Rheumatoid arthritis (RA) is a systemic inflammatory disorder characterized by pain, swelling, and destruction of the affected joints. The exact mechanism of RA pathogenesis is not well understood. Recently, remarkable progress in the area of anticytokine therapy has provided an alternative and successful approach for therapeutic intervention in RA. However, methotrexate (MTX) still plays a central role in the treatment of RA, although administration of this agent sometimes causes serious side effects, such as interstitial pneumonia, renal failure, and myelosuppression. Intraarticular injection of MTX is thought to be safe compared with the systemic administration of this agent, although the clinical effectiveness in controlling synovitis in RA patients is controversial. Most studies have documented insufficient antiinflammatory effects (1–3). Mechanisms of resistance to MTX are considered to consist of 3 parts: decreased transport, impaired polyglutamylation, and increased dihydrofolate reductase enzyme activity (4–6). It has been reported that there is a significant correlation between reduced levels of folate carrier protein (one of the MTX transporters) at diagnosis and the histologic responses to preoperative MTX chemotherapy for osteosarcoma (6). From these observations, it is possible to conclude that the efficacy of MTX is limited by its transport into cells. Thus, we hypothesized that poor delivery of MTX into RA synovial cells would lead to poor clinical efficacy of intraarticular MTX injection therapy.
To facilitate uptake of MTX into synovial cells, we chose an ultrasound (US) treatment technique (sonoporation) with enhancement by the use of an echo-contrast microbubble agent. Previous reports have indicated that US exposure increases transfection efficiency of gene constructs, due to increased cell membrane porosity and acoustic cavitation (7, 8), which is enhanced with the use of microbubble agents. This is one of the best techniques for in vivo work and clinical applications because it is simple and noninvasive. Additionally, there are no viral components, although the success rate for induction is lower than that found with viral technologies in vitro and in vivo, as previously reported (9–12). Our findings in the present study indicate that US irradiation treatment with a microbubble agent enhances the antiinflammatory effect of intraarticular MTX injection in an ovalbumin (OVA)–induced arthritis model in rabbits.
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- MATERIALS AND METHODS
In vitro MTX induction into synovial cells. To confirm the promotion of MTX induction into RA synovial cells by US irradiation and to determine the optimum concentration of Optison, fluorescence-conjugated MTX was administered into RA synovial cells by sonoporation in vitro. In the absence of US, MTX was administered into a few synovial cells. With US exposure, the administration rate was increased to almost 5% for MTX alone, and ∼31% with 5% Optison (Figure 1). Further increases in the concentration of this reagent elevated the induction rate to a maximum of 48%. For the studies described here, 5% Optison was used in each of the experiments.
Figure 1. Fluorescence photomicrographs of synovial cells and tissue treated with Texas Red–conjugated methotrexate (MTX). Texas Red–conjugated MTX was administered to rheumatoid arthritis (RA) synovial cells by sonoporation in vitro alone (A) or with 5% Optison (B). Synovial tissue was collected from joints after injection of Texas Red–conjugated MTX with 5% Optison, with (C) or without (D) ultrasound (US) treatment. The upper parts of C and D show the joint cavity. Without Optison, almost 5% of cells were fluorescence positive (A). The ratio of fluorescence-positive cells was elevated to ∼31% with the addition of 5% Optison (B). In several layers of the synovial lining, with US treatment, fluorescence-positive cells were observed (C). Without US, only a weakly positive area was observed (D). (Original magnification × 200.)
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In vivo MTX induction into synovial cells. To confirm the promotion of MTX induction in vivo into RA synovial cells by US treatment, fluorescence-conjugated MTX was administered into RA synovial cells by sonoporation in vivo. In the presence of US, fluorescence-positive cells were observed in several synovial lining layers, while only a weakly positive area was observed in the absence of US (Figure 1). The Texas Red–positive area was restricted to synovial cells, and was not observed in other tissue such as tendon or muscle in either group.
