Preclinical evaluation of 99mTc labeled chondroitin sulfate for monitoring of cartilage degeneration in osteoarthritis

    Preclinical evaluation of 99m Tc labeled chondroitin sulfate for monitoring of cartilage degeneration in osteoarthritis Grazyna Sobal, Kavitha Velusamy, Siegfried Kosik, Johannes Menzel, Marcus Hacker, Maximilian Pagitz PII: DOI: Reference:

S0969-8051(16)30043-9 doi: 10.1016/j.nucmedbio.2016.02.009 NMB 7806

To appear in:

Nuclear Medicine and Biology

Received date: Accepted date:

16 February 2016 29 February 2016

Please cite this article as: Sobal Grazyna, Velusamy Kavitha, Kosik Siegfried, Menzel Johannes, Hacker Marcus, Pagitz Maximilian, Preclinical evaluation of 99m Tc labeled chondroitin sulfate for monitoring of cartilage degeneration in osteoarthritis, Nuclear Medicine and Biology (2016), doi: 10.1016/j.nucmedbio.2016.02.009

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ACCEPTED MANUSCRIPT Preclinical evaluation of 99mTc labeled chondroitin sulfate for monitoring of cartilage degeneration in osteoarthritis.

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Grazyna Sobal, Kavitha Velusamy, Siegfried Kosik*, Johannes Menzel§, Marcus Hacker, Maximilian Pagitz*

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Department of Nuclear Medicine, Medical University of Vienna, §Department of Immunology, Medical University of Vienna,*Department of Small Animals and Horses, University of Veterinary Medicine, Vienna, Austria

ABSTRACT

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Purpose: In previous in-vitro and ex-vivo studies we proved the specific uptake of 99mTc radiolabeled chondroitin sulfate (CS) in human articular cartilage. As a logical next step for the clinical use for imaging osteoarthritis we investigated in-vivo uptake of 99mTcCS in dogs.

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Procedures: The radiolabeling of CS Condrosulf (IBSA, Lugano, Switzerland) was performed using 25 mg of CS and 20-40 MBq/kg body weight of 99mTc by means of the tin method. In-vivo uptake of 99mTcCS was evaluated in dogs (n=12, castrated males, 4-9 years, with 15-51 kg body weight). 6 healthy dogs served as controls and 6 with clinical and radiological signs of osteoarthritis in the carpal, elbow, and tarsal joint were examined. The tracer was i.v. injected into the external cephalic vein. The uptake was monitored after 2, 4 ,6 and 24h in healthy and osteoarthritic dogs using a planar gamma camera by regional planar or whole body ventral and dorsal acquisition. For whole body scintigraphy animals were under general anaesthesia, for planar under sedation only. Results: In healthy control dogs we did not detect any specific uptake of 99m TcCS in the cartilage. In contrast, in the diseased dog suffering from osteoarthritis a significant, specific, persistent uptake between 4-6h in tarsal, carpal and cubital joints was documented. Median target (joint) to background (mid antebrachium) ratio (T/B) in the OA joints after 4, 6, and 24h was significantly higher than in healthy controls. Target to background ratio using soft tissue as a background (T/S) a similar significantly higher than in healthy controls

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In all osteoarthritic joints we found a significant positive correlation (r=0.8, n =20) between grade of disease (I-III) and T/B. When matching radiographic (X ray) changes in osteoarthritic joints (grade II and III) we found also a maximal uptake of 99mTcCS at the specific anatomical site of highest cartilage degeneration. None of the dogs experienced any side effects.

Keywords: Osteoarthritis scintigraphy

99m

Tc-chondroitin sulfate Chondrocytes Cartilage

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1. Introduction

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Conclusion: These results suggest that 99mTcCS might become a promising diagnostic tool for imaging osteoarthritis. More extensive and detailed examinations are required, however, before extending this methodology for application in humans.

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Chondroitin sulfate (CS), a complex glycosaminoglycan, is a major component of cartilage matrix proteoglycans (PG) which are progressively and irreversibly degraded in osteoarthritis (OA). OA represents the most frequent form of joint disorder in developed countries with over one-half of the people aged more than 65a showing radiographic changes in painful knees [1]. Eventually, PG loss also engenders damaging and disruption of the collagen network in the matrix. Interestingly, CS exhibits a number of biological functions such as activation of monocytes and B-cells [2], complex formation with fibronectin [3] and modulation of integrin function [4]. Several clinical studies also clearly demonstrated therapeutic effects of orally administered CS (Condrosulf, IBSA, Lugano, Switzerland) in OA patients such as reduction of pain as well as improvement of articular functions [5-6]. Imaging with a PG-specific ligand allowing quantification of PG loss in cartilage would be useful for the early detection of OA, because anatomic imaging by 3-T delayed gadolinium-enhanced MRI, while showing the spatial distribution of GAGs in cartilage, allows only indirect evaluation of PG content [8]. Due to the chondrotropic character of CS and its uncomplicated labeling with 99mTc [9] this GAG was proposed as a promising tracer for imaging and quantifying of cartilage degradation especially in early OA [10]. Thus, several

