Abstract
Quaternary ammonium compounds are known to highly concentrate in articular cartilages after i.v. administration. This property was used to synthesize new potential radiodiagnostic agents for joint imaging. Pharmacokinetic study was performed in rats for three new compounds:N-pyridinium-propyl-cyclam (NPPC),N-triethylammonium-propyl-cyclam (NTPC), andN-[triethylammonium]-3-propyl-[15]ane-N5 (NTP 15–5). After i.v. administration, [3H]NPPC and [3H]NTPC highly and rapidly concentrated in articular cartilage, this uptake being followed by a single exponential decrease with half-lives of, respectively, 75 and 82 min. Except cartilage, only the kidney was highly labeled. After complexation of 99mTc by NPPC, NTPC, and NTP 15–5, only 99mTc-NTP 15–5 exhibited a high affinity for cartilage. On the other hand, the pharmacokinetic behavior of 99mTc-NTPC and99mTc-NPPC was very different from those of their3H-labeled analogs. Concentration in cartilaginous tissues was strongly diminished, and liver and bone were highly labeled. For all labeled species, the major route of excretion was urine, and HPLC analysis showed that [3H]NTPC and [3H]NPPC were excreted under their unchanged form. On the other hand, no 99mTc-NTPC and 99mTc-NPPC were found in the urine, the radioactivity being mainly due to free technetium, contrary to 99mTc-NTP 15–5, which was excreted in the urine under the complexed form. These data can explain the striking differences observed between the three99mTc-labeled molecules, the lack of concentration of 99mTc-NTPC, and 99mTc-NPPC in cartilages in comparison with their 3H-labeled analogs due to an instability in vivo of these technetiated complexes.
The radionuclide imaging of articulations is actually assessed by bone imaging agents such as99mTc-methylene diphosphonate and inflammation imaging agents such as 67Ga citrate (Fogelman et al., 1994). These methods are more sensitive than radiography, but their specificity toward osteoarthritis is low. The use of a radiopharmaceutical concentrating in the cartilaginous tissue could be able to enhance the efficacy of scintigraphy in joint disease imaging. To date, 75Se derivatives have been developed for this purpose (Yu et al., 1989), but the clinical use of such radionuclide is precluded by its long period (120 days). A radiopharmaceutical useful for cartilage imaging in humans should have both a high affinity for this tissue and a chemical group able to bind a short period radionuclide as 99mTc or123I.
In previous works, we have demonstrated that the acetylcholinesterase reactivators quaternary ammoniums-oximes rapidly and strongly concentrated in cartilaginous tissues such as intervertebral disks and articular cartilages after i.v. injection to rats (Garrigue et al., 1990, 1991). This property is due to a binding of the basic quaternary ammonium group of the drugs to the acidic functions (COO−, SO3−) of the cartilage proteoglycans (Maurizis et al., 1992). These results allowed us to synthesize molecules owning a quaternary ammonium function able to bind the cartilaginous tissue and another function able to bind99mTc with a view to obtain new radiodiagnostic agents for the articular cartilage imaging.
In this work, we describe the disposition in rats of three new molecules (Fig. 1),N-pyridinium-propyl-cyclam (NPPC)1 andN-triethylammonium-propyl-cyclam (NTPC), labeled with3H on the polyaza cycle and labeled by99mTc complexation, andN-[triethylammonium]-3-propyl-[15]ane-N5 (NTP 15–5) labeled by 99mTc complexation.
Materials and Methods
Chemicals.
[3H]NPPC, [3H]NTPC, and NTP 15–5 were prepared as described previously (Nicolas et al., 1999). Specific radioactivities were 2.4 mCi/mmol for [3H]NPPC and [3H]NTPC. Radiochemical purities were >95% for both products.99mTc-NPPC, 99mTc-NTPC, and99mTc-NTP 15–5 were prepared as described previously (Nicolas et al., 1999) by reducing99mTcO4−with stannous chloride in saline at 85°C for 30 min. The yield was about 100% and the pH equivalent was 7. The specific radioactivity was 600 to 700 mCi/mmol and the technetiated compounds were stable at room temperature more than 24 h at pH 7.
Animal Experiments.
