WO2014046631A1 - CONTROLED DRUG DELIVERY SYSTEMS FOR ANTI-TNF-α - Google Patents

CONTROLED DRUG DELIVERY SYSTEMS FOR ANTI-TNF-α Download PDF

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WO2014046631A1
WO2014046631A1 PCT/TR2012/000148 TR2012000148W WO2014046631A1 WO 2014046631 A1 WO2014046631 A1 WO 2014046631A1 TR 2012000148 W TR2012000148 W TR 2012000148W WO 2014046631 A1 WO2014046631 A1 WO 2014046631A1
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pcl
anti
peg
microspheres
intra
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PCT/TR2012/000148
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French (fr)
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Aysen TEZCANER
Dilek Keskin
Seza OZEN
Bulent ATILLA
Ali USANMAZ
Cetin KOCAEFE
Ozge ERDEMLI
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Tezcaner Aysen
Dilek Keskin
Ozen Seza
Atilla Bulent
Usanmaz Ali
Kocaefe Cetin
Erdemli Ozge
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005Wall or coating material
    • A61K9/5021Organic macromolecular compounds
    • A61K9/5031Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241Tumor Necrosis Factors

Abstract

The present invention relates to intra-articular controlled delivery of protein based anti-TNFα drugs with polymeric microcarrier systems for the treatment of juvenile and adult Rheumatoid Arthritis. Presently, anti-TNFα drugs used in the treatment of rheumatic diseases have indications for "systemic applications" (intramuscular or subcutaneous injections).

Description

CONTROLED DRUG DELIVERY SYSTEMS FOR ANTI-TNFa

Field of the Invention

The present invention relates to intra-articular controlled delivery of protein based anti-TNFa drugs with polymeric microcarrier systems for the treatment of juvenile and adult Rheumatoid Arthritis. Presently, anti-TNFa drugs used in the treatment of rheumatic diseases have indications for "systemic applications" (intramuscular or subcutaneous injections).

Background of the Invention

Inflammatory rheumatic diseases are characterized by inflammation of synovial tissues, often leading to destruction of joint cartilage and bone. Rheumatoid arthritis (RA), a systemic autoimmune disease, is the most common chronic inflammatory and is characterized by pain, swelling, stiffness, and inflammation of synovial membrane (synovitis) which often results in joint destruction and disability (CHOY, 2001,FIRESTEIN, 2003,DOAN, 2005,BASRA, 2012). Ankylosing spondylitis (AS) is a common inflammatory rheumatic disease that affects the spine (vertebral joints), hip and sacrum and sacroiliac joints, causing chronic inflammatory back pain and stiffness in this area (LOKWANI, 2011 Psoriatic arthritis (PsA) is an inflammatory rheumatic disorder associated with psoriasis in which scaly red and white patches develop on the skin (CANTINI, 2010). Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease of childhood (HASHKES, 2005) and begins before the age of 16 years and persists for at least 6 weeks (WEISS, 2007). Subtypes of JIA are oligoarticular JIA (50%-60%), polyarticular JIA (30-35%), systemic-onset JIA (10-20%), juvenile psoriatic arthritis (2-15%) and enthesitis-related arthritis (1-7%) (WEISS, 2007). Oligoarticular JIA affects five or fewer joints during the first 6 months, whereas polyarticular JIA affects five or more joints within the first 6 months. Systemic-onset JIA is the most severe type of the disease. Beside joints internal organs are also affected in this disease. Juvenile psoriatic arthritis is an asymmetric arthritis that often affects the knees and ankles and the small joints of the hands and feet. Enthesitis-related arthritis subtype includes patients with juvenile ankylosing spondylitis and arthritis associated with inflammatory bowel disease.

0.5-1% of the population is affected by RA. Its etiology is unknown but genetic susceptibility, environmental factors (smoking) or infectious agents are suggested to play a role in its etiology (SOMMER, 2005). Although pathophysiology of JIA and RA are not yet fully understood, cellular hyperplasia, pronounced angiogenesis, changes in the expression of cell-surface adhesion molecules, proteinases, proteinase inhibitors, an influx of inflammatory leucocytes and many cytokines are shown in inflamed synovium. Communication between different inflammatory cells, especially macrophages and T lymphocytes, and resident cells in the joint is important for the development of RA and JIA. These cells communicate via network of cytokines, some of which exert pro-inflammatory actions and others provide anti-inflammatory or immunoregulatory effects. Pro-inflammatory cytokines particularly interleukin-1 (IL-1), tumor necrosis factor alpha (TNFa), interleukin-6 (IL-6) and interleukin-17 (IL-17), and interleukin-18 (IL-18) and other cytokines such as interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) are all produced by the synovial membrane in RA (FELDMANN, 1996,AREND, 2001).

The main objectives targeted in the treatment of RA and JIA are reduction of pain, control of inflammation and prevention of joint destruction. After the discovery of the pathogenic roles of pro-inflammatory cytokines secreted by synovial membrane in RA formation, biologic therapies, including targeting of those cytokines have been developed (FELDMANN, 1996,DOAN, 2005,FAN, 2007). Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFa) play major roles in the pathogenesis of RA. Therefore, many studies are focused on the development of drugs that target these two cytokines. Clinically, soluble TNFa receptor (etanercept) and anti-TNF monoclonal antibodies (infliximab and adalimumab) have been used for treatment of JIA and RA by subcutaneous or intravenous injection. Clinical trials with TNFa blocking agents show high efficacy in RA patients who have failed traditional non-biologic disease modifying anti-rheumatic drugs (DMARDs)(DOAN, 2005,FAN, 2007). Blocking TNFa significantly reduced the production of other pro-inflammatory cytokines such as IL-1, IL-6, IL-8 and GM-CSF in cultures of synovial cells from patients with RA (CHOY, 2001). Therefore, anti-TNFa drug administration therapy shows more extensive impact against inflammation than therapies against other proinflammatory cytokines.