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studies have demonstrated dose-dependent uptake of CS by cartilage in vitro and in vivo [11-14]. Oral administration, however (in contrast to i.v. administration), resulted in partial degradation of CS [13-15]. More than 70 percent of orally administered radioactivity were absorbed and found in urine and tissues. After 24 h the labeled CS was present in higher amounts in liver, kidneys, intestine, synovial fluid and cartilage than in other tissues. This is an indication that CS is bioavailable at the joints after oral administration.

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For our preclinical study we decided to use a canine model for the following reasons: Spontaneous degenerative arthritis develops in dogs such as german shepherds, labrador retrievers and beagles [16-18]. Canine models offer advantages over rodent models because matrix metalloproteinases (MMP) and PG degradation products reflect to a higher degree those of human OA patients [19,20]. In addition, growth plates in dogs as in humans close with maturity unlike those of rats. Joint laxity in dogs engenders OA and spontaneous rupture of anterior cruciate ligaments. Joint degeneration and features of early OA have been observed in the canine „groove“ model as a consequence of damaged weight-bearing zones of the articular cartilage in the femoral condyles of the knee. In this model collagen is damaged and PG turnover is impaired after 10 weeks, resulting in a diminished PG content [21-23] and indicating a slow though steady progression of OA features. Interestingly, characteristic changes of OA, such as fibrillation of the articular surface and chondrocyte clustering indicating moderate cartilage destruction were noted in the tibial plateau, although this cartilage surface was not attacked by surgical intervention. In general, long-term changes in cartilage can be monitored more conveniently in larger animals than in rodents with non-invasive methods such as MRI or scintigraphic imaging. The purpose of the present study was to obtain the pharmacokinetics and whole-body distribution of i.v. administered 99mTc labeled CS (99mTcCS) in dogs with special emphasis on its chondrotropic quality, i.e. its cartilage targeting potential. This is a conditio sine qua non for using this tracer as a means to detect OA-associated cartilage degeneration and breakdown. 2. Materials and methods 2.1. Radiolabeling In previous in-vitro and ex-vivo studies we proved uptake of 99mTcCS in human articular cartilage [10,11]. As a logical next step for the clinical use of

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imaging OA we investigated uptake of the tracer in dogs afflicted with OA and in healthy control dogs. The radiolabeling of CS, as Condrosulf (IBSA, Lugano, Switzerland) was performed using 25 mg of CS and 20-40 MBq/kg body weight of 99mTc by means of the tin method [9].

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2.2. Quality Control

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Quality control of the tracer was performed using ITLC-SG chromatography and 0.2 M saline in 10% ethanol as solvent to detect colloid content. Aluminium oxide IB-F TLC-sheets and ethanol as solvent were used to estimate free pertechnetate. 2.3. Animals

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In-vivo uptake of 99mTcCS was evaluated in dogs (n=12, castrated males, 4-9 years, with 15-51 kg body weight). The evaluation was performed according to the project “GZ 68.205/0119-II/3b/2012“approved by the institutional ethics committee and the national authority according to § 8ff of Law for Animal Experiments, Tierversuchsgesetz – TVG.

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Six healthy dogs served as controls (C) and six with clinical and radiological signs of OA in the carpal, elbow, and tarsal joint were examined. The tracer was i.v. injected into the external cephalic vein. The uptake was monitored after 2, 4, 6 and 24h in healthy and 4, 6 and 24h in OA dogs using a planar gamma camera (Diacam, MiE GmbH, Germany) with low energy high resolution collimator and dedicated software (Scintron, MiE GmbH, Germany) by regional planar or whole body ventral and dorsal acquisition. For whole body scintigraphy animals were under general anaesthesia, for planar under sedation only. 2.4. Statistical analysis Statistical analysis was performed using Student’s T-test. A value of p<0.05 was considered as significant. 3. Results 3.1. Radiolabeling and quality control

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ACCEPTED MANUSCRIPT Labeling efficiency ranged 88.5-93.5%, specific activity of 99mTcCS was 8.5-8.9 mCi/µMol. The tracer was stable over a 6h period after labeling (95.494.90.91%, n=12 of the radiochemical purity).