Sprague-Dawley male rats (Iffa-Credo, l'Arbresle, France) weighing 100 to 120 g were housed in facilities with an ambiant temperature of 20 ± 2°C, relative humidity of 40 ± 20%, a natural cycle light, and access to food and water ad libitum. Whole body autoradiography was carried out according to the technique described byUllberg et al. (1971). [3H]NTPC or [3H]NPPC dissolved in 0.9% NaCl at 10 mg/kg were given i.v. The radioactive dose was 100 μCi/animal. Rats were sacrificed by ether inhalation at 10 min, 30 min, 1 h, and 6 h after administration and immediately frozen in liquid nitrogen. Tissue slices (20 μm) were performed with a cryomicrotome (Cryo-polycut; Reichert-Jung, Heidelberg, Germany) and applied to Hyperfilm 3H (Amersham). For biodistribution studies, rats were given a 10 mg/kg dose (10 μCi/animal) of [3H]NTPC or [3H]NPPC supplemented with 200 μCi/kg dose of99mTc-labeled molecule (additional weight negligible as compared with the tritiated molecule), and for99mTc-NTP 15–5, 50 μCi of the technetiated molecule. Five animals were sacrificed by ether inhalation at 10 min, 30 min, 1, 2, 6, and 24 h after dosing, and blood was withdrawn by cardiac puncture. Samples of different tissues were taken and the radioactivity of 99mTc was directly determined by counting in a Packard Autogamma 5000 spectrometer.3H radioactivity was measured using a Wallac Winspectral 1414 scintillation spectrometer after combustion in a Packard 306 sample oxidizer. In separate experiments, animals were kept in metabolic cages enabling collection of feces and urine (Iffa-Credo, L'Arbresle, France). Urine radioactivity was directly measured with a Packard Autogamma 5000 spectrometer for 99mTc and after dilution of aliquots in scintillation cocktail (Packard Ultima Gold) for 3H. Feces radioactivity was measured as described for tissues.
Urine Analysis.
To separate complexed molecule from free TcO4−, urine obtained for 24 h after injection was pooled and an aliquot poured on to a DOWEX 50WX4 column (1 × 5 cm) eluted by distillated water. The basic complex was bound to the resin and free TcO4− was eluted in the aqueous eluent. Elution of the complex was performed with 2 N NH4OH. After evaporation to dryness under reduced pressure, one part of the dry residue was dissolved in the HPLC eluent and the other part was submitted to NMR analysis.
NMR Analysis.
The dry residue obtained as described above was dissolved in dimethyl sulfoxide-D6.1H spectra were recorded at 200 MHz on a Bruker AM200 apparatus, and the chemical shifts were expressed in ppm using tetramethylsilane as internal standard.
HPLC Analysis.
HPLC was performed using a Chromatem model (Altex; Touzart et Matignon, Paris, France) equipped with two pumps, a solvent programmer, a 250-nm UV detector, a Flow-one radioactive detector (Radiomatic), and an automatic fraction collector. Partisil SCX 10-μm analytical column (20 × 0.4 cm) (Whatman Inc., Clifton, NJ) was eluted at 0.7 ml/min with 0.2 M KH2PO4acidified to pH 2.8 by H3PO4. For [3H]NTPC and [3H]NPPC, fractions were dissolved into Packard Ultima-Gold scintillation cocktail before counting. For the 99mTc-labeled molecules, fractions were directly counted in the autogamma spectrometer.
In Vitro Study of Binding to Proteoglycans.
Rabbit articular chondrocytes were cultured as described in a previous paper (Maurizis et al., 1992). [3H]NTPC or [3H]NPPC at a concentration of 1 mg/107 cells, or 50 μCi of99mTc-NTPC, -NPPC, or -NTP 15–5 per 107 cells were incubated for 24 h in the culture medium. The supernatant was then dialyzed against buffers of increasing pH from 5 to 11 and radioactivity bound to proteoglycans measured by Sepharose 2B chromatography as described previously (Maurizis et al., 1992).
Data Analysis.
Experimental points (six time points between 10 min and 24 h) were fitted using the Siphar program (S.I.M.E.D. Créteil, France) on an Apple Macintosh computer. The elimination half-time (t1/2) was calculated from the formula:t1/2 = ln 2/K where Kis the slope of the elimination phase. The area under the curve (AUC) was calculated using the trapezoidal rule with extrapolation to infinity. Total blood clearance was determined by the ratio of administered dose to AUC. Statistical analysis was conducted using the Kruskal-Wallis nonparametric test.
Results
Excretion of Radioactivity.