Anti-TNFa drug therapy has now become a standard therapy for RA patients who fail to respond to disease modifying anti-rheumatic drugs since 2000. Five anti-TNFa drugs such as infliximab, etanercept, adalimumab, golimumab and certolizumab pegol have been widely used in the treatment of JIA and RA and they differ in composition, mechanism of action and pharmacokinetics. All of these anti-TNFa drugs inhibit the inflammatory cascade by binding TNFa to avoid triggering the events that lead to pain, inflammation, and cartilage and bone destruction (DOAN, 2005). Infliximab, a recombinant human murine chimeric immunoglobulin- Gl (IgGl) antibody, is Food and Drug Administration (FDA) approved for PsA, AS and RA. It is given by infusion at 0, 2, and 6 weeks then 8 weekly for RA (at dose 3 mg/kg) whilst for AS and PsA then 6-8 weekly (at dose 5 mg/kg). Etanercept, a dimeric fusion protein consisting of the

5 extracellular ligand-binding portion of the human 75 kDa TNFa receptor linked to the Fc portion of the human IgGl, is approved FDA approved for RA, polyarticular JIA, PsA and juvenile and adult AS. It is given as a subcutaneous injection either 50 mg once weekly or 25 mg twice weekly (3-4 days apart) for RA, AS, PsA and polyarticular JIA (0.8 mg/kg to maximum 25mg twice weekly dose). Adalimumab, a human-derived recombinant IgGl monoclonal antibody, is

L0 FDA approved for RA, polyarticular JIA, PsA and AS. It is given as a subcutaneous injection of 40 mg fortnightly for RA, AS and PsA and in polyarticular JIA the dose is weight dependent; for patients less than 30 kg the dose is 20 mg subcutaneously fortnightly; for those who weigh 30 kg or more the dose is 40 mg subcutaneously fortnightly. Golimumab, human IgGlK monoclonal antibody, is FDA approved for RA, PsA and AS. It is given as a subcutaneous injection of 50 mg

L5 on the same date each month for RA, AS, and PsA. Certolizumab pegol, a pegylated Fab-9 fragment of a humanized anti-TNF-a antibody, is FDA approved for RA. It is given as a subcutaneous injection of 2 x 200 mg at weeks 0, 2, 4, followed by a maintenance dose of 200 mg every 2 weeks.

The general principle is to treat patients with RA and JIA with systemic drugs along with local to injections to the inflamed joints for more targeted therapy. Systemic application of anti-TNFa drugs at frequent intervals can cause patient discomfort by creating pain and possible side effects. Therefore, anti-TNFa drugs are used for patients who fail to respond to conventional medicines. Additionally, systemic application of these drugs is not preferred in oligoarticular JIA treatment. Systemically administered conventional treatments such as methotrexate and !5 subcutaneous administration of anti-TNFa drugs could be less effective in the treatment of some local inflammations. Joint damage after a less effective treatment might limit patient's life quality and expensive orthopedic operations could be needed in future. For local treatment of the joint the current practice is to use intra-articular steroids to control local joint disease. Furthermore, local joint injections are the main treatment for oligoarticular JIA, which is the 10 most common form of JIA. Therefore, an effective local treatment approach to suppress the inflammation at defined joints is needed. Combination of conventional treatment of JIA, RA and AS with a more effective local treatment will be beneficial when considering of the cost and side effects of systemic application of anti- TNFa drugs. Recently, a small number of preliminary clinical studies have tested the efficacy of the existing formulations of anti-TNFa drugs such as as etanercept, infliximab and adalimumab for intra-articular use in the RA patients (NIKAS, 2004,CONTI, 2005,BLIDDAL, 2006a,BLIDDAL, 2006b,BOESEN, 2008,ROUX, 2008,BOUYSSET, 2009). However, they were small scale double- blind studies, open-label and case reports conducted with a small number of patients and they have only used the existing formulations. Only systemically administered form of anti-TNFa drugs was applied intra-articularly to the patients in these preliminary clinical studies and intra- articular administration dose was as high as subcutaneous administration dose to increase duration of drug activity. In some preliminary clinical studies following the intra-articular injection, improvements in the patient's clinical findings (i.e., reduction in joint stiffness and swelling, unconstraint movements, etc.) were observed for a short period (BLIDDAL, 2006a, BLIDDAL, 2006b,ROUX, 2008). However, in some studies no differences were observed between intra-articularly etanercept administrated patients and placebo group (buffer injected into joints of patients) (BOESEN, 2008). Therefore, these preliminary studies do not give conclusive information about the reliability and effectiveness of this treatment approach. Effects of intra-articular administration of anti-TNFa drugs are short and inadequate. Therefore, controlled drug delivery systems are needed to suppress the inflammation effectively and to improve the potential for clinical use of anti-TNFa drugs. Additionally, dose of drug loading as well as size of this controlled drug delivery system must be limited into the joint of children. Studies related with the development and optimization of various drug delivery systems are needed to improve the potential for clinical use of anti-TNFa drugs and reduce the adverse effects arising from the use of high doses. These objectives were provided by the developed drug delivery systems presented in this invention. Another important aspec of local therapy is the need for prolonged suppression of inflammation. The joint disease of RA and JIA is characterized by chronic inflammation and thus an ongoing anti-inflammatory effect is needed for effective termination of the local disease. Thus one of our main aims was to have a prolonged effect of the drug with a sustained release.

