Autologous conditioned serum was developed in the mid 1990s as an expeditious, practical, and relatively inexpensive means of generating the interleukin-1 receptor antagonist, a naturally occurring inhibitor of the cytokine interleukin-1. The latter is thought to be an important mediator of inflammation, pain, and tissue destruction in musculoskeletal conditions. ACS has been widely and successfully used in the local treatment of human and equine osteoarthritis and radicular compression; it has also shown promise in treating tendinopathies, muscle injuries, and tunnel widening after reconstruction of the anterior cruciate ligament. Experience suggests that autologous conditioned serum is safe and effective.
Key points
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Autologous conditioned serum is prepared by the incubation of whole blood with surface-treated glass beads within a special syringe.
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During incubation, the serum is enriched in products synthesized and released by peripheral blood platelets and leukocytes including, but not limited to, the interleukin-1 receptor antagonist.
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Randomized, controlled trials find that locally injected autologous conditioned serum is effective in treating osteoarthritis, radicular compression, and tunnel widening after reconstruction of the anterior cruciate ligament.
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Additional studies suggest utility in treating tendinopathies and muscle injuries.
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Further studies are required to confirm clinical effectiveness in specific indications, to determine the composition of autologous conditioned serum, to determine its mode of action, to understand individual responses to therapy, and to explore potential synergies with other therapeutic agents.
Introduction
Autologous conditioned serum (ACS) is an autologous blood product enriched in the interleukin-1 receptor antagonist (IL-1Ra), a naturally occurring inhibitor of interleukin-1 (IL-1). ACS is administered locally to treat conditions in which IL-1 is thought to be an important agent of pathologic conditions. Several reviews have been written on this topic.
IL-1Ra has been produced in Escherichia coli as the recombinant molecule anakinra, marketed as Kineret. Anakinra, in combination with methotrexate, is approved by the US Food and Drug Administration for the treatment of rheumatoid arthritis (RA), self-administered subcutaneously at a daily dose of 100 mg. However, the therapeutic efficacy of anakinra in RA has generally been disappointing, and it is not widely used in this context. Clinical responses in sepsis have also been weak. However, systemic anakinra is effective in systemic juvenile idiopathic arthritis and a variety of rare autoinflammatory disorders; it is also of benefit in gout and pseudogout.
There is considerable interest in using anakinra intra-articularly in the treatment of osteoarthritis (OA) and injured joints. An initial, open-label clinical trial in patients with OA of the knee provided highly encouraging results with sustained clinical improvement after intra-articular injection of 100 mg of anakinra. However, a subsequent multicenter, randomized controlled trial (RCT) showed no sustained benefit of intra-articular Anakinra. Nevertheless, there was transient improvement, observed at day 4, in certain parameters, notably pain. The temporary nature of the beneficial effects probably reflects the rapidity with which proteins are removed from joints. An additional clinical trial administered anakinra intra-articularly to patients after rupture of the anterior cruciate ligament (ACL) and again found improvement in certain parameters during the 2-week study period. In a further small, uncontrolled, unblinded study of 6 patients with persistent postsurgical knee effusions, a single 200-mg injection of anakinra decreased pain and swelling, improved range of motion, and permitted return to sporting activities.
There is, thus, optimism that IL-1Ra could prove efficacious in injured and arthritic joints if there were a way to maintain therapeutic concentrations intra-articularly. Gene delivery provides one technology for achieving this, and proof of principle has been established in animal models and human clinical trials for RA. Genetic delivery of IL-1Ra into human knee joints with OA is at an advanced preclinical stage of development.
Introduction
Autologous conditioned serum (ACS) is an autologous blood product enriched in the interleukin-1 receptor antagonist (IL-1Ra), a naturally occurring inhibitor of interleukin-1 (IL-1). ACS is administered locally to treat conditions in which IL-1 is thought to be an important agent of pathologic conditions. Several reviews have been written on this topic.