The cumulative excretion of the radioactivity in the urine and feces of rats given the labeled molecules is shown in Table1. These results show that [3H]NTPC and [3H]NPPC were excreted mainly in the urine. Fecal excretion was very low (less than 3% within 72 h). For the 99mTc-NTPC and -NPPC urinary excretion was also observed, but important amounts of radioactivity remained in the body after 48 h. For99mTc-NTP 15–5, excretion was mainly in the urine, but was more complete after 48 h than for the99mTc-NTPC and -NPPC.
Autoradiographs.
Figure 2 shows an example of autoradiographs obtained with [3H]NTPC 30 min after injection of a 100-μCi dose to rat. Cartilages of the intervertebral discs and articulations, and kidney were strongly labeled. No other tissues exhibit high radioactivity levels, except liver, which was less firmly labeled than cartilages and kidney. Results obtained with [3H]NPPC (not shown) were identical with those of [3H]NTPC.
Quantitative Biodistribution.
Doubly labeled NTPC and NPPC
Tables 2 and3 show the biodistribution of the labeled molecules 10 min and 1 h after injection. Striking differences were observed between the 3H and99mTc labels. The tritiated molecules highly concentrated in the cartilaginous tissues and very poor amounts of radioactivity were found in the surrounding tissues, bone, and muscle. Kidney was strongly labeled, which agrees with the urinary elimination of these molecules. The 99mTc molecules were significantly less concentrated in the cartilage both 10 min and 1 h after injection (P < .05), but the concentration in bone and liver was higher after 1 h (P < .01). Figures 3 and4 show the evolution of the radioactive concentrations between 10 min and 6 h after injection, and Fig.5 shows the comparative AUC values (0 → ∞). For the tritiated molecules, the blood concentration kinetic agreed with a single exponential decrease witht1/2 = 42 min for [3H]NTPC and 45 min for [3H]NPPC, and the blood clearance was 1.54 ml/min for [3H]NTPC and 1.45 ml/min for [3H]NPPC (no significant difference). The maximal concentration in cartilages was obtained before 10 min and a single exponential decrease was observed with at1/2 (elimination) = 75 min for [3H]NTPC and 82 min for [3H]NPPC (no significant difference). In contrast with the target tissues, liver slowly concentrated radioactivity, the maximal value being obtained 2 h after injection, followed by a very slow elimination rate (t1/2 > 8 h for both labeled molecules). In the kidney, the main excretory organ, the radioactive concentration exhibited a behavior close to that of the blood [t1/2 (elimination) = 52 min for [3H]NTPC and 55 min for [3H]NPPC]. The technetiated molecules exhibited absorption and elimination rates close to those of the tritiated molecules, but cartilage exhibited a significant decrease of the AUC (P < .01) contrary to liver and bone, which exhibited a significant increase (P < .05). As for tritiated molecules, pharmacokinetic behavior in kidney was similar to that observed in the blood.
99mTc-NTP 15–5.
Table 4 shows the biodisposition of radioactivity 10 min and 1 h after administration. Contrary to the99mTc-NTPC and -NPPC, this molecule strongly concentrated in cartilages, and the surrounding tissues, bone, and muscle were weakly labeled. Kidney was highly labeled, in accordance with the urinary elimination of this molecule. Figure6 shows the radioactive concentrations in blood, liver, cartilage, and bone between 10 min and 6 h after injection. The blood concentration agreed with a one exponential decrease kinetic with t1/2 = 49 min and an elimination clearance = 1.80 ml/min. The maximal concentration in cartilage was obtained before 10 min and an exponential decrease was observed with t1/2 (elimination) = 92 min. Liver concentrated less radioactivity than [3H]NTPC and [3H]NPPC, but the elimination was slower (t1/2=12 h). Kidney exhibited an elimination rate close to the one observed in blood (t1/2 = 56 min).
In Vitro Binding to Proteoglycans.
After a 24-h incubation of the labeled molecules in the culture medium of cultured rabbit articular chondrocytes (1 mg/107 cells for [3H]NTPC and [3H]NPPC, or 50 μCi/107 cells for99mTc-NTPC, NPPC, and NTP 15–5), chromatographic analysis showed significant amounts of radioactivity bound to proteoglycans. Dialysis of the bound radioactivity against buffers of increasing pH showed half dissociation pH = 8.2 for [3H]NTPC and 8.3 for [3H]NPPC, identical for the3H molecules and 99mTc molecules, and 8.5 for the 99mTc-NTP 15–5.
Urine Analysis.