MTX concentration. The MTX concentrations in synovial tissue were measured in the MTX+/US+ and MTX+/US− groups (8 samples each) 12 hours after the injection of MTX and Optison. The mean ± SD weight of the tissue from right knees (the MTX+/US+ group) was 454 ± 37.0 mg, while that from left knees (the MTX+/US− group) was 467 ± 44.4 mg. There was no significant difference between these groups. The mean ± SD MTX concentration in the MTX+/US+ group was estimated at 7.575 ± 1.590 × 10−7 mg/dl, while that in the MTX+/US− group was 2.875 ± 0.7889 × 10−7 mg/dl (P < 0.01).
Histologic findings. In the MTX+/US+ group, moderate inflammation (inflammatory cell infiltration, thickened synovial lining layers, and hypervascularity) was observed after 3 days, but it became less severe over a period from 7 to 56 days. Evidence of inflammation decreased in the synovial lining layer and the vascular network. In the MTX+/US− group, moderate inflammation similar to that seen in the MTX+/US+ group was observed after 3 days, continued until 28 days, and became less severe after 56 days.
After 7 days (Figure 2), in the MTX+/US+ group, minimal inflammatory cell infiltration and slight hypervascularity (minimal inflammation) were observed. In the MTX+/US− group, marked inflammatory cell infiltration, proliferating synovial lining layers (as many as 5), and proliferating blood vessels (moderate inflammation) were observed.
Figure 2. Day-7 histologic images of synovial tissue in the MTX+/US+ group and the MTX+/US− group. A and B, Hematoxylin and eosin staining. C and D, Immunohistochemical staining with anti–α-smooth muscle actin antibody. A and C, In the MTX+/US+ group (histologic score 3), minimal inflammatory cell infiltration, several synovial lining layers, no villus formation, and slight hypervascularity were observed. B and D, In the MTX+/US− group (histologic score 9), marked inflammatory cell infiltration, proliferating synovial lining layers (as many as 5 layers), no villus formation, and general occurrence of a large number of blood vessels were seen. See Figure 1 for definitions. (Original magnification × 40.)
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After 28 days (Figure 3), in the MTX+/US+ group, moderately proliferating synovial lining layers and focal proliferation of a small number of blood vessels (minimal inflammation) were observed. In the MTX+/US− group, marked inflammatory cell infiltration, proliferating synovial lining layers (>5 cell layers), and general proliferation of blood vessels (moderate inflammation) were observed.
Figure 3. Day-28 histologic images of synovial tissue in the MTX+/US+ group and the MTX+/US− group. A and B, Hematoxylin and eosin staining. C and D, Immunohistochemical staining with anti–α-smooth muscle actin antibody. A and C, In the MTX+/US+ group (histologic score 5), minimal inflammatory cell infiltration, a moderate number of synovial lining layers, no villus formation, and focal occurrence of a small number of blood vessels were seen. B and D, In the MTX+/US− group (histologic score 10), marked inflammatory cell infiltration with marked edema, >5 pronounced synovial lining layers, no villus formation, and a broadly distributed large number of blood vessels were seen. See Figure 1 for definitions. (Original magnification × 40.)
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After 56 days, both groups showed moderately proliferating synovial lining layers and focal proliferation of a small number of blood vessels (minimal inflammation). There was no significant difference between the 2 groups.
Articular cartilage was examined histologically for signs of damage caused by US exposure and/or MTX injection. A very slight decrease in the intensity of metachromatic staining (histologic scoring 0 or 1) in the surface area was observed in some samples of the MTX+/US+ and MTX+/US− groups on days 7 and 28.
Histologic scores. The mean ± SD histologic scores at 3, 7, 14, 28, and 56 days after treatment were 9.00 ± 0.31, 5.42 ± 0.30, 4.71 ± 0.18, 4.57 ± 0.37, and 5.57 ± 0.30, respectively, in the MTX+/US+ group, and 8.42 ± 0.20, 8.57 ± 0.37, 8.43 ± 0.20, 8.29 ± 0.36, and 5.71 ± 0.29 in the MTX+/US− group. The scores in the MTX+/US+ group and the MTX+/US− group were significantly different by two-way factorial ANOVA (P < 0.05). In addition, when we compared them at each time point using Student's paired t-test, the scores in the MTX+/US+ group at 7, 14, and 28 days were significantly better than those in the MTX+/US− group (Figure 4).