Various natural and synthetic degradable polymeric devices have been developed for controlled drug delivery to eliminate adverse effects, increase therapeutic efficacy of the drug by delivering the drug effectively to the target tissue (EDLUND, 2002). Biodegradable polymeric biomaterials can provide a sustained drug release at therapeutic dosage and activity of the drug in the body environment can be maintained within these systems for a long time. Poly(£-caprolactone) (PCL), a synthetic aliphatic polyester, had been approved by the U.S. FDA for human clinical use

5 in vivo and it has been frequently used in studies related with controlled drug delivery applications due to its biodegradability and biocompatibility (SI HA, 2004b,LUCIANI, 2008,KARATAS, 2009,ZHANG, 2009). PCL has been also used to develop controlled peptide and protein delivery systems (LIN, 2001,SINHA, 2004b,COCCOLI, 2008). Some properties of PCL such as high permeability to many drugs, ability to be fully excreted from the body once

LO bioresorbed and excellent biocompatibility make PCL suitable for controlled drug delivery (WOODRUFF, 2010). Ability of PCL to form compatible blends with other polymers can affect the degradation kinetics which in turn can be tailored to fulfill desired release profiles (WOODRUFF, 2010). Degradation of PCL homopolymer is very slow as compared to other polyesters such as poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) making it more suitable for long

L5 term delivery systems and slow degradation of PCL provide an advantage with a negligible tendency to generate an acidic environment during degradation as compared to PLA and PLGA (WOODRUFF, 2010). However, its slow degradation kinetics in vivo can limit its application when used as a versatile matrix material for a drug delivery system. Introduction of hydrophilic poly(ethylene glycol) (PEG) blocks into PCL chains is a mean to enhance their hydrophilicity, io biodegradability and mechanical properties as compared with the parent homopolymer and much wider application field for a drug delivery can be obtained (WEI, 2009). Poly(ethylene glycol) (PEG) a hydrophilic, non-immunogenic, non-antigenic, U.S. FDA approved polymer, has been widely used in biomedical field. PEG is the gold standard for stealth polymers in the field of polymer based drug delivery for prevention of protein absorption and improvement of

:5 biocompatibility of the blood contacting compound (KNOP, 2010). In recent years, modification of drugs and drug delivery systems by attaching one or more PEG chains covalently has been widely used in drug delivery technology (KNOP, 2010). Fabrication of microparticles with PEGylated surfaces can be prepared by addition of PEG and PEG-derivatives during spray-drying or emulsion-based solvent evaporation techniques, copolymerization of PEG with other matrix o forming polymers, and formation of microparticles from PEG block or random copolymers (WATTENDORF, 2008). PEG and hydrophobic biodegradable polymer, such as PCL, poly(l-lactic acid), poly(d, l-lactic acid) and PLGA triblock and diblock co-polymers are used to produce PEGylated nanospheres and microspheres for drug delivery (SINHA, 2004a). In literature, PEG- PCL copolymers were used to prepare serum albumin, human basic fibroblast growth factor (bFGF) loaded nanospheres and microspheres (ZHOU, 2003,KIM, 2006,GOU, 2008a,GOU, 2008b,WEI, 2009). Micelle, nanoparticule and microsphere forms of PEG-PCL copolymeric drug delivery systems have been mostly prepared by using methoxy poly (ethylene glycol)-block- poly(s-caprolactone) (MPEG-PCL) diblock and triblock copolymers (KIM, 2001,SHI, 2005,CUONG, 2008,BAIMARK, 2009,LI, 2009).

Hutchinson et al. (WO 2011/049979 A2) have disclosed injectable formulations for intraarticular or peri-articular administration of compound that inhibits synthesis of leukotrienes and other 5-lipoxygenase products in the treatment or prevention of joint pain. In some embodiments of this invention, it has been stated that biocompatible and biodegradable polymers can be used to encapsulate and coat the leukotriene inhibitor compound in the form of microspheres. In this invention, PCL was recommended as an example of biocompatible, biodegradable for preparing biodegradable nanoparticles. However, this intervention was only aimed for the specific use of leukotriene inhibitors for the treatment of pain. Thus the results for only leukotriene inhibitor compound loaded PLA and PLGA microcarriers were given in this invention and PCL microcarriers were not studied. Therefore, results related with PCL were unknown due to its different degradation kinetics, degradation products and degree of bioefficacy. In other embodiments of this invention, intra-articular formulation of a leukotriene synthesis inhibitor compound in combination with anti-TNF alpha agents was described. However, intra-articular application of anti-TNF alpha agents and combination of leukotriene synthesis inhibitor compound and anti-TNF alpha agents were not studied in their invention and results related with these applications were unknown. Therefore, their invention was only concerned with intra-articular application of leukotriene loaded PLA and PLGA microcarriers. Additionally, prevention or elimination of inflammation was not studied in their invention and only a treatment of rat model of joint pain and clinical trials in which reduction of joint pain of osteoarthritis patients were assessed.