Table 5 shows the results of the chromatographic analysis of the urine collected between 0 and 24 h after injection of the labeled molecules. For doubly labeled [3H, 99mTc]NTPC and NPPC, more than 90% of the urinary 3H radioactivity was eluted from DOWEX 50WX4 column by the 2 N NH4OH eluent. HPLC analysis of this fraction showed only one radioactive peak with the same retention time as those of unchanged NTPC (4.7 min) and NPPC (5.3 min). NMR analysis of these fractions identified them to authentic NTPC and NPPC. On the other hand, for 99mTc label, more than 80% of the urinary radioactivity was eluted from the DOWEX 50WX4 column in the aqueous eluent for both compounds. This result clearly indicates extensive dissociation of the complex between the polyaza cycle and the TcO2 residue. For 99mTc-NTP 15–5, about half of the urinary radioactivity was obtained in the 2 N NH4OH eluent, which proves a greater stability of the complex as compared with NTPC and NPPC. HPLC analysis of this fraction showed only one radioactive peak with a retention time (6.2 min) identical with that of the unchanged NTP 15–5.
Discussion
The results described above show that the polyazamacrocycles bound to quaternary ammonium are rapidly distributed in the kidney and the cartilages after i.v. injection. The blood concentration rapidly decreases according to a one exponential model kinetic. The excretion is mainly in the urine and the compounds are eliminated as the unchanged form without metabolic degradation. The maximal concentration in the target tissue cartilage is reached less than 10 min after injection and followed by an exponential decrease with elimination half-lives close to 1 h 30 min. No significant differences were found between aliphatic (NTPC) or aromatic (NPPC) quaternary ammoniums, indicating that the nature of the substituents has no influence on the pharmacokinetic parameters. For the tritiated molecules, the ratio between the radioactive concentration of cartilages and the surrounding tissues, bone, and muscle, is >10 between 10 min and 1 h 30 min after injection. At the opposite, the affinity for cartilage of the99mTc complexes of the cyclam derivatives is strongly reduced and the affinity for the bone significantly increased. This result clearly shows that the complex between the TcO2 residue and the polyazamacrocycle is unstable in vivo, giving after oxidation TcO4− that exhibits no affinity for cartilage and concentrates in bone and liver. This hypothesis is confirmed by the fact that the rats given the99mTc complex of NTPC and NPPC excreted only free technetium in the urine. This low stability of the complex in vivo is probably due to the fact that the polyazamacrocycle has three secondary and one tertiary amine functions. For a maximal stability of the TcO2 complex, the complexing structure should have four symmetric atoms able to bind with the pyramidal structure of the TcO2 bonds (Kung et al., 1990; Riché et al., 1993). Contrarily to NTPC and NPPC, NTP 15–5 exhibits four symmetric secondary amine functions on its polyaza cycle. This structure explains that the 99mTc complex of this compound is able to highly concentrate in cartilages with an affinity close to that of nontechnetiated NTPC and NPPC. The stability of this molecule in vivo is confirmed by the presence of important amounts of99mTc-NTP 15–5 in the urine. The in vitro study conducted on chondrocyte culture shows that these compounds are able to bind to the acidic functions of the proteoglycans, as it was demonstrated for other quaternary ammoniums (Olsen et al., 1975;Larsson et al., 1981; Maroudas et al., 1989; Yu et al., 1989). The half-dissociation pH identical before and after complexation shows that this structure modification does not change the affinity of the molecules toward the acidic functions of the cartilage.
In conclusion, among the three molecules studied, NTP 15–5 appears to be able to serve as a radiopharmaceutical for joint imaging after99mTc complexation. The stability of the complex is good in vivo, and the ratio between cartilage and surrounding tissues is sufficient to produce a convenient image without using a background subtraction, and the rapid elimination process considerably reduces the irradiation risk. With this molecule, preclinical scintigraphic studies are actually in progress on animal models.
Footnotes
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Send reprint requests to: J.C. Maurizis, Institut National de la Santé et de la Recherche Médicale, U484, rue Montalembert, B.P 184, 63005 Clermont-Ferrand Cedex, France. E-mail:MAURIZIS{at}Inserm484.u-clermont1.fr
- Abbreviations used are::
- NPPC
- N-pyridinium-propyl-cyclam
- NTPC
- N-triethylammonium-propyl-cyclam
- NTP 15–5
- N-[triethylammonium]-3-propyl-[15]ane-N5
- AUC
- area under the curve
- Received July 15, 1999.
- Accepted November 22, 1999.
- The American Society for Pharmacology and Experimental Therapeutics