Biodistribution of a Nonpeptidic Fluorescent Endothelin a Receptor Imaging Probe

Abstract

Biodistribution studies are essential for understanding the biologic behavior of novel fluorochrome-based molecular imaging agents. In this study, the biodistribution of a recently developed fluorescent imaging probe with high affinity to the endothelin A (ET_A) receptor was evaluated by fluorescence reflectance imaging (FRI). CD-1 mice were injected with 2 nmol of the probe intravenously and sacrificed at various time points. Tissue samples of the heart, spleen, lung, kidneys, liver, brain, and muscle were removed and imaged by FRI. Initially, the signal intensity (SI) was highest in lung, kidney, and liver tissue, followed by the heart, whereas spleen, muscle, and brain showed the lowest SI. In the kidneys, the SI decreased rapidly. In the heart, an initial SI increase was observed, followed by SI attenuation, whereas in the lung, the SI steadily increased. Competition experiments showed a significant (p #x003C; .005) degree of specific binding in the heart, with a reduction in SI of > 50%. In conclusion, FRI allows us to perform biodistribution studies of novel fluorescent tracers. The developed imaging probe can be exploited to image ET_A receptor expression ideally 30 minutes to 3 hours after injection.

THE ENDOTHELIN (ET) AXIS is known to play a major role in the modulation of vascular tone,^1–5 as well as in the progression of various human cancer types.^6–12 Three isoforms of ET exist (ET-1, ET-2, and ET-3), exerting their effects via two different G protein–coupled receptors (ET_AR, ET_BR). Whereas ET_ARs are primarily located on vascular smooth muscle cells and are responsible for vasoconstriction and cell proliferation, ET_BRs are located on smooth muscle cells and vascular endothelial cells, causing vasodilation by the release of nitric oxide and prostacyclin, and are responsible for the clearance of ET-1 from plasma in lung tissue.¹³ In oncology, ET-1 in particular, acting primarily through the ET_AR, is associated with the neoplastic growth of multiple tumor types. ET antagonism inhibits tumor cell proliferation, invasiveness, and new vessel formation. Hence, therapeutic inhibition of ET_AR is a concept that is currently under investigation for tumor treatment, especially in breast and prostate cancers.¹⁴ In this context, a method for the visualization of receptor density in affected tissue would be invaluable for clinical diagnosis and the evaluation of therapy. Choosing optical imaging as the detection modality can be an attractive alternative to scintigraphic imaging techniques since it is relatively inexpensive, free of ionizing radiation and isotope decay, and applicable for tomographic (eg, fluorescence-mediated tomography [FMT]) and surface-weighted (eg, fluorescence reflectance imaging [FRI]) imaging. These techniques allow the detection of molecules in picomolar concentrations, which is comparable to conventional scintigraphic imaging techniques.^15–17 The developed photoprobe, termed ET_A-Cy5.5 (2; Figure 1), is based on PD 156707, a potent ET receptor antagonist with high affinity and ETA selectivity.^18–21 The tracer was synthesized by reaction of Cy5.5 N-hydroxysuccinimidyl- (NHS-) ester with amino-precursor 1 (see Figure 1).²² In this study, we examined the biodistribution of the tracer in wild-type CD-1 mice to assess the applicability of the probe to image ET_AR expression in vivo.

Materials and Methods

General

All chemicals, reagents, and solvents for the preparation of the compounds were analytical grade and purchased from commercial sources. PD 156707 (3-benzo[1,3]dioxol-5-yl-5-hydroxy-5-(4-methoxyphenyl)-4-(3,4,5-trimethoxyben-zyl)-5H-furan-2-one) was prepared as reported and used as the sodium carboxylate salt.²³ Compound analysis was done by mass spectrometry using a Quattro LCZ spectrometer (Waters-Micromass, Manchester, UK) with a nanospray capillary inlet.

Figure 1.

Amino precursor (TFA-salt) 1 reacts with Cy5.5 NHS-ester to yield the endothelin-A receptor affine photoprobe ET_A-Cy5.5 (2).