Wallach et al. (US 6,083,534) disclose pharmaceutical compositions for controlled delivery of soluble forms of receptors from a polymer matrix. In some embodiments of their invention, p55 sTNF receptor and anti-TNFo antibody, a mouse monoclonal antibody raised against human recombinant TNFa in laboratory conditions were studied. However, they have not used the currently approved formulations for anti-TNFa agents. For example, etanercept is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kDa TNFa receptor linked to the Fc portion of the human IgGl. Other clinically applied anti-TNFa agents are recombinant human murine chimeric IgGl antibody (infliximab), human-derived

5 recombinant IgGl monoclonal antibody (adalimumab), human IgGlK monoclonal antibody (golimumab), and pegylated Fab-9 fragment of a humanized anti-TNF-a antibody (certolizumab pegol). Therefore, results related with the applications of these clinically applied anti-TNFa agents were unknown. In some embodiments of their invention, a controlled release of a soluble receptor from a biocompatible polymer matrix was described and they studied with p55 sTNF

.0 receptor loaded PLGA, PLLA and EVac microspheres. However, PCL microspheres were not prepared and no related results were presented. Additionally, particle size of PLGA, PLLA and EVac microspheres was not given explicitly in their invention. The diameters of different types of microparticles tested previously for intra-articular administration in literature were compared in the review article of Butoescu et. al and they concluded that the most suitable size is between 5

.5 and 10 pm (BUTOESCU, 2009). The size of microcarriers plays an essential role on the phagocytosis of these systems once they are injected into the joint. Small particles are easily phagocytosed by synovial macrophages and larger particles tend to produce a multinuclear giant cell response. In their invention, the amount of loading of p55 sTNF receptor loaded microspheres was low and the amount of sTNF receptor released from this system could be

Ό insufficient during the subsequent stages of disease. In their invention, no sterilization procedure was applied to the microspheres. After the sterilization procedure, release kinetics of drugs released from polymeric matrix could change and drugs could lose their bioactivity. Therefore, the type of sterilization for human use was not presented and findings concerning the effect of sterilization to the polymeric matrix and the biological activity of the drug were not

5 presented in their invention. In our invention, bioactivity of anti-TNFa drug was protected after our sterilization procedure.

Lee et al. (EP 1 237 575 Bl) disclose treatment of arthritis with an LFA-I antagonist and a TNFa antagonist. In some embodiments of their invention, LFA-I antagonist and a TNFa antagonist formulation microencapsulated in biodegradable polymers was described. However, o efficacy of administrations of LFA-I antagonist and a TNFa antagonist, separately or together, were compared in collagen-induced arthritis model and they did not study any microencapsulation technique. Dose of drug use and bioactivity of the released drug from a microcarrier will change when an encapsulation technique is used. Therefore, it is not known what the results concerning microcarrier systems would be. Additionally, a TNFa antagonist (Enbrel) was administered through intra-peritoneal route to mice but no intra-articular 5 administration of TNFa antagonist was studied in their invention.

AIM OF THE INVENTION

Aim of this invention is to produce intra-articularly injectable polymeric microcarrier systems of anti-TNFa drugs that will provide long term controlled release of the drugs used to suppress the o inflammation in the joints of juvenile and adult rheumatoid arthritis as a local treatment approach. High treatment efficacy and clinical applicability are targeted with the presented biodegradable polymeric delivery systems for controlled release of anti-TNFa drugs that we have developed. In this invention, main objectives in developing microcarrier systems are

• to provide high anti-TNFa drug encapsulation efficiency of microcarrier systems,

5 · to produce a drug delivery system that provides a sustained-prolonged release of anti- TNFa drug for at least 3 months after intra-articular administration

• to protect the bioactivity of anti-TNFa drug throughout the entire release period and to ensure sufficient amount of anti-TNFa drug release to suppress the local inflammation,

• to obtain optimum particle size that should not cause any inflammatory responses and o engulf by phagocytic cells in joint,

• adjustable release rates and released amounts of anti-TNFa drug according to selected polymer,

• to deliver the drug in an amount that can be used in pediatric joints or small joints of adults as well.

5 With the controlled release of anti-TNFa drug from polymeric delivery systems it is aimed:

• to reduce the levels of pro-inflammatory cytokines and matrix metalloproteinases

released from the synovial fibroblasts at joints,

• to treat the signs and symptoms with a single injection not requiring re-injections, to eliminate adverse effects of high dose injection.

SUMMARY OF THE INVENTION

The invention provides the preparation of anti-TNFa drug loaded intra-articularly injectable, 5 biocompatible and biodegradable polymeric microcarriers that can provide a sustained release of anti-TNFa drug at therapeutically efficient doses for more than 3 months, wherein polymeric microcarriers are produced by using PCL homopolymer or PEG-PCL copolymer. Anti-TNFa drug loaded PCL and PEG-PCL microspheres according to this present invention are prepared by using the anti-TNFa drug at lower doses (equal to the dose of one systemic injection) compared to L0 that of anti-TNFa drug administered subcutaneously and intra-articularly. Application period of these microspheres are longer than that of anti-TNFa drug administered subcutaneously or and intra-articularly. Longer injection periods of PCL and PEG-PCL microspheres loaded with anti- TNFa drug provide more prolonged release of anti-TNFa drug at therapeutically effective doses to increase the effectiveness of the treatment.

L5

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows general view of scanning electron microscopy (SEM) images of γ-irradiated anti- TNFa drug loaded PCL microspheres.

FIG. IB shows general view of SEM images of γ-irradiated anti-TNFa drug loaded PEG-PCL-PEG !0 microspheres.

FIG. 2 presents the cumulative pg anti-TNFa drug/ mg microspheres release profiles of γ- irradiated anti-TNFa loaded type II PCL and PEG-PCL-PEG microspheres determined by using ELISA in phosphate tampon solution for 90 days: · type II PCL and A type II PEG-PCL-PEG microspheres.