IL-1Ra has been produced in Escherichia coli as the recombinant molecule anakinra, marketed as Kineret. Anakinra, in combination with methotrexate, is approved by the US Food and Drug Administration for the treatment of rheumatoid arthritis (RA), self-administered subcutaneously at a daily dose of 100 mg. However, the therapeutic efficacy of anakinra in RA has generally been disappointing, and it is not widely used in this context. Clinical responses in sepsis have also been weak. However, systemic anakinra is effective in systemic juvenile idiopathic arthritis and a variety of rare autoinflammatory disorders; it is also of benefit in gout and pseudogout.
There is considerable interest in using anakinra intra-articularly in the treatment of osteoarthritis (OA) and injured joints. An initial, open-label clinical trial in patients with OA of the knee provided highly encouraging results with sustained clinical improvement after intra-articular injection of 100 mg of anakinra. However, a subsequent multicenter, randomized controlled trial (RCT) showed no sustained benefit of intra-articular Anakinra. Nevertheless, there was transient improvement, observed at day 4, in certain parameters, notably pain. The temporary nature of the beneficial effects probably reflects the rapidity with which proteins are removed from joints. An additional clinical trial administered anakinra intra-articularly to patients after rupture of the anterior cruciate ligament (ACL) and again found improvement in certain parameters during the 2-week study period. In a further small, uncontrolled, unblinded study of 6 patients with persistent postsurgical knee effusions, a single 200-mg injection of anakinra decreased pain and swelling, improved range of motion, and permitted return to sporting activities.
There is, thus, optimism that IL-1Ra could prove efficacious in injured and arthritic joints if there were a way to maintain therapeutic concentrations intra-articularly. Gene delivery provides one technology for achieving this, and proof of principle has been established in animal models and human clinical trials for RA. Genetic delivery of IL-1Ra into human knee joints with OA is at an advanced preclinical stage of development.
Autologous conditioned serum
Background
Wehling and colleagues developed ACS in the mid-1990s as an expeditious, practical, and relatively inexpensive means of generating IL-1Ra for local, therapeutic application in musculoskeletal diseases. ACS is based on studies that found that macrophages and monocytes are major endogenous sources of IL-1Ra. Production of IL-1Ra can be enhanced by a variety of stimuli, including adhesion to certain surfaces. Based on this information, Meijer and colleagues developed a method for stimulating IL-1Ra synthesis by whole human blood. According to their method, peripheral blood is drawn into a syringe containing treated glass beads to which blood monocytes and other adherent cells have the opportunity to attach. The syringe and its contents are then incubated at 37° for several hours, during which time platelets degranulate and mononuclear cells synthesize and secrete IL-1Ra along with a variety of additional anti-inflammatory products. During this period, synthesis of the inflammatory cytokines IL-1β and tumor necrosis factor-α (TNF-α) does not increase greatly. After incubation, the ACS is recovered and sterilized by filtration. ACS is then injected locally into sites of injury or disease.
Stimulation of blood cells by the glass beads is not specific to IL-1Ra, and ACS contains a variety of growth factors and cytokines ( Table 1 ). Indeed, it has not been formally demonstrated that IL-1Ra is responsible for the therapeutic properties of ACS. The composition of ACS shown in Table 1 is in rough agreement with that reported by Darabos and colleagues and Rutgers and colleagues ; the only major discrepancy is the approximately 10-fold higher TNF-α concentrations noted by the latter investigators. These authors also reported the presence of osteoprotegerin, interferon-γ, and oncostatin M. The data in Table 1 , including the TNF-α levels, are in agreement with the data of Wright-Carpenter and colleagues who also noted the presence of IL-7. Darabos and colleagues detected epidermal growth factor in ACS.