Preparation of ETA-Cy5.5 (2)

The ligand-dye conjugate ET_A-Cy5.5 (2) was prepared according to the described procedure.²² Briefly, amino-precursor 1 was dissolved in bicarbonate buffer (0.1 M, pH 8.6) at 1.0 mM concentration, Cy5.5 NHS-ester was dissolved in dimethylsulfoxide at 1.0 mM concentration, and equal amounts of both solutions were mixed and stirred for 60 minutes at room temperature in the dark. The reaction mixture was then transferred to semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC), using a Knauer system (Knauer Wissenschaftliche Gerätebau GmbH, Berlin, Germany) with two K-1800 pumps, an S-2500 ultraviolet (UV) detector, and a RP-HPLC Nucleosil 100–5 C18 column (250 × 4.6 mm). The conditions used were as follows: eluent A (water, 0.1% trifluoroacetic acid [TFA]); eluent B (acetonitrile, 0.1% TFA); gradient from 90% A to 40% A over 40 minutes at a flow rate of 1.5 mL/min, detection at Λ = 254 nm. The appropriate fractions (t_R = 31 minutes) were collected, lyophilized, redissolved in phosphate-buffered saline, and finally stored at −20°C. The average content of 2 was 0.8 ± 0.3 μmol/mL (≈ 60% yield) as determined by fluorometer measurements with Λ_abs = 678 nm and Λ₆₇₈ = 250,000 M⁻¹cm⁻¹ (Hitachi U3310 UV/VIS spectrophotometer and Hitachi F-4500 fluorescence spectrometer, Tokyo, Japan).

Animals

Three- to 5-week-old female CD-1 mice (25-30 g; Charles-River WIGA, Sulzfeld, Germany) were used for all in vivo experiments. Animal studies were approved by the institutional animal care committee. Mice were anesthetized by intraperitoneal injection of a combination of ketamine and xylazine (90/10 mg per kilogram of body weight), and ETA-Cy5.5 (2.0 nmol) was injected into the tail vein. For injection of the photoprobe, a small insulin syringe was used (BD Micro-Fine +,0.33 (29G) × 12.7 mm). For predosing experiments, 2.0 μmol of PD 156707 was injected 10 minutes prior to injection of ET_A-Cy5.5. At given time points (10 and 30 minutes and 1, 3, 5, 24, and 48 hours) after injection, mice (n = 6–8 for each time point) were sacrificed by overdosing anesthesia. To clear blood from organs and tissue, the animals were perfused with ice-cold saline solution via the left ventricle of the heart.²⁴ Organs were rapidly removed, weighed, and subjected to FRI. For selected time points (1 hour and 24 hours; n = 6-8), displacement experiments were performed, where 2.0 μmol of PD 156707 was given 10 minutes prior to sacrifice.

Fluorescence Reflectance Imaging

Explanted organs were cleaned from surrounding tissue and placed on a petri dish. Optical reflectance imaging was performed using the In-Vivo FX Imaging System (Kodak Molecular Imaging Systems, New Haven, CT). Illumination was provided by a 150 W halogen lamp with selectable bandpass excitation and emission filters. For Cy5.5 excitation, Λ = 630 ± 10 nm and emission Λ = 700 ± 17.5 nm filters were employed. Light from the fluorescence screen is captured with the 4-million pixel cooled charge-coupled device camera equipped with a 10× zoom lens. Image acquisition times were 30 seconds at the emission wavelength band. Optical images were coregistered with background (phase contrast) images, and regions of interest were selected and analyzed with the Kodak Molecular Imaging 4.0 software. Signal intensity (SI) data are expressed as photon counts per milligram of wet tissue weight (pc/mg).

Plasma Stability

Mouse plasma (75 μL) was incubated with 20 μL (12 nmol) of ET_A-Cy5.5 solution at 37°C using an Eppendorf Thermomixer comfort (Eppendorf AG, Hamburg, Germany). At different time points (0 and 30 minutes and 1, 3, 5, and 24 hours; n = 3), proteins were precipitated by addition of acetonitrile and centrifugation at 13,000 rpm (10 minutes, 16,000g) using an Eppendorf centrifuge (5415D, Eppendorf AG). Supernatants were filtered and subjected to RP-HPLC analysis (for conditions, see above), and the area under the corresponding peak was measured. Average values were determined in three independent experiments with probe duplicates. Values at time point 0 minutes were assigned to 100% tracer amount.

Statistics

All data are presented as means ± standard error of the mean (SEM). Statistical differences between the predosed and the control group of animals were analyzed with an unpaired, two-tailed Student t-test using the InStat-program (GraphPad Software, San Diego, CA). A p value of #x003C; .05 was considered to indicate a statistically significant difference.