[5 FIG. 3 shows the cumulative pg anti-TNFa drug/ mg microspheres release profiles of γ- irradiated anti-TNFa loaded type II PCL and PEG-PCL-PEG microspheres determined by using ELISA in cell culture medium containing 5 % serum for 60 days: ▲ type II PCL and · type II PEG-PCL-PEG microspheres. FIG. 4 is a graphic representation of cumulative g anti-TNFa/ mg microspheres release profiles of γ-irradiated anti-TNFa loaded type II PCL and PEG-PCL-PEG microspheres determined by using ELISA in synovial fluids of healthy or A patients for 14 days: A type II PCL microspheres incubated in healthy synovial fluid, Δ type II PCL microspheres incubated in synovial fluid of RA 5 patients, · type II PEG-PCL-PEG microspheres incubated in healthy synovial fluid, o type II PEG- PCL-PEG microspheres incubated in synovial fluid of RA patients.

FIG. 5 shows ratios of the percent cell viability of RA synovial fibroblast cells incubated with free anti-TNFa drug or γ-irradiated anti-TNFa drug loaded type II PCL and PEG-PCL-PEG microspheres to the percent cell viability control RA synovial fibroblast cells.

LO FIG. 6 represents the comparison of %-production levels of physiologically active TNFa produced by RA synovial fibroblast cells incubated with free anti-TNFa drug or γ-irradiated anti- TNFa drug loaded type II PCL or PEG-PCL-PEG microspheres to %-production levels of physiologically active TNFa produced by control synovial RA fibroblast cells.

FIG. 7A is a graphic representation of the comparison of %-production levels of IL-6 produced L5 by RA synovial fibroblast cells incubated with free anti-TNFa drug or γ-irradiated anti-TNFa drug loaded type II PCL or PEG-PCL-PEG microspheres to %-production levels of IL-6 produced by control synovial RA fibroblast cells.

FIG. 7B shows the comparison of %-production levels of MMP-3 produced by RA synovial fibroblast cells incubated with free anti-TNFa drug or γ-irradiated anti-TNFa drug loaded type II »o PCL or PEG-PCL-PEG microspheres to %-production levels of MMP-3 produced by control synovial RA fibroblast cells.

DETAILED DESCRIPTION OF THE INVENTION

In this invention, intra-articularly injectable polymeric microcarrier systems of anti-TNFa drugs

[5 that will provide long term controlled release of anti-TNFa drugs were produced by using PCL homopolymer or PEG-PCL copolymer. Commercially available PCL homopolymer (Sigma-Aldrich, Germany) was used to prepare anti-TNFa drug (Enbrel, Wyeth, England) loaded PCL microspheres. PEG-PCL-PEG triblock copolymer was synthesized to prepare anti-TNFa drug (Enbrel) loaded PEG-PCL-PEG microspheres. Synthesis of Polv (ethylene glycol) - Polv (ε-caprolactone Copolymer

PEG-PCL-PEG triblock copolymer was synthesized by ring-opening polymerization ε-caprolactone initiated by methoxy poly(ethylene glycol) (MPEG). Firstly, MPEG-PCL diblock copolymer was synthesized by ring-opening polymerization of ε-caprolactone initiated by MPEG in the presence

5 of catalyst (dibutyltindilaurate) (NIKLES, 2010). Then, PEG-PCL-PEG triblock copolymer was synthesized by the reaction of synthesized MPEG-PCL diblock copolymer with coupling reagent (hexamethyl diisocyanate) (FU, 2009,GONG, 2009). Briefly, pre-determined amount of MPEG (Aldrich, Germany), ε-caprolactone (Aldrich, Germany) with a monomer mol ratio 200:1, respectively were kept at 140°C and stirred under vacuum. Then, pre-determined amount of

LO dibutyltindilaurate (Aldrich, Germany) with a concentration of 0.5% of total reactants was added and the reaction mixture was maintained at 140°C for 9 hours under nitrogen atmosphere to synthesize MPEG-PCL diblock copolymer. After MPEG-PCL diblock copolymer synthesis, hexamethyl diisocyanate (HMDI, Aldrich, Germany) with a concentration 10% of total reactants was added to the reaction vessel and the polymerization reaction was continued at 80°C for 6

L5 hours under nitrogen atmosphere. To remove the unreacted monomer and oligomer, the crude triblock copolymers were dissolved in dichloromethane (Merck, Germany) and n-hexane (Sigma- Aldrich, Germany) was added to the point of imminent precipitation. The solution was chilled at 4°C overnight. The precipitates were then filtered and washed with n-hexane several times before thoroughly dried under reduced pressure at 40°C for 3 days. The thermal properties of

!0 copolymers were characterized by differential scanning calorimetry (DSC, Perkin Elmer, USA) and thermogravimetric analysis (TGA, Perkin Elmer, USA). The melting temperature of PEG-PCL- PEG triblock copolymer was found 66.64°C and differential thermogravimetric curve of PEG-PCL- PEG triblock copolymer showed two weight loss steps confirming the triblock copolymer formation. First weight-loss step reflected thermal degradation of the less stable PCL block chain

!5 (350°C) and the second weight-loss stages were relevant to the degradation of the PEG blocks (420°C). Additionally, proton nuclear magnetic resonance (1H NMR) spectroscopy (Bruker Biospin, Germany) and Fourier transform infrared (FT-IR) spectroscopy (Bruker IFS 66/S, FRA 106/S, Germany) were used to characterize chemical composition of copolymers. Both of the characteristic proton and FT-IR peaks of PEG segment and PCL segment were observed when

;o the 1H NMR and FT-IR spectra of PEG-PCL-PEG triblock copolymers were examined.