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3.2. Tracer biodistribution

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The whole body biodistribution after 6h in the healthy and OA dogs (Fig. 1) showed tracer accumulation in both kidneys, spleen, liver, and stomach and cartilage (Table 1). When comparing tracer specificity by 99mTc injection in OA versus 99mTcCS we found that only 99mTc accumulates in the salivary and thyroid glands (Fig. 2). Thus, also the 99mTcCS tracer is stable in vivo under scintigraphy conditions and free 99mTc does not dissociate from the tracer. 3.3. Tracer uptake

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In healthy control dogs we did not detect any specific uptake of 99mTcCS in the cartilage. In contrast, in the diseased dogs suffering from OA a significant, specific, persistent uptake between 4-6h in tarsal, carpal and elbow joints was documented (Figs. 3-6). Median target (joint) to background (mid antebrachium or mid lower leg) ratio (T/B) in osteoarthritic joints after 4, 6, and 24h was 2.13, 2.08, and 2.41, (n=6) and 1.54, 1.31, and 1.02 in controls (n=6) i.e. significantly higher (p= 0.004, 0.007 and 0.004), (Table 2). Target to background ratio (soft tissue) (T/S) was 1.42, 2.38, and 1.91 in osteoarthritic and 0.98, 1.58 and 0.09 in control joints i.e. significantly higher (p=0.005, 0.025 and 0.005), respectively. Uptake in contralateral osteoarthritic joints using T/B and T/S versus controls was always significantly higher (p= 0.0035, p=0.0052), (Table 2). We observed a similar uptake pattern as in ex-vivo human arthroplasty tissues correlating with the grade of degeneration [11]. Uptake in contralateral OA joints (Figs. 4-6) using T/B and T/S versus control ipsilateral joints was also significantly higher (p= 0.0035, p=0.0052). In all investigated OA joints we found a significant positive correlation (r=0.8, n =49) between grade of disease (I-III) and T/B (Fig. 7). When matching radiographic (X ray) changes in OA joints (grade II and III) we found also a maximal uptake of 99mTcCS at the specific anatomical site of highest cartilage degeneration (Fig. 8). None of the dogs experienced any side effects. 4. Discussion

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Early OA is characterised by altered patterns of synthesis of extracellular matrix (ECM) - macromolecules which are, however, clinically silent in humans, so that unfortunately the extent of cartilage damage is generally diagnosed only at an advanced stage of the disease. Damage then is irreversible, patients become symptomatic (pain!) or exhibit radiographic changes such as osteophytes or joint space narrowing [24-26].

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Among the earliest biochemical alterations occurring in OA PG degradation is the most prominent. Therefore, these changes should be detected in vivo to allow early diagnosis, monitoring of the pathological process and initiate therapeutic intervention [27-28]. The most promising way to do so is to develop a cartilage targeting strategy relying on carriers to deliver therapeutic drugs or radioactive tracers selectively to cartilage tisues. In this context, our team and others have observed the chondrotropic qualities of labeled CS [11,14] qualifying this PG-GAG for cartilage imaging /purposes. Thus, cartilage (PG) was found to bind to negatively charged soluble 99mTcCS in vitro and in vivo.

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As an inverse function of the integrity of PG in OA joints, uptake in vitro [10,11] and in vivo of 99mTcCS was found in the present study to interact increasingly with degenerated cartilage as compared to intact cartilage. The hallmark of early pathological changes in the OA cartilage matrix is a reduction in negatively charged CS groups on the PG molecules, i.e. the fixed charge density (FCD) is lowered [11]. Positively charged groups on the collagen network are thus less effectively shielded and 99mTcCS can more easily diffuse into the cartilage and reach chondrocytes. In spite of the low mass of local chondrocytes the interaction of CS with these cells plays an additional role in CS uptake by cartilage as the affinity of CS-receptors was found to be high [10]. This effect might be over represented, however, in dog models because adult dogs have a cell density five times higher than that found in human cartilage [29]. For SPECT analysis of OA-dependent cartilage degeneration of lower limbs intravenous administration of 99mTc as labeled CS and as pertechnetate (control) was used only in two healthy human volunteers [14]. It was shown by these authors that after 2h radioactivity of 99mTcCS progressed increasingly to the knee joints of human healthy volunteer to a more significant degree than in case of the pertechnetate controls. However, in contrast to our studies in dogs, no studies with OA patients or OA animals for nuclear medicine diagnostics were undertaken so far.