Results

We investigated the biodistribution of the previously developed ET_AR selective optical imaging probe ET_A-Cy5.5 (2)in wild-type CD-1 mice to assess its capability for in vivo imaging studies. The results of the examinations are summarized in Table 1 and Table 2 and illustrated in Figure 2, Figure 3, and Figure 4. Figure 2 shows the time courses of organ fluorescence intensities after administration of 2 nmol of the tracer. At 10 minutes after injection, the highest SIs are found in the lung (> 1.3·10⁵ pc/mg), in the liver (> 7.5·10⁴ pc/mg), and in the kidneys (> 7.4·10⁴ pc/mg), whereas the brain shows the lowest SI (< 1.4·10⁴ pc/mg). After 30 minutes, any organ except the spleen shows a higher fluorescence intensity compared with 10-minute values. The highest ratio of increase is found in muscle (> 1.3), heart (> 1.2), and liver (> 1.2). Values after 60 minutes reveal a high increase of fluorescence intensities in the lung and heart, whereas the liver and kidney show attenuation of the signal. The results at time points 3 and 5 hours show a noticeably high increase in fluorescence SI in the lung (> 2.8·10⁵ pc/mg). Also, a slight increase in fluorescence SI is found in spine and muscle, but, overall, intensities are lower than for the heart, kidney, or liver. Comparing the kidneys and the liver, it is noticeable that values for the liver stay nearly constant at ≈ 8.7·10⁴ pc/mg, whereas the signal in the kidneys decreases from 7.3·10⁴ pc/mg to #x003C; 5.5·10⁴ pc/mg over this period of time. The heart shows a decrease in fluorescence intensity from 7.3·10⁴ pc/mg at 1 hour to #x003C; 5.3·10⁴ pc/mg at 3 and 5 hours. After 24 and 48 hours, SIs have attenuated to values ≤ 5.0·10⁴ pc/mg in all organs except for the lung, where the tracer seems to accumulate, and SI values are still > 2.4·10⁵ pc/mg.

Figure 3 shows the effect of predosing on mean fluorescence intensities of selected organs. The specificity of tracer uptake is best described when looking at the ratio of total to nonspecific binding in the heart (see Figure 3A). In contrast to the values when the tracer was given alone (see above), the intensities after predosing drop from 5.2·10⁴ pc/mg to #x003C; 3.5·10⁴ pc/mg in the first 60 minutes postinjection and do not rise markably above this value until 48 hours after administration. The ratio of total to nonspecific binding rises from 1.5 (p #x003C; .01) at 30 minutes to 2.1 (p #x003C; .0001) after 60 minutes and at 3 and 5 hours is still high at 1.6 (p #x003C; .005) and 1.5 (p #x003C; .0001), respectively. The predosing effect in the lung (see Figure 3B) is significant at time points from 1 to 5 hours, with ratios of total to nonspecific binding of 1.4 (p #x003C; .05), 1.4 (p #x003C; .005), and 2.0 (p #x003C; .001). After 24 and 48 hours, predosing had no influence on the fluorescence intensities anymore. In the liver, the effect of predosing is not as accentuated as in other organs (see Figure 3C). At 30 minutes, the ratio of total to nonspecific binding is > 1.7 (p #x003C; .005), but at 60 minutes and 3 hours, it is reduced to 1.3 and 1.1, respectively. At 5 hours, the ratio rises to > 1.7 (p ≤ .0005) again. When examining the values for the kidneys, it is most noticeable that at 10 minutes, the SIs are significantly higher (p ≤ 001) after predosing, indicating a higher amount of free tracer owing to PD 156707-occupied sites resulting in an enhanced excretion. Later on, the values attenuate constantly over time, showing no differences with respect to predosing (see Figure 3D). Figure 4 shows selected results of the displacement experiments at time point 60 minutes in comparison with the results from the predosing studies. Whereas in the heart, predosing and displacement show significant effects on fluorescence signal reduction with ratios of total to nonspecific binding of 2.1 (p #x003C; .0001) and 1.5 (p #x003C; .01), the effect of predosing in the lung (ratio of total to nonspecific binding = 1.4, p < .05) cannot be reproduced by displacement experiments. Here, the reduction in signal intensity is #x003C; 10%.