Additionally, PEG-PCL-PEG triblock copolymer formation was confirmed with the proton peaks (1.4 and 3.3 ppm) coming from the coupling agent (HMDI) seen in the 1H NMR spectrum and with N-H bending vibration peak (1470.61 cm ) related with the coupling agent (HMDI) observed in the FT-IR spectrum. The macromolecular weight and mass distribution of copolymer was determined by using gel permeation chromatography (GPC, Agilent 1200, USA). The 5 parameters of the copolymer synthesis such as polymerization time, mol ratio of monomers and temperature can be changed to obtain PEG-PCL-PEG triblock copolymers with various molecular weights within a range 10 000 - 100 000 g/mol. The number average molecular weight (Mn) and weight average molecular weight (Mw) values of PEG-PCL-PEG triblock copolymer chosen for an example of this invention was 47038 and 14051 g/mol, respectively.

o Preparation of Anti-TNFo Loaded PCL Microspheres

Anti-TNFa drug (Enbrel, Wyeth, England) loaded PCL microspheres were prepared by the double emulsion-solvent evaporation method. Briefly, 100 μΙ of the internal aqueous phase (phosphate buffered saline; PBS, 10 mM sodium phosphate, 145 mM NaCI, pH 7.2) containing 50 μΙ anti- TNFa drug solution with different concentrations (10, 25 and 50 mg/ml) and 50 μΙ 1% poly

.5 (vinyl alcohol) (PVA; Aldrich, Germany) was added to 5% w/v PCL (Sigma-Aldrich, Germany) in dichloromethane (Merck, Germany). The mixture was sonicated for 60 s on ice using Sonorex sonicator (Bandelin, Germany). The resulting primary water-in-oil (w/o) emulsion was added to external 40 ml PVA solution (1% w/v) and stirred with a magnetic stirrer (Schott, Australia) at 1100 rpm for 15 min. The double emulsion (w/o/w) was poured into 140 ml of PVA solution and o stirred at 14000 rpm for 3 min in an ice bath with a homogenizer (Ultraturrax T-25, IKA, Germany). After this homogenization step, emulsion was stirred at 1100 rpm for 3 hours to evaporate the dichloromethane. Finally, formed microspheres were collected by filtration through a 0.45 μιη hydrophilic PVDF membrane (Millipore, Ireland), washed with distilled water and stored at 4°C. Microspheres w prepared with 10, 25 and 50 mg/ml anti-TNFa drug stock

5 solutions were named as type I, II and III, respectively. Anti-TNFa loaded PCL microspheres were sterilized by exposure γ-irradiation (25 kGy) obtained from 60Co γ-source (Gamma- cell 220, MDS Nordion, Canada) at ambient temperature and fixed dose rate (1.74 kGy/h) in Turkish Atomic Energy Authority.

o Preparation of Anti-TNFa Loaded PEG-PCL-PEG Microspheres

25 mg/ml anti-TNFa drug loaded PEG-PCL-PEG microspheres (type II PEG-PCL-PEG microspheres) were also prepared by the double emulsion-solvent evaporation method. Briefly, 100 pi of the internal aqueous phase (PBS) containing 50 μΙ 25 mg/ml anti-TNFa stock solution and 50 μΙ 1% PVA was added to 2.5% w/v PEG-PCL-PEG solution in chloroform (Sigma, Germany)/dimethyl sulfoxide (DMSO; AppliChem, Germany) mixture (1:1). The mixture was sonicated for 60 s on ice using sonicator. The resulting primary water-in-oil (w/o) emulsion was added to external 40 ml Pluoronic F-68 (PLF-68, Sigma, Germany) solution (3% w/v) and stirred with a magnetic stirrer at 1100 rpm for 15 min. The double emulsion (w/o/w) was poured into 168 ml of PLF-68 solution and stirred at 14000 rpm for 3 min in an ice bath with a homogenizer. After this homogenization step, emulsion was stirred at 1100 rpm for 3 hours to evaporate the organic solvent. Finally, formed microspheres were collected by filtration through a 0.45 μηι hydrophilic PVDF membrane washed with distilled water and stored at 4°C. After preparation, microspheres were sterilized by γ-irradiation as described before.

Encapsulation Efficiency

The encapsulation efficiency of anti-TNFa drug loaded PCL and PEG-PCL-PEG microspheres was determined by modifying the method including the hydrolysis of the microspheres by strong base and the extraction of the protein with sodium dodecyl sulfate (SDS) (WONG, 2001). Briefly, microspheres were dissolved in DMSO at 37°C for 1 hour. 2150 μΙ of 0.25 N NaOH (Riedel de- Haen, Germany) solution containing 0.5% SDS (Bio-Rad, USA) was added to the test tube and gently mixed in water bath at 37°C for 4 hours. The mixture was then centrifuged (EBA-20, Hettich, Germany) at 3500 rpm for 5 min. The amount of protein encapsulated was determined by using bicinchoninic acid (BCA) assay (WONG, 2001). The calibration curve of BOA assay was constructed with different concentrations of anti-TNFa drugs (0-20 pg/ml) treated with DMSO and NaOH/SDS solution to determine the amount of encapsulated protein in microspheres. The encapsulation percentage of drug in microparticles was calculated from the ratio of the actual loading of the drug in the microsphere to the theoretical loading. The encapsulation efficiency of the system can be altered by changing the parameters used during the protein encapsulation into a polymer matrix and it should be higher than 50 % for developing an effective drug delivery system. Protein encapsulation efficiencies of PCL microspheres loaded with different concentrations of anti-TNFa drug (10, 25 and 50 mg/ml) are compared in Table 1 to examine the effect of amount of loaded anti-TNFa drug to the encapsulation efficiency. Encapsulation efficiencies of type II and type III PCL microspheres were significantly higher than that of type I PCL microspheres (p<0.05). However, no significant difference was observed for the encapsulation efficiencies of type II and type III PCL microspheres. According to the results

