Bioconjugate Chem. 2009, 20, 1547–1552
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Optimal 99mTc Radiolabeling and Uptake of Glucosamine Sulfate by Cartilage. A Potential Tracer for Scintigraphic Detection of Osteoarthritis Grazyna Sobal,* Johannes Menzel,† and Helmut Sinzinger* University Clinic of Nuclear Medicine and Institute of Immunology, Medical University of Vienna, Vienna, Austria. Received March 3, 2009; Revised Manuscript Received May 26, 2009
Glucosamine sulfate (GS) is used in treatment of human osteoarthritis, but no data for99mTcGS scintigraphy are available. Radiolabeling of GS was performed using the 99mTcO4-/tin method. We applied two procedures for separation of free 99mTc using PD10 and G10 columns. In each eluted fraction, GS content was estimated by the Elson/Morgan method. For optimal radiolabeling, we varied the amount of GS, tin, 99mTc activity, and pH. For uptake age matched human rib cartilage (males, 78 and 63 years old) and 5-10 µCi/well of 99mTcGS were used. Uptake was monitored up to 72 h. Also, washout of the tracer 3 h and 24 h after uptake was investigated. At pH 2, using PD10 column, the uptake of 99mTcGS amounted to 100.8 ( 2.9%, n ) 6 at saturation time of 72 h. Uptake was age-dependent; at pH 5, it amounted to 99.8% as compared to 66.1% at 78 vs 63 years old. When the amount of tin was varied at pH 2, the tracer uptake amounted to 21.37% (1 mg) vs 45.99% (2.5 mg) at saturation. At pH 7, the amount of needed tin was much lower and amounted to 42.50 ( 2.50% using 0.1 mg vs 25.11 ( 1.90% using 0.05 mg. Although the uptake at pH 7 (0.1 mg tin) is comparable with that at pH 2 (2.5 mg tin), the washout of the tracer amounted only to 4.10 ( 1.25% and 2.05 ( 0.65% after 3 h and 24 h, respectively. During degeneration of cartilage, incorporation of 99mTcGS is high and could therefore be a promising tracer not only to target osteoarthritis but also to monitor the effects of therapy.
INTRODUCTION Glucosamine (GA) is a natural sugar and a key component of the extracellular matrix of cartilage. These monomeric sugars possess charged side chains, which absorb water and provide lubrication for cartilage that covers the bones in the joints. With aging or under pathological conditions, GA, like other sugars, declines in concentration and loses the ability to absorb water. Treatment with GA affects the symptoms but not the disease, since after ingestion, GA has been shown to be absorbed in the joints as a component of the cellular matrix of cartilage. Glucosamine sulfate (GS) is the sulfate derivative of the naturally occurring aminomonosaccharide, GA. GA and chontroitin sulfate (CS) are widely used drugs for treatment of osteoarthritis (1-4) as symptomatic slow-acting drugs for osteoarthritis (SYSADOA). Osteoarthritis (OA) represents an advanced stage of disease caused in part by injury, loss of cartilage structure, and function and is an inflammatory process. The results of an in vitro study (5) indicated that GS and CS can regulate gene expression and synthesis of nitric oxide (NO) and prostaglandin PGE2, thereby modulating antiinflammatory activity. Bone scintigraphy is generally regarded as very sensitive but nonspecific. Because of their sensitivity and advantage over MRI (6, 7), noninvasive isotopic methods including SPECT and PET (8, 9) play an important role in clinical studies of arthritis. Although imaging techniques using radiolabeled polyphosphonates have been a major clinical tool for imaging osteoblastic activity and tumor involvement of the skeleton during the past decades, radiolabeled probes specific for the pathophysiology * Address all correspondence and reprint requests to Dr. G. Sobal, Department of Nuclear Medicine, Medical University of Vienna, A-1090 Vienna, Wa¨hringer Gu¨rtel 18-20, Austria. Tel. +431/ 40400-5558, Fax +431 /40400-5552, E-mail: Grazyna.Sobal@ meduniwien.ac.at. † Institute of Immunology.