Table 1.

Fluorescence Intensities, Expressed as the Photon Counts per Milligram of Wet Tissue Weight (Mean ± SEM, n = 6-8) of Explanted Organs Measured at the Given Time Points

	10 min	30 min	60 min	3 h	5 h	24 h	48 h
Organ	Mean ± SEM	Mean ± SEM	Mean ± SEM	Mean ± SEM	Mean ± SEM	Mean ± SEM	Mean ± SEM
Heart	50,142 ± 5,190	61,071 859	72,993 ± 3,482	52,177 ± 4,091	52,501 ± 1,577	54,481 ± 3,166	37,639 ± 6,486
Spleen	44,004 ± 1,546	40,906 ± 1,157	49,537 ± 1,616	41,753 ± 1,837	54,312 ± 5,972	33,803 ± 1,250	49,893 ± 3,204
Lung	133,100 ± 1,4554	149,461 ± 3,518	197,726 ± 4,464	232,244 ± 12,804	282,094 ± 21,768	245,844 ± 13,818	243,169 ± 4,126
Spine	34,113 ± 2,378	34,509 ± 1,622	37,308 ± 1,029	41,635 ± 1,537	45,806 ± 2,336	23,438 ± 2,008	31,006 ± 2,507
Kidney, left	74,102 ± 4,657	83,012 ± 5,525	73,299 ± 1,739	53,980 ± 4,293	54,639 ± 5,182	32,113 ± 1,259	28,321 ± 1,501
Kidney, right	83,840 ± 4,085	100,249 ± 4,086	80,798 ± 2,892	65,894 ± 5,351	65,127 ± 7,810	36,492 ± 1,242	29,956 ± 1,612
Liver	78,616 ± 6,208	97,850 ± 2,451	87,554 ± 2,934	87,302 ± 5,848	88,680 ± 4,595	48,135 ± 4,511	42,413 ± 3,601
Brain	13,749 ± 1,185	14,672 ± 235	14,979 ± 660	13,330 ± 695	14,201 ± 529	12,853 ± 787	12,884 ± 828
Muscle	31,169 ± 1,600	40,532 ± 815	41,016 ± 1,173	39,339 ± 2,109	44,588 ± 1,301	28,105 ± 399	25,060 ± 1,088

Table 2.

Fluorescence Intensities, Expressed as the Photon Counts per Milligram of Wet Tissue Weight (Mean ± SEM, n = 6–8) of Explanted Organs after Predosing Measured at the Given Time Points

	10 min	30 min	60 min	3 h	5 h	24 h	48 h
Organ	Mean ± SEM	Mean ± SEM	Mean ± SEM	Mean ± SEM	Mean ± SEM	Mean ± SEM	Mean ± SEM
Heart	51,604 ± 1,921	39,826 ± 4,474	34,770 ± 4,072	32,887 ± 1,700	34,793 ± 1,807	41,954 ± 4,185	31,314 ± 1,283
Spleen	39,981 ± 2,632	36,992 ± 3,489	32,836 ± 2,025	42,580 ± 3,996	33,734 ± 1,679	37,996 ± 2,900	38,774 ± 3,414
Lung	169,773 ± 23,917	160,834 ± 6,829	142,993 ± 20,160	161,247 ± 14,532	142,686 ± 7,010	272,183 ± 28,506	200,403 ± 18,290
Spine	47,905 ± 7,766	37,122 ± 4,150	39,646 ± 3,922	38,208 ± 3,330	35,964 ± 2,193	28,180 ± 3,114	25,294 ± 757
Kidney, left	125,476 ± 10,471	109,037 ± 18,217	66,444 ± 8,803	60,254 ± 7,957	43,537 ± 1,239	27,254 ± 3,017	20,633 ± 1,920
Kidney, right	147,988 ± 10,059	108,994 ± 18,059	73,441 ± 8,703	71,193 ± 9,696	51,669 ± 1,217	33,835 ± 4,293	20,892 ± 1,475
Liver	78,649 ± 12,635	56,505 ± 6,652	66,641 ± 9,172	80,463 ± 12,591	51,317 ± 1,239	43,088 ± 6,040	35,253 ± 3,583
Brain	15,148 ± 500	12,814 ± 795	12,293 ± 873	12,351 ± 459	13,338 ± 423	11,744 ± 394	11,779 ± 915
Muscle	41,738 ± 1219	41,828 ± 4,090	44,641 ± 4,727	36,315 ± 2,274	34,030 ± 2,320	22,465 ± 2,166	18,739 ± 1,467