5 obtained for PCL microspheres loaded with different anti-TNFa drug concentrations, 25 mg/ml was chosen to prepare anti-TNFa drug loaded microspheres for release studies in different media. These microspheres were also incubated with the synovial fibroblast cells isolated from the synovial membrane of RA patients. 25 mg/ml anti-TNFa drug loaded PEG-PCL-PEG microspheres (type II PEG-PCL-PEG microspheres) were also prepared and their capsulation

L0 efficiency was found 75.91±1.16%. Existence of hydrophilic PEG segments in polymer chains improved the affinity of polymer with protein molecules and this caused high encapsulation efficiency.

Table 1. Protein encapsulation efficiencies of PCL microspheres prepared with different .5 concentrations of anti-TNFa drug

Protein Encapsulation

Efficiency, %

Type I PCL microspheres 50.29 ± 3.03 *, #

Type II PCL microspheres 65.37 ± 1.80 *

Type III PCL microspheres 60.09 ± 4.56 #

*, # Statistical significances between groups (p < 0.05)

Surface Morphology and Particle Size

The surface morphologies of anti-TNFa drug loaded PCL and PEG-PCL-PEG microspheres were o examined by scanning electron microscopy (SEM, JSM - 6400 Electron Microscope, Japan).

Microspheres were mounted onto metal stubs using carbon tape, vacuum-coated with gold (25 nm) by using Hummle VII sputter coating device (Anatech, USA) for SEM analysis. As seen from FIG. 1A and IB, microspheres possessed a spherical shape with a rough surface without pores. The mean particle sizes of γ-irradiated microspheres were determined from SEM images by measuring the diameters of 500 microspheres for each group using Image 3 analysis software (NIH, USA). As described before, the size of microcarriers plays an essential role on the phagocytosis of these systems once they are injected into the joint. In the review article of Butoescu et. al, diameters of different types of microparticles tested previously for intra-articular administration in literature were compared and they concluded that the most suitable size is between 5 and 10 pm (BUTOESCU, 2009). In this invention, mean particle sizes of anti-TNFa drug loaded PCL and PEG-PCL-PEG microspheres were found 5.22±0.15 pm and 4.96±0.09 pm, respectively.

In Vitro Release Study

In vitro anti-TNFa drug release profiles from PCL and PEG-PCL-PEG microspheres were evaluated in different release media such as phosphate buffered saline, cell culture medium containing 5% serum and synovial fluid of healthy or RA patients.

In vitro anti-TNFa drug release profiles from γ-irradiated type II PCL and PEG-PCL-PEG microspheres were evaluated by incubating microspheres in 0.01 M phosphate buffered saline (PBS, pH 7.4) containing 0.01% Tween 20 (Sigma, Germany) and 0.05% sodium azide (Sigma, Germany) as bacteriostatic agent in a shaking water bath at 37°C for 90 days (PISTEL, 2000). At defined time intervals, the release medium was completely removed after centrifugation and the amount of anti-TNFa drug released from PCL and PEG-PCL-PEG microspheres was determined in the supernatant by BCA method. Additionally, Q-ETA Etanercept Enzyme-linked immunosorbent assay (ELISA) (Matriks Biotek, Turkey) kit was used to measure the bioactivity of released anti-TNFa from γ-irradiated type II PCL and PEG-PCL-PEG microspheres.. The amount of biologically active released anti-TNFa (pg) from 1 mg type II PCL and PEG-PCL-PEG microspheres was determined and the cumulative pg anti-TNFa drug/mg microsphere release profile is evaluated in FIG. 2. After 4 days, amounts of biologically active anti-TNFa drug released from 1 mg PEG-PCL-PEG microspheres were found significantly higher than those of biologically active anti-TNFa drug released from 1 mg PCL microspheres. It was thought that higher encapsulation efficiency of type II PEG-PCL-PEG microspheres (75.91±1.16%) compared to that of type II PCL microspheres (65.37±1.80 %) cause more release of anti-TNFa drug from PEG-PCL-PEG microspheres. Besides, the presence of hydrophilic PEG segment could induce easier penetration of water into the polymer matrix and promote the protein release into the release media. In a previous study, they investigate the microspheres based on (AB)n type amphiphilic multiblock copolymers for sustained and complete release of a model protein, bovine serum albumin (BSA) (KIM, 2004). They concluded that amount of released BSA increased with increasing the ratio of PEG segment in the PEG-PCL and PEG-PLLA copolymers

5 since, microspheres that are more hydrophiiic are expected to have a larger swelling ratio causing higher release rates. To examine the bioactivity loss of released anti-TNFa during 90 days, the ratio of the amount of biologically active anti-TNFa drug released from γ-irradiated PCL (determined by ELISA) and PEG-PCL-PEG microspheres to the total amount of anti-TNFa microspheres (determined by BCA) are given in Table 2. Bioactivity of anti-TNFa drug released

) from PCL and PEG-PCL-PEG microspheres was protected completely during 7 days. At the end of 90 day release study, 93% bioactivity of anti-TNFa released from PCL microspheres and 94.6% bioactivity of anti-TNFa released from PEG-PCL-PEG microspheres were protected. As described in the Enbrel® etanercept US Prescribing Information of Wyeth, anti-TNFa drug (Enbrel) also contains mannitol, sucrose and trometamol to protect the bioactivity of anti-TNFa drug. j Therefore, bioactivity of anti-TNFa drug released was largely protected during 90 days. In literature, incorporation of additives such as proteins, sugars, polyols and heparin into protein delivery systems have been widely used to provide stabilizing of proteins in microsphere formulation and in release solution (SAEZ, 2008).