of synovium and articular cartilage are just at the early stages of exploration. For this reason, more specific probes for articular cartilage disease would be preferable. GS as an endogenous cartilage component is one of these candidates. GS is widely used for the treatment of human osteoarthritis, but no data on GS labeling with 99mTc or studies of radiolabeled 99mTc GS in the cartilage are available. The aim of this study was to elaborate the optimal radiolabeling of 99mTc GS for future SPECT analysis by providing the optimal radiolabeling method, quality control of the tracer, its stability, and evaluation of uptake of radiolabeled compound by cartilage and to verify the degree of GS internalization.
EXPERIMENTAL PROCEDURES Materials. GS was provided by Rottapharm, Monza, Italy. Radiolabeling of GS was performed using 99mTcO4-/stannous chloride according to the methods (8, 10) analogous to chondroitin sulfate labeling. We found that, because of colloid formation, pH is a limiting factor for the labeling procedure and tracer uptake. Radiolabeling Procedure. Briefly, 25 mg of GS was dissolved in 0.75 mL of 0.3 M HCl, pH 2; then, 1 mg of stannous chloride was added followed by the addition of 1-3 mCi (37-121 MBq) of 99mTcO4-/(0.25 mL). We also performed experiments by buffering final reaction mixture with 0.50 M sodium acetate buffer at pH 5.0 or 0.01 M sodium citrate buffer at pH 7 to obtain more physiological conditions. The reaction mixture was gently stirred and incubated for 1 h at room temperature. Thereafter, the radiolabeled product was applied to the Sephadex PD10 chromatography column (Bio-Rad, California, USA) for separation from free technetium. The radiolabeled GS was eluted from the column with 0.3 M HCl in 10% ethanol or buffer in the void volume (3-5 mL), while free technetium was eluted from the column with the total volume (6-10 mL). In parallel, we measured 99mTc activity in each fraction to obtain the elution profile from the column. For
10.1021/bc9000883 CCC: $40.75 2009 American Chemical Society Published on Web 07/17/2009
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optimal radiolabeling, we varied the amount of GS (2.5 mg versus 25 mg), tin (1-5 mg), and also 99mTc activity (1.5-4 mCi, 55.5-148 MBq). We also applied two different procedures for separation of free 99mTc after radiolabeling by using PD10 and G10 (self-prepared, 15 mL) columns. In each eluted fraction after labeling, GS content was estimated by the Elson and Morgan method (11). For each of the radiolabeling variations, the radiolabeling efficiency, maximal uptake, and washout of the tracer were estimated. Uptake Studies. For uptake studies, human rib cartilage (males, 78 and 63 years old, post mortem) cut into pieces of about 5 mg wet weight was used. For uptake, we incubated with 5-10 µCi/well of 99mTcGS. Uptake was monitored every 10-30 min, and thereafter every few hours until saturation was achieved (up to 72 h). After each incubation period, the tissue was separated from incubation medium by simple differential centrifugation and rapidly washed twice with PBS buffer. The net (after subtraction of residual activity and activity related to adhesion of tracer to the tubes) cellular uptake of 99mTcGS in vitro was measured by a gamma counter (Packard, Meridien, CT, USA). Also, washout of the tracer after uptake was investigated. The washout of the tracer was performed by incubation of tissue after uptake experiments (3 h and 24 h) with PBS buffer for different times (10 min to 3 h). Derivatisation of GS. GS measurement was perfomed according to the modified method by Elson and Morgan (11). Briefly, 1 mL of column eluent containing GS was mixed with 1 mL of the acetylacetone reagent (2 mL of acetylacetone dissolved in 98 mL of 1 N sodium carbonate) and closed with a cap. The tubes were then placed in a water bath at 90 °C for 45 min. Thereafter, they were cooled in running water. Then, 4 mL of ethanol (95%) was added and tubes were vortexed. Finally, 1 mL of PDMAB (p-dimethylaminobenzaldehyde) for derivatization was added. After mixing again, the tubes were allowed to stand at room temperature for 1 h, getting a characteristic pink color. The absorbance was read at 540 nm. GS content of the samples was calculated using a standard curve with defined GS amount (5-50 µg/mL). Statistical Analysis. The results are expressed as mean values ( SD. Statistical analysis was performed using one-way ANOVA; p < 0.05 was considered statistically significant.