A representative organ panel from biodistribution studies is displayed in Figure 5 (A + B, white light and color-coded near-infrared fluorescence, 3-hour values). Organs are placed on a petri dish. Figure 5, C to E, shows white light and FR images (raw and color coded) of hearts 60 minutes after injection of the tracer (left to right: C, ET_A-Cy5.5 alone; D, predosing experiment; E, displacement experiment). Fluorescence is highest in the ET_A-Cy5.5 group and clearly decreases after predosing or displacement. Figure 6 shows the results of the in vitro stability assay with mouse plasma. The percentage of intact tracer in the supernatant after given time points is recorded. The values show an exponential decrease to approximately 20% after 24 hours of incubation.

Figure 2.

Time courses of fluorescence intensities of explanted organs, expressed as photon counts per milligram of wet tissue weight (pc/mg, mean ± SEM). A, Fluorescence intensities of heart (♦), spine (▲), muscle (■), and brain (•). B, Fluorescence intensities of lung (♦), liver (▲), kidneys (■), and spleen (•) (note the difference in scale).

Figure 3.

Time courses of fluorescence intensities for selected organs without (♦) and with predosing (■) expressed as the photon counts per milligram of wet tissue weight (pc/mg, mean ± SEM; A, heart; B, lung; C, liver; D, kidney). Stars indicate significant differences in intensities (*p ≤ .01; ^**p ≤ .005; ^***p ≤ .0001; note the difference in scale). Between 30 minutes and 5 hours, there is a highly specific signal in heart tissue, whereas in the lung, intensities rise constantly from 10 minutes to 5 hours in nonpredosed animals. After 1 day, the tracer has accumulated in lung tissue. The liver shows specific tracer uptake after 30 minutes and 5 hours. Attenuation of the signal to background values is observed after 24 and 48 hours. Signal intensity in the kidneys attenuate constantly over time. In particular, the highly significant signal yield after predosing at 10 minutes indicates a first fast washout via these organs.

Figure 4.

Results of the displacement experiments (textured gray bars) for heart and lung (60-minute values) compared with the predosing experiments (light gray bars). Black bars represent values where the tracer was given alone. Fluorescence intensities are expressed as the photon counts per milligram of wet tissue weight (pc/mg, mean ± SEM). Significant differences are found in the heart after predosing (p #x003C; .005) and after displacement (p #x003C; .05). In the lung, the significant difference after predosing (p #x003C; .05) cannot be reproduced in the displacement experiment. Here, the reduction in signal intensity is < 10%.

Discussion

Our biodistribution data reveal that it is feasible to image ETARs in vivo using the previously developed highly affine fluorescent tracer. Binding specificity was shown by both competition and displacement experiments with specific tracer uptake in target tissue such as heart and lung. This reflects the biology of receptor distribution in vivo with high abundance of ETARs in these organs.^25,26 A phenomenon well known from other target-specific imaging approaches is that specific probe binding is overlaid by unspecific tracer distribution in the early “perfusions phase.”^27,28 According to our data, imaging 1 to 5 hours after injection of the probe revealed the best results with respect to imaging ETAR expression. At later time points, the specificity of the data is spoiled by clearance of the probe via internalization, as well as by probe degradation, as shown by the plasma stability assay.