| Cytokine | N | Basal Concentration | Concentration in ACS |
|---|---|---|---|
| IL-1Ra | 224 | 236 | 2015 |
| IL-1β | 224 | UD | 7.9 |
| IL-6 | 200 | UD | 28.7 |
| TNF-α | 92 | UD | 10.1 |
| IL-10 | 92 | UD | 33.4 |
| FGF-2 | 92 | 14.6 | 26.6 |
| VEGF | 92 | 61 | 508.6 |
| HGF | 92 | 431 | 1339 |
| IGF-1 | 92 | 86,000 | 117,209 |
| PDGF AB | 92 | 205 | 39,026 |
| TGF-β | 80 | 1165 | 97,939 |
Clinical Development
ACS was first used clinically in 1997. Beginning in 2001, ACS was manufactured as Orthokine in a Good Manufacturing Process (GMP) facility. Participating physicians were provided with syringes, known as Orthokine syringes , containing treated glass beads. The beads are 3.5 mm in diameter, comprise borosilicate glass of maximum hydrolytic resistance, and are polished according to a proprietary process. Blood was drawn into Orthokine syringes and shipped to the GMP facility for the production of Orthokine. Typically, 6 injections of Orthokine, each 2 mL, were injected into knee joints over a period of 3 to 6 weeks. For epidural use, 3 injections, each 1 mL, were injected over a 3-week period. The number of injections and their timing were determined empirically.
This means of distribution proved cumbersome, time consuming, and geographically limiting and could not be performed in all markets because of regulatory hurdles. For these reasons, the system was changed in 2004 to one in which the physician was provided with a smaller, disposable Orthokine syringe and an incubator so that ACS could be produced locally in the individual clinic or physician’s office. Although the original 50-mL syringe required a 24-hour incubation at 37° for optimal production of ACS, the newer 10-mL syringe allows a shorter incubation time of 6 to 9 hours.
Clinical Experience with Autologous Conditioned Serum —Intra-Articular Delivery
Baltzer and colleagues published the first clinical use of ACS, describing treatment of 1000 patients with OA of the knee and Kellgren Lawrence (KL) scores of 1 to 3 ( Table 2 ). In this prospective, nonrandomized, uncontrolled study, Western Ontario MacMaster Universities (WOMAC) arthritis scores improved by ≥50% after 3 months in ≥70% of patients receiving intra-articular ACS. Each subcategory of the WOMAC scoring system improved, and improvements were maintained for 3.5 years in ≥35% of patients. There were no infections or allergic reactions to injection of ACS; 3.5% of patients reported joint swelling or pain immediately after injection, but these symptoms subsided spontaneously over the course of a few hours. Subsequently, there have been 2 large, double-blind RCTs evaluating the efficacy of ACS for the treatment of OA of the knee.
| Indication | No of Patients | Study Design; Entry Criteria | Outcome Measures | Main Findings | Reference |
|---|---|---|---|---|---|
| Human knee OA | 1000 | Retrospective, uncontrolled, unblinded Established OA KL grades 1–3 | WOMAC | WOMAC scores improved ≥50% in ≥70% of patients Improvement sustained for 3.5 y in ≥35% patients | |
| Human knee OA | 376 | Randomized, placebo controlled ACS compared with saline and HA Established OA KL grades 2–3 Follow-up: 7 wk, 13 wk, 6 mo, and 2 y | WOMAC VAS SF-8 HRQL GPA | Sustained, statistically significant improvement in all outcome measures compared with saline and HA No severe adverse events | |
| Human knee OA | 167 | Randomized, multicenter, placebo controlled ACS compared with saline KL grades 1–3 Follow-up: 3, 6, 9, 12 mo | WOMAC KOOS VAS KSCRS | Statistically significant improvements in KOOS symptoms and KOOS sport compared with saline No difference in WOMAC Trend toward improvement in other parameters Two severe adverse events, one of which attributed to ACS | |
| Human knee OA | 20 | Patients who received placebo in the above study were given ACS | WOMAC KOOS VAS KSCRS | Improvement to the same degree as previously seen when receiving placebo | |