Figure 4. Histologic scores of synovial tissue from patients in the MTX+/US+ and MTX+/US− groups at each time point. Values are the mean and SD. P values were determined using two-way factorial analysis of variance. See Figure 1 for definitions.
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To confirm that the antiinflammatory effect was not due to US exposure, we injected the right knees with Optison only and exposed them to US (MTX−/US+ group). Scores in the MTX+/US+, MTX+/US−, MTX−/US+, and MTX−/US− groups were compared on days 7 and 28, and a significant difference was observed between the MTX+/US+ group and the other 3 groups (P < 0.05) (Figure 5).
Figure 5. Histologic scores of synovial tissue from patients in the MTX+/US+, MTX+/US−, MTX−/US+, and MTX−/US− groups 7 and 28 days after treatment. Values are the mean and SD. See Figure 1 for definitions.
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Findings of real-time PCR. Using real-time PCR, we examined the quantitative gene expression of IL-1β in synovial tissue obtained 7 and 28 days after US irradiation. The mean ± SD expression 7 and 28 days after treatment was 1.3 × 10−3 (1.5 × 10−4) and 3.3 × 10−3 (2.5 × 10−4), respectively, in the MTX+/US+ group, and 8.6 × 10−2 (9.0 × 10−3) and 8.4 × 10−2 (1.2 × 10−3) in the MTX+/US− group. There was a significant difference (P < 0.01) between the groups at each time point.
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- MATERIALS AND METHODS
Our results show that, in a rabbit arthritis model, microbubble-enhanced US treatment promotes uptake of MTX into synovial cells, which results in acceleration of antiinflammatory effects following intraarticular MTX injection. The antiinflammatory effect was confirmed by histologic scoring and expression of IL-1β mRNA in synovial tissue. The increased uptake of MTX into synovial cells was confirmed histologically by analysis of MTX concentration in synovial tissue and induction of Texas Red–conjugated MTX in vitro and in vivo. To confirm that the antiinflammatory effect was not due to US irradiation treatment with or without Optison, we injected Optison only (without MTX), and administered US to the right knees in the rabbits. Because there was no antiinflammatory effect, we concluded that neither microbubble injection with US exposure nor MTX injection has an antiinflammatory effect. However, the combination of MTX, microbubble injection, and US exposure was very effective. To our knowledge, this is the first report to describe the use of US in conjunction with antiinflammatory joint therapy in vivo. US gained attention through its use in gene therapy and tissue engineering. It is safe, minimally invasive, and can accommodate different therapeutic applications.
An antiinflammatory effect of this procedure (significantly reduced histologic inflammation and IL-1β gene expression in the synovial tissue) was observed from 7 to 28 days, but not at 56 days. All of the inflamed synovium of the joint cannot be covered by the use of a US probe applied at the skin surface. Indeed, histologic evaluation of inflammation of synovial tissue in the femorotibial joint, where US was not administered, revealed no difference from that in the control group (data not shown). Another explanation is that 56 days (exactly 66 days after immunization) may be enough to decrease the inflammation of synovial tissue in AIA naturally.
One hypothesis regarding the mechanism for the efficacy of this procedure is that the bioeffects are consequences of inertial cavitation, violent oscillations, and the collapse of bubbles in the surrounding fluid. The Optison stock concentration is ∼6.5 × 108/ml, and 5% Optison (3.25 × 107/ml) was injected into the joint. The microbubbles consist of hollow albumin filled with octafluoropropane. They are collapsed by the cavitation produced by US, and are considered to be eliminated quickly by distribution or phagocytosis. Physical and chemical phenomena related to inertial cavitation include microstreaming, shock waves, microjets, extremely high localized temperatures, pressures inside the bubbles, and generation of free radicals (16). If US causes promotion of induction through increased membrane porosity, as described above, it is reasonable to expect that there would be some limitations for transducible agents in terms of molecular size, 3-dimensional structure, and chemical compositions. In this study, we chose MTX (MW 454.45) as a transducive agent. Because we showed that Texas Red–conjugated MTX (MW 1,257.49) was also incorporated into cells in an in vitro experiment, molecules of this size should be readily incorporated into cells with this method. Uptake into cells of some genes or other pharmacologic agents that have an antiinflammatory effect can be promoted by microbubble-enhanced US exposure.