The excretion route of the tracer is found to be divergent between liver and kidney, with a first, fast washout via the kidneys and a later elimination by the liver (see Figure 2A and Figure 3, C and D). The latter interestingly seems to appear in a period from 1 to 3 hours, where the specificity of tracer binding in liver tissue is diminished, emphasizing a high amount of metabolites present at this time point. This is in accordance with findings for radiolabeled ET^AR ligands, and it has been explained by the assumption that metabolization in the liver is a rapid process.²⁹ In particular, the high value of fluorescence in the kidneys after predosing at time point 10 minutes indicates that the hydrophilic nature of the tracer promotes renal excretion. This is in contrast to experiments using radioabeled nonpeptidic compounds, which usually are much more lipophilic. A high degree of specific binding in the kidneys was not observed, reflecting the predominance of the ET_BR subtype in these organs.^30,31 Lung tissue plays an important role in ET receptor ligand elimination.³² In normal and pathologic conditions, lung ET_BRs mediate the clearance of ET-1 from plasma by internalization of the ligand-receptor complex. Whereas ET_AR is recycled to a significant extent after internalization, ET_BR usually is degraded by lysosomal processing.^33,34 In one case, the internalization of ET_ARs after incubation with an ET_AR-selective antagonist has also been observed,³⁵ indicating that this processing pathway is also possible for the applied fluorescent tracer. In lung tissue, a large amount of ET_ARs and ET_BRs are present. The predominance of one subtype and concentration of receptors differ greatly with species and tissue type. For instance, airway smooth muscle cells from porcine origin express ranges from 7:3 to 3:7 for ET_A to ET_B ratios.^36–38 The tracer uptake in lung tissue is found to be significantly specific at medial time points from 1 to 5 hours, where a high predosing effect can be observed. Interestingly, displacement experiments 1 hour after injection of the tracer do not significantly reduce the fluorescence intensity in this tissue. This can be explained by an elevated degree of internalization, which was already discussed in an investigation with the radiolabeled tracer [¹⁸F]-ET-1.³⁹ Here, the investigators found rapid clearance of the endogenous ligand by lung and kidney via ET_B-mediated internalization, preventing visualization of target tissue such as the myocardium. In addition, a fast accumulation in the liver is described, relating to ET_AR binding and metabolic activity.

Compared with traditional biodistribution studies, working with fluorescent probes and FRI is fundamentally different with respect to probe quantification. Given that FRI is only a semiquantitative imaging method, differences in the fluorescence intensities of the organs may in part be explained by varying absorption and scattering properties of the tissues examined. We intended to reduce the absorption by hemoglobin by tissue perfusion prior to organ harvesting. However, lung tissue itself is per se more transparent to light compared with liver or muscle, for example, which in part explains the different fluorescent signal gains for different organ subtypes.^40,41 The assessment of tracer specificity (ie, ratios of total to nonspecific binding), however, should remain unchanged despite different scattering and absorption properties. More advanced fluorescence imaging techniques such as FMT^15–17,42 or modified imaging systems, for example, systems using polarized fluorescent light,^43,44 may correct at least in part for this systematic shortcoming. Thus, further studies are warranted to elucidate this point. Furthermore, direct in vivo to ex vivo correlations should be possible, for example, by fluorescence microscopy. In conclusion, we successfully developed a new fluorescent ET_AR tracer that shows specific binding to murine ET_ARs in vivo. Further studies are currently in progress to investigate the “diagnostic performance” of this new molecular imaging probe in different pathologies, for example, tumor tissues, where ET_AR density is regulated.

Figure 5.

Petri dish with harvested organs 3 hours postinjection (A, white light; B, color-coded near-infrared fluorescence [NIRF] images; numbers indicate photon counts per pixel). High fluorescence values are observed in the lung (4), liver (8), kidneys (6), spine (5), and heart (1); lesser to no intensity is found in the spleen (3), brain (7), and muscle (2). C–E, White light and NIRF images (raw and color coded, left to right; numbers indicate photon counts per pixel, 60-minute images) of explanted hearts after administration of 2 nmol of the tracer (C, ET_A-Cy5.5 alone; D, predosing experiment; 2.0 μmol PD 156707 10 minutes prior to tracer administration; E, displacement experiment, 2.0 μmol PD 156707 50 minutes after tracer administration).

Figure 6.

Plasma stability determination in vitro. Percentage of intact fluorescent probe (mean ± SEM) left after incubation at given time points (0, 30, and 60 minutes; 3, 5, and 24 hours). The measured value at time point 0 minutes was assigned to 100% tracer amount.

Footnotes

Acknowledgments

Financial disclosure of authors: Financial support from the Deutsche Forschungsgemeinschaft (DFG SCHA 758-5-1, SFB 656 A4, and SFB 656 Z2) and the Interdisciplinary Center for Clinical Research, University of Muenster (IZKF ZPG 4b and FG 3) is gratefully acknowledged.

We would like to thank Mrs. Susanne Greese for technical assistance and Dr. Heinrich Luftmann (Department of Organic Chemistry, University of Muenster) for mass spectrometry. Financial disclosure of reviewers: None reported.

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