| Human knee OA | 118 | Uncontrolled study; subjects received ACS (2 mL/wk for 4 wk) with physiotherapy Follow-up: 2 y Painful OA of knee, KL grades 1–4 | WOMAC Pain | Rapid and sustained improvement in pain and WOMAC scores Only 1 patient progressed to total knee joint replacement | |
| Human knee OA | 30 | Uncontrolled study; low-dose ACS (1 mL/wk for 3 wk) Follow-up: 3 mo Painful OA of knee, KL grades 1–3 | WOMAC | Rapid and progressive improvement in WOMAC scores Maintained for at least 3 mo | |
| Human hip OA | 119 (150 hips) | Retrospective, nonrandomized, no placebo KL grades 2–4 ACS ± cortisone ± anakinra Follow-up: 14 mo | VAS | Statistically significant improvement in VAS in all groups No adverse events | |
| Tunnel widening after single bundle ACL reconstruction (human) | 62 | Randomized, double blind, placebo controlled Isolated ACL rupture Outerbridge up to grade 2 Surgical reconstruction of ACL Follow-up: 6 and 12 mo | CT WOMAC IKDC 2000 | Statistically significant reduction in tunnel widening Lower effusion at 6 mo Improved range of motion Improved WOMAC stiffness No serious adverse events | |
| Tunnel widening after double bundle ACL reconstruction (human) | 62 | Randomized, double blind, placebo controlled Isolated ACL rupture Outerbridge up to grade 2 Double-bundle surgical reconstruction of ACL Follow-up: 6 and 12 mo | CT Lysholm IKDC 2000 | Statistically significant reduction in tunnel widening Better Lysholm and IKDC 2000 scores No serious adverse events | |
| Equine OA | 262 | Nonrandomized; no control Lameness unresponsive to intra-articular glucocorticoid or HA Follow-up: 6 and 12 wk | Lameness | Elimination or improvement in lameness in 221 horses at 6 wk No lameness in 178 horses at 12 wk No adverse events | |
| Equine OA | 20 | Nonrandomized Lameness unresponsive to intra-articular PSGAG or HA Follow-up: 3 mo | Lameness | Full activity restored in 10/10 PSGAG failures and 7/10 HA failures | |
| Equine OA | 54 | 27 lame horses in each group received either ACS or a mixture of HA and betamethasone Follow-up: 6 mo | Lameness | ACS produced a stronger reduction in lameness | (abstract) |
| Canine OA | 11 dogs 15 joints | OA confirmed by clinical examination and radiology 2–4 injections of 1.4 mL ACS Observational study; no control group | Lameness | Clinical signs improved within 1 wk of the first or second injection Decrease in lameness in all dogs | (abstract) |
The first of these, also by Baltzer and colleagues, compared ACS (6 injections, twice per week, 2 mL per injection) with standard of care (hyaluronic acid; HA) and placebo (saline). Subjects in the HA group received 1 injection (2 mL) per week for 3 weeks of a 1% solution of HA with a molecular weight of 1.4 × 10 6 Da (HYA-Ject Ormed, Freiburg, Germany). Entry criteria included age greater than 30 years, established OA of the knee, a KL score of 2 to 3, a pain score of at least 50 mm on a 100-mm visual analog scale (VAS), and a willingness to discontinue nonsteroidal anti-inflammatory drugs (NSAIDs) and other analgesics for 6 months. Outcome measures included VAS, WOMAC scores, Short-Form 8 Health-Related Quality-of-Life (SF-8 HRQL) survey, and the global patient assessment of treatment efficacy. Outcomes were measured at 7 weeks, 13 weeks, and 6 months; traceable patients were recalled after 2 years and reassessed in an observational, prospective, cohort study with a new, blinded observer.
A total of 376 patients were randomly assigned to 1 of the 3 study groups; 345 completed the initial, 6-month study, and 310 were traceable after 2 years. Patients receiving ACS experienced considerable, statistically significant improvement, beyond that obtained with placebo, in all outcome measures. Remarkably, these improvements were maintained for at least 2 years ( Fig. 1 ). HA, in contrast, was no more effective than placebo. ACS produced no severe adverse events. At 3 months, 71% of subjects had greater than 50% reduction in VAS pain score; at 6 months, this response was 67%.