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Figure 1. Uptake of 99mTcGS after separation on PD10 column as a function of time, at different pH values (A). The washout of 99mTcGS from cartilage at pH 2, after 3 h and 24 h of uptake as a function of time (B). Each point represents the mean value of three radiolabeling experiments with two measurements each.
RESULTS Radiolabeling Procedure and Uptake Studies. The radiolabeling yield was strongly influenced by pH because of colloid formation. At higher pH 5, the labeling efficiency was very low (10.1-12.5%) and the colloid content high (31.6-89.9%), detected on Sephadex PD10 chromatography column by separation after labeling. In contrast, at pH 2.0 almost no colloid or only a few percent of colloid was formed (maximal 4.5%; data not shown), and a very high labeling efficiency (72.0-95.6%) was achieved. During the incubation with 99mTcGS, we observed continuously increasing uptake of tracer over the time up to saturation. At pH 2, after separation on a PD10 column, uptake was very high, amounting to 100.8 ( 2.9%, n ) 6, at saturation of 72 h (Figure 1A). This means that the obtained saturation of the tracer at this pH was achieved when the total of introduced 99mTc GS tracer was completely taken up into the cartilage. At pH 5, the uptake was much lower, but increasing with time until saturation amounting to 50.1 ( 5.5%, n ) 6 (Figure 1A). After 3 h uptake, the washout of the tracer from cartilage at pH 2 amounted to 45.7 ( 2.5% and was completed after 30 min. After 24 h uptake, the washout was unchanged at 44.1 ( 7.5%, but it was completed after 3 h (Figure 1B). These results indicate that in both experiments more that 55% of the
Figure 2. Age-dependent uptake of 99mTcGS at ph 2. Each point represents the mean value of three radiolabeling experiments from two specimens.
introduced tracer is retained in the cartilage and is not eluted. Uptake was age-dependent and amounted to 99.8% as compared to 66.1% at 78- vs 63-year-old individuals at saturation by 72 h (Figure 2). The nonspecific uptake in the presence of a 50-fold excess of cold GS in all experiments increased with time up to a maximum of 12-15% at saturation by 72 h. Because of similar molecular weights of both components (GS and pertechnetate), the separation of free 99mTc after radiolabeling by a chromatography column or ITLC-SG chromatography was difficult. For radiolabeling of GS using 99m TcO4-/1 mg tin method at pH 2, we evaluated the separation by PD10 and G10 columns. By estimation of GS content in
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Figure 3. Chromatography procedure on PD10 vs G10 column, after radiolabeling at pH 2. In parallel, GS content in each eluted fraction was estimated. Figure 5. Dose-dependent uptake of 99mTcGS at pH 2 (A) and washout of the tracer 3 h vs 24 h after uptake (B), at constant condition: pH 2, 2.5 mg tin. Each point represents the mean value of three radiolabeling experiments with two measurements each.
Figure 4. Dose-dependent (tin) estimation of radiolabeling efficiency at pH 2. Each point represents the mean value of three radiolabeling experiments with two measurements each.