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The canine knee joint (stifle) is similar to the human knee. Dogs are large enough to undergo arthroscopy and MRI as well as scintigraphy. These methods can therefore accurately assess long-term progression of OA in dogs. [30,31]. In the present study, we documented by scintigraphic analysis in diseased dogs a significant, persistent uptake of 99mTcCS between 4-6h after iv administration in tarsal, elbow and carpal joints. In contrast, we did not detect any or very low specific uptake of 99mTcCS in the cartilage in healthy dogs not afflicted by OA (Fig. 1,3). Ronca [14] et al. also compared i.v. and oral administration in rats using H-labeled CS. It was found by them that oral administration of CS results in a much more significant degree of CS breakdown to polysaccharides, oligosaccharides, monosaccharides and tritiated water than after iv administration. This fact could be accounted for by increasing the oral dosage of CS to five times that of the alterative application route. In this way, similar quantities of CS in blood, synovia and articular cartilage were obtained in both administration procedures. Irrespectively of the methodology used a high content of CS in the synovial fluid as well as in cartilage was detected as a consequence of the obvious cartilage «chondrotropism» of CS. In dogs, Palmieri et al. [32] also observed a rapid increase of serum radioactivity after oral administration, followed by a large plateau with a maximum at 28h. Tropism to joint cartilage was noted. Taken together, these results prove that in vivo CS is bioavailable and targets cartilage.

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There are other models of OA imaging using different agents. Such agents obviously bind with high affinity to the negatively charged PG molecules [33-36]. A significant decrease in uptake was found before the onset of radiologic abnormalities in the early stage of the disease. However, these agents are artificial constructs in contrast to our approach using endogenous CS as structural element of cartilage PG for imaging OA. It is important to underline that discrepancies between animal models of OA and the human disease exist, keeping in mind the late onset and slow progression of idiopathic human OA. The animal models show very early OA and severe chondrodysplasia [37]. On the other hand, spontaneous OA models, such as the dog model used in the present study, can be used to assess cartilage/tissue changes as OA develops. These changes can be compared with matched controls and with changes in human disease. In the long run, however,

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ACCEPTED MANUSCRIPT experiments with human volunteers using 99mTcCS as an imaging agent for early SPECT diagnosis of OA seem to be inevitable. 5. Conclusion

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In vivo imaging of cartilage degeneration in an animal model of OA using TcCS as tracer was demonstrated. CS uptake correlated in vitro in cartilage tissue of human origin with the degree of cartilage degeneration [11]. These results provide data in favour of imaging agents such as 99mTcCS, targeting cartilage as diagnostic tools in early OA as well as for monitoring therapy of advanced OA. 99mTcCS could also be helpful in the search for and developing of new drugs for treating OA, especially for evaluating the efficacy of diseasemodifying OA drugs (DMOAD) such as the CS itself, a symptomatic slowacting OA drug [38]. 99mTcCS seems to be a valuable specific radiotracer to image OA. It allows to differentiate between OA grades (I-III) and could be used to diagnose early OA. This could be particularly important for treatment of OA before an irreversible joint damage takes place.

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Acknowledgments

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We are grateful to IBSA (Lugano, Switzerland) for supplying the CS preparation Condrosulf.

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Fig. 1 Whole body biodistribution of 99mTcCS in OA versus healthy control. Visibly increased 99mTcCS uptake in both elbows and the left carpal joint in OA but no uptake in the healthy dogs.

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Fig. 2 Comparison of 99mTcCS uptake versus 99mTc only in salivary glands and thyroid. We see only 99mTc uptake in the salivary and thyroid glands but no definable 99mTcCS uptake in the salivary and thyroid glands.

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Fig. 3 99mTcCS uptake in OA beagle in elbow joint as compared to control with no uptake of tracer. Fig. 4 99mTcCS uptake in OA beagle in tarsal joint as compared to control with no/very low uptake of tracer. Fig. 5 99mTcCS uptake in OA rottweiler in left tarsal joint as compared to control with no uptake of tracer.

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Fig. 6 99mTcCS uptake in OA golden retriever in right elbow joint (grade III) as compared to left elbow joint (grade II) with much lower uptake of tracer.

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Fig. 7 Correlation of 99mTcCS uptake with clinical grade of disease I-III (n=20) by all investigated joints including controls (n=49).

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Fig. 8 An example of 99mTcCS uptake correlation by SPECT with X ray investigation. When matching radiographic (X ray) changes in OA joints from dog (grade III) we found a maximal uptake of 99mTcCS at the specific anatomical site of highest cartilage degeneration. Table 1 The whole body biodistribution after 4-6h in the healthy and OA dogs showed tracer accumulation in both kidneys, spleen, liver, and stomach and cartilage. Table 2 Tracer biodistribution calculated as ratio: median target (joint) to background (mid antebrachium) T/M or target to background ratio using soft tissue as a background (T/S) in the osteoarthritis versus healthy joints.

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Healthy controls- vs O.A. animals (6h) in % I.D. Controls Patients Thyroid Urinary Bladder 19,0 26,4 Kidney left 2,1 2,8 Kidney right 2,1 2,3 Liver 18,2 13,6 Spleen 1,4 1,1 Target 0,2

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Table 1

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Table 2

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