In vivo US and Optison conditions were selected based on in vitro data, and it is clear that there are significant differences between in vitro and in vivo conditions. Since the power of US is reduced by the long distance between the joint and the skin in vivo, it may be necessary to elevate the US power to a higher setting, provided there is no resultant heat production on the skin. In this study, output power (1–2W), duration (30 seconds to 2 minutes), and duty cycle (10–50%) were elevated.
Electroporation is also an effective MTX transduction technique. It has been widely used for transduction of genes and pharmacologic agents in vivo and in vitro. It does not require viral vector construction or virus preparation. Many reports describe electroporation as a technique for promoting electrochemotherapy through the uptake of MTX into cancer cells (17–19). We also showed that electrochemotherapy was effective in digital chondrosarcoma (20). However, the electrical fields created also affect normal tissue beyond the target site. Furthermore, cell anomalies or tissue damage after electroporation have often been observed. Thus, it is not an appropriate technique for the treatment of joints in RA patients. The encapsulation of MTX into cells with the use of a viral vector, recombinant polyomavirus–like particle, has been described (21), but this is a complex procedure and may not be suitable for clinical application.
MTX injection and US treatment can be used together in the clinic because of their low-risk safety profile. This technique may reduce synovitis in human arthritis and take the place of surgical synovectomy. It has been reported that surgical synovectomy of RA joints may offer short-term symptomatic relief but no retardation of the bone destruction or the disease process (22). While the benefits of our procedure might not match those of surgical excision of inflamed synovium, it is much less invasive than surgery and it can be performed frequently to obtain symptomatic relief. Further experiments are needed to determine whether this procedure alters the course of the disease. Furthermore, this technique can be applied in other inflammatory diseases.
For broader application in the clinic, there are some additional considerations that must be addressed. First, the intraarticular injection of MTX, which at high doses is sometimes used as an immunosuppressive agent, may cause adverse effects. The risk of iatrogenic infection warrants close attention. But MTX is reported to suppress production of superoxide and nitric oxide, and not to affect glycosaminoglycan synthesis of chondrocytes in vivo and in vitro (23, 24).
Second, US may cause adverse effects. It has been reported that US increases matrix synthesis and that it does not affect the viability or proliferation of chondrocytes (25, 26). The procedure reported here caused no harm to joint tissue apart from the proliferation zone in the synovium. However, the thick skin of the human joint may restrict the penetration of the ultrasonic vibration into synovial cells, which may cause a decrease in the uptake of MTX. The Sonitron is not approved for human use, but there is other equipment with greater power that is approved for human use in Japan (not for sonoporation). Longer exposure or higher power may cause local heat production. In such a case, a lower percentage setting of the duty cycle will help to prevent adverse effects. The optimal conditions necessary for the effective administration of US in humans need further investigation.
Third, the echo-contrast microbubbles may cause local or systemic side effects. These may include drug-induced allergic shock or joint pain because of high osmotic pressure. Moreover, another concern is that the overdosage of microbubbles or overexposure to ultrasonic waves may cause irreversible tissue injury. A minimum concentration of microbubbles would be desirable for clinical use. Optison has been used in the US and other countries for cardiac applications only, but it is not allowed for use in humans in some countries, such as Japan. Alternative clinical echo-contrast agents are now available in Japan. Levovist is one of these agents, although its gene transduction efficacy is reported to be inferior to that of Optison (27). Both are classified as second-generation microbubble agents. Optison consists of micrometer-sized (mean diameter 2–5 μm), denatured hollow albumin microspheres with a shell thickness of ∼15 nm. The microbubbles are filled with octafluoropropane. Levovist is a galactose-based, air-filled microbubble agent, 99% of which is smaller than 7 μm. Further studies are planned to compare the performance of these agents in vivo.
This method can be used to introduce not only MTX but also other agents, such as steroids and some genes, into synovial cells. The procedure can be used not only for intraarticular injection but also for a systemic approach with intravenous or oral administration of the therapeutic drug. Studies are under way to examine these approaches. This technique enables a very targeted application of reagents to the diseased tissue, thus enabling healthy tissue to be spared from treatment.