each eluted fraction, we found a shift between the maximum of GS content peak and the radioactivity peak by PD10 (Figure 3A); therefore, we applied a G10 column for more precise separation of components after radiolabeling (Figure 3B). Varying the amount of tin (0.1-5 mg), at pH 2 and using G10 column for separation, we found the optimal conditions at 2.5 mg tin and 25 mg of GS, 4 mCi, for radiolabeling. This factor not only influences the labeling efficiency (22.95-42.73%, 1 mg vs 2.5 mg tin) (Figure 4), but also the 99mTcGS uptake in cartilage. While tracer uptake using 1 mg tin for labeling achieved a maximum of 21.37% at saturation (72 h), in contrast using 2.5 mg tin the maximal uptake amounted to 45.99% at saturation (Figure 5A). However, using 3-5 mg of tin the labeling efficiency and uptake could not be further increased and achieved a comparable amount to that using 1 mg, amounting to 28.13% at saturation at 72 h. Tracer washout after 3 h incubation using 2.5 mg tin for radiolabeling amounted to a maximum of 17.44 ( 6.60% and after 24 h to 4.94 ( 3.60%, respectively (Figure 5B). Finally, we found that buffering of the radiolabeling mixture with 0.01 M citrate at pH 7, reflecting more closely physiological
conditions, gave the best results of highest labeling efficiency and specific activity of 1.6 MBq/mg GS. However, the amount of tin was much lower than at pH 2. Using 0.1 mg tin for radiolabeling, we get the highest labeling efficiency and specific activity (Figure 6A), followed by 0.05 mg tin, getting slightly worse results (Figure 6B). Also, the corresponding uptake at this pH 7 (0.1 mg tin) showed a higher uptake (42.50 ( 2.50%) than using 0.05 mg tin for radiolabeling (25.11 ( 1.90%) (Figure 7A). Although the uptake at this pH 7 (0.1 mg thin) is comparable to that at the uptake at pH 2 (2.5 mg tin) (Figure 5A), the washout of the tracer at pH 7 was much lower, amounting only to (4.10 ( 1.25%) and 2.05% ( 0.65%), after 3 h and 24 h, respectively (Figure 7B).
DISCUSSION GA is an endogenous component of cartilage and is a monomeric part of glucosaminoglycans in cartilage matrix and synovial fluid (12). Chondrocytes use it for CS proteoglycan synthesis. GS has been found to be of benefit in OA in a number of studies (13-15) for the symptomatic treatment beneficially affecting the radiographic progression of OA (16-19). Therefore, 99mTc GS could be such a specific tracer for uptake in articular cartilage. The mechanism of action of GS has not been fully elucidated yet. Cartilage-unrelated effects such as inhibition of superoxideradical generation (20) or the inhibition of nitric oxide synthesis (21) have been suggested to explain the fast onset of action on symptoms reported even in short-term clinical trials (22). In contrast, the long-term effects of GS reported effects on cartilage metabolism are by stimulation of anabolic activities on proteoglycan synthesis (23, 24) and depression of catabolic activity, such as the effect of metalloproteases (24, 25). Clinical studies have shown that GS is fairly safe (22, 26, 27) and definitely far more safe than standard nonsteroidal anti-
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Figure 6. Radiolabeling of GS at pH 7 using 0.1 mg tin (A), as compared to 0.05 mg tin (B). Each point represents the mean value of three radiolabeling experiments with two measurements each.
Figure 7. Uptake of 99mTcGS at pH 7 using 0.1 mg tin vs 0.05 mg tin (A) and corresponding minimal washout of the tracer (0.1 mg tin) 3 h vs 24 h after uptake. Each point represents the mean value of three radiolabeling experiments with two measurements each.
