This difference appears to be due to the limited region of interest scanned by MSI as compared to the broader region of interest observed in the QWBA image (Figure 1). The low levels observable in the uveal tract is most likely related to melanin binding of chloroquine and insufficient extraction by the matrix solvent. The high concentration of the drug in the stomach was limited in the QMSI evaluation due to the potential of saturation of the signal, crystallization differences, or suppression effects impacting the linearity of the method. Another representation to correlate the quantification data of WBA (µg equivalents/g of tissue) with MSI is presented in the Table 2 and Figure 4 where the liver was normalized to 1 (100%) and all the organs were compared to this level. Figure 4 highlights the closeness of the results between the two methods for relative quantification purposes.
MSI of m/z 320.1894 (1H-Chloroquine) MSI of m/z 292.1581 (1H-desethyl chloroquine)
Choroid
Retina
Ciliary Body
Lens
Harderian gland
Cornea
Skin
Vitreous humor
QWBA image of LE rat at 24h dosed with 3H-Chloroquine
Figure 1: Distribution of 1H-Chloroquine and 1H-desethyl chloroquine at T24h
*1-Desethyl Chloroquine not detected or under the LOD
Table 2 Figure 4: Relative quantification in whole-body region by QMSI and QWBA (Reference organ: Liver)
Organ Relative QWBA (µg Equivalents [3H]Chloroquine/g of tissue)
Relative QMSI (µg [1H]Chloroquine/g of
tissue)
Relative QMSI (µg [1H]Desethyl Chloroquine/g of tissue)
Liver 1.00 1.00 1.00 Kidney, Cortex 0.17 0.16 0.57
Kidney, Medulla 0.29 0.26 0.50 Kidney 0.23 0.20 0.53
Stomach* 1.69 2.91 BLOD Lung 0.40 0.28 0.60
Uveal tract* 0.25 0.02 BLOD Lens* 0.01 0.00 BLOD
Harderian gland* 0.09 0.02 BLOD Skin* 0.03 0.01 BLOD
Whole-body mid region In the whole-body mid region, both drug parent and metabolite were detected in various tissues from the prepared T4h sections (Figure 2). The two molecules were mainly co-localized within the different tissues analyzed and the quantitative distribution data obtained by QMSI matched QWBA parent-equivalent concentration data. The parent drug data from QMSI within the stomach contents was compared directly to the QWBA data since the bulk of the contents would be parent drug at T4h; whereas the liver can provide a good understanding of drug metabolite distribution.
MSI of m/z 320.1894 (1H-Chloroquine)
Figure 2: MSI vs WBA in the whole-body region at T4h
9.1mm QWBA image of LE rat at 4h dosed with
3H-Chloroquine
MSI of m/z 292.1581 (1H-Desethyl Chloroquine)
Optical image T4h whole-body
Combining the strengths of QWBA and MALDI QMSI allowed for the sensitive detection of drug related material (QWBA) and the quantitative differentiation of parent and metabolite (QMSI). Discriminate images were generated for chloroquine and desethyl-chloroquine so that each compound could be directly quantified to generate tissue concentrations.
Distribution results Eye region 1H-Chloroquine was clearly detected and identified by MALDI-FTICR at the first time-point T4h in the different regions of the eye, especially the uveal tract. 1H-desethyl-Chloroquine was also detected and identified in ocular tissues. Figure 1 below demonstrates the tissue distribution at the time-point T24h to allow for a better visualization of the drug related metabolite.
Table 1: QMSI vs QWBA
QMSI (µg/g of tissue)
Organ 1H-Chloroquine 1H-Desethyl Chloroquine TOTAL QMSI TOTAL QWBA (µg eq/g
of tissue) Liver 111.4 3.1 114.5 103.0
Stomach 293.8* BLOQ 293.8 174.0 Kidney, Cortex 13.5 BLOQ 13.5 17.6
Kidney, Medulla 25.2 BLOQ 25.2 29.6 Kidney 17.7 BLOQ 17.7 23.3 Lung 26.3 0.7 27.0 41.7
Uveal tract 0.6 BLOD 0.6 26.0 Harderian gland 0.6 BLOD 0.6 9.6
Lens BLOD BLOD BLOD 0.9 Skin BLOQ BLOD BLOQ 3.0
Y = 3468716.833x + 726297.7096 R²= 0.9955
LOD (1H-Chloroquine) =0.056 µg/g of tissue LLOQ (1H-Chloroquine) = 0.406 µg/g of tissue
Optical image T24h eye
Choroid Retina
Ciliary Body
Lens
Cornea
Skin
Vitreous humor
Harderian gland
Choroid Retina
Ciliary Body
Lens
Harderian gland
Cornea
Skin
Vitreous humor
Figure 3: Calibration curve on a Liver section of 1H-Chloroquine
00.5
11.5
22.5
33.5
Ratio
QWBA eq 1H Chloroquine
QMSI 1H Chloroquine
QMSI 1H-DesethylChloroquine
Spleen Stomach content 1 Kidney Lung Liver
Heart Stomach content 2 Intestines
Spleen Stomach content Kidney Lung Liver
Heart Stomach content Intestines
Spleen Stomach content 1 Kidney Lung Liver
Heart Stomach content 2 Intestines