inflammatory drugs (NSAIDs), especially concerning the gastrointestinal side effects (26, 27). Short-term studies indicate that a daily dose of 1.5 g GS for at least 7 days is sufficient to achieve significant improvement in OA symptoms, being as effective as a 400 mg daily dose of ibuprofen (27). Of course, the treatment with GS results in only mild side effects, if any,
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as compared to ibuprofen. In addition, although the improvements occurred more rapildy with ibuprofen, a more persistent benefit was achieved in patients receiving GS (28). Many studies report that GS is significantly more effective than placebo and even as effective as NSAIDs (17). Other studies show that GS has an even significantly greater influence on pain reduction than ibuprofen itself (18, 19). In accordance with these findings, a meta-analysis revealed a 40.2% benefit from GS treatment concerning global pain scores or Lequesne’s index as compared to placebo (28). Due its low molecular weight, radiolabeling of GS is difficult, as is the separation after radiolabeling from 99mTc, which also has low molecular weight; in contrast to other GAGs, e.g., chondroitin sulfate with much higher molecular weight. For this reason, it was very important to elaborate the optimal radiolabeling condition considering labeling efficiency, uptake of 99m TcGS in degenerated cartilage itself, and washout of the tracer with time to estimate the net uptake. It was important and interesting for us to evaluate the in vitro uptake of 99mTcGS by cartilage itself. We found a very high persistent cartilage uptake. The age-dependent uptake of 99m TcGS might reflect the changes in pathologically affected cartilage by dynamic stress during life span. In healthy cartilage, chondrocytes are able to maintain a dynamic equilibrium between anabolic and catabolic processes. However, with increasing age and degeneration this equilibrium is seriously unbalanced causing a dramatic decrease of matrix material, especially of the PG fraction. The major source of structural variation in PG is due to glycosaminoglycan (GAG) chains. They become extended with highly negative charged sulfate and carboxylate groups. With progressing age and degeneration, the number of main and side chains as well as their length and sulfation pattern varies (29). This results in a loss of negative charge density and can increase charge-mediated diffusion of GS to compensate this negative charge deficiency. This tendency was also observed in our uptake studies. Of course, this needs to be proven in a higher number of samples. Apparently, pH plays a very important role not only for the radiolabeling yield, but also with respect to later 99mTc GS uptake by cartilage. While the radiolabeling at pH 5.0 resulted in very high colloid formation and lower uptake of the tracer, in contrast, at pH 2.0 only few percent colloid were formed and the uptake was very high. The obtained saturation of the tracer at pH 2.0 was achieved when all the 99mTc GS was completely incorporated into the cartilage. This means that GS has a high tropism for cartilage. At the physiological condition of pH 7.0, the uptake is comparable to that at pH 2, but the washout is decreased, amounting only to a maximum of 4%. Only experiments undertaken at pH 7.0 are representative for in vivo conditions. The amount of Sn2+ influences the labeling procedure of GS. The reason for the highly different uptake depending on the labeling conditions is more complex. For each pH value and each Sn2+ amount, there seems to be an optimal reduction state of Tc7+ in pertechnetate to Tc+5,+4,+3 and the ability to form a more or less stable complex with GS. At pH 7.0, the higher amount of tin (0.1 mg versus 0.05 mg) as a reducing agent influences most likely the efficiency to create the most stable complex with high availability of negatively charged GAGmoieties of cartilage proteoglycans. Our study supports other findings (23, 24) indicating effects of GS on cartilage metabolism including stimulation of anabolic activities such as synthesis of proteglycans. Experimental evidence indicates that GS and CS have a particular effect on cartilage where they serve as substrates in the biosynthesis of glycosaminoglycans (GAGs) (30). GS stimulates chondrocytes to synthesize the cartilage GAGs and proteoglycans (PGs), but
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also inhibits lysosomal enzymes degrading cartilage (31). Animal and human studies have shown that GS after oral administration rapidly appears in the articular cartilage and is retained there for long time periods (32-35). These findings indicate that endogenous GS has an affinity for cartilage as a substrate for PG biosynthesis. Also, in this respect 99mTc GS could be used for scintigraphy of joints to monitor effects of therapy by pre- and post-therapy scintigraphy.
CONCLUSION Using 0.1 mg tin for radiolabeling, at pH 7 (G10 column for separation), we found the highest radiolabeling efficiency and specific activity of GS, a quite high uptake of the tracer, and only a very small washout, resulting in a high “net” uptake of tracer into the degenerated cartilage. Due to high incorporation into cartilage,99mTc-GS could become a promising tracer to target osteoarthritis, to detect cartilage degeneration, and also to monitor therapeutic effects.
ACKNOWLEDGMENT The authors would like to thank Rottapharm, Monza, Italy, for providing of glucosamine sulfate.
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