) Table 2. The ratio of the amount of biologically active anti-TNFa drug released from y-irradiated type II PCL and PEG-PCL-PEG microspheres (detected by ELISA) to the total amount of anti- TNFa drug released from (detected by BCA) at the end of 6 hour, 7 and 90 day.

Ratio of the amount of biologically active released protein to the amount of total released protein

6 hour 7 day 90 day

Type II PCL microspheres 0.996 ± 0.002 0.993 ± 0.006 0.930 ± 0.032

Type II PEG-PCL-PCL microspheres 0.998 ± 0.00 0.991 ± 0.007 0.946 ± 0.015

All groups were significantly different from each other (p < 0.05). In vitro anti-T Fa drug release profiles from γ-irradiated type II PCL and PEG-PCL-PEG microspheres were evaluated by incubating microspheres in cell culture medium (Dulbecco's Modified Eagle Medium (DMEM); PAA, Austria) containing 5 % fetal bovine serum (FBS; PAA, Austria) under 5% C02 atmosphere at 37°C for 60 days. At defined time intervals, cell culture medium was completely removed and replaced by fresh medium. Removed sample aliquots at defined time intervals were examined by Q-ETA Etanercept ELISA for anti-TNFa quantitation. Amount of biologically active released anti-TNFa (pg) from 1 mg type II PCL and PEG-PCL-PEG microspheres was determined in cell culture medium containing 5 % serum and the cumulative pg anti-TNFa drug/mg microsphere release profile is presented in FIG. 3. Amounts of biologically active anti-TNFa drug released from 1 mg PEG-PCL-PEG microspheres were found significantly higher than those of biologically active anti-TNFa drug released from 1 mg PCL microspheres after 4 days. As explained before, high encapsulation efficiency of PEG-PCL-PEG and presence of hydrophilic PEG segment cause more release of anti-TNFa drug from PEG-PCL- PEG microspheres. Besides, PEG segment at PEG-PCL-PEG microspheres prevent adsorption of serum proteins in the cell culture medium to microsphere surfaces and anti-TNFa drug can be released more easily. In a previous study, blend films based on PCL and PEG grafted PCL were prepared with different blend ratios and protein adhesion on the films were studied (CHO, 2009). They concluded that the level of the protein adsorption decreased when PEG component was introduced.

According to the results of in vitro release studies in phosphate buffered saline and cell culture medium containing 5% FBS, anti-TNFa loaded type II PCL and PEG-PCL-PEG microspheres provide a sustained-prolonged release of biologically active anti-TNFa drug for at least 3 months after intra-articular administration. Therefore, once weekly or twice weekly administration period of anti-TNFa drugs in conventional treatment can be extended to longer periods with this microcarrier approach, preferably at least 3 months interval or longer.

For prediction of in vivo bioactivity of anti-TNFa drug released from type II PCL and PEG-PCL- PEG microspheres, they were incubated in synovial fluids of healthy and RA patients at 37°C under 5% C02 atmosphere for 14 days. At defined time intervals, samples were taken from the synovial fluids and the biologically active amount of anti-TNFa released from microspheres was measured with Q-ETA Etanercept ELISA. The cumulative pg anti-TNFa drug/mg PCL and PEG- PCL-PEG microsphere release profiles in synovial fluids of healthy and RA patients are given in FIG. 4. The amount of biologically active anti-TNFa drug released from PCL microspheres in RA synovial fluids was significantly higher than that of biologically active anti-TNFa drug released from PCL microspheres in healthy synovial fluid at the end of 14 days (p<0.05). However, no significant difference was observed between the amounts of anti-TNFa drug released from PEG- PCL-PEG microspheres incubated in healthy synovial fluids and synovial fluids of RA patients at the end of 7 and 14 days (p > 0.05). At each time point, the amount of biologically active anti- TNFa drug released from PEG-PCL-PEG microspheres was found significantly higher than that of biologically active anti-TNFa drug released from PCL microspheres. As mentioned before, presence of hydrophilic PEG segment and high encapsulation efficiency of PEG-PCL-PEG cause more release of anti-TNFa drug from PEG-PCL-PEG microspheres. Besides, anti-TNFa drug can be released from PEG-PCL-PEG microspheres more easily due to PEG segment since its presence prevents adsorption of serum proteins in the cell culture medium to the surface of microspheres.

The amounts of biologically active anti-TNFa drug released from PCL or PEG-PCL-PEG microspheres in PBS, synovial fluids of healthy and RA patient's synovial fluids and cell culture medium containing 5% fetal bovine serum at the end of 7 days are compared in Table 3. In all release media, amount of biologically active anti-TNFa drug released from PEG-PCL-PEG microspheres was found significantly higher than that of released from PCL microspheres. It was thought that high encapsulation efficiency of PEG-PCL-PEG and presence of hydrophilic PEG segment cause more release of anti-TNFa drug from PEG-PCL-PEG microspheres. For both microsphere group, amount of biologically active anti-TNFa drug released in cell culture medium with 5% FBS was higher than those of determined in synovial fluids of healthy and RA patients at the end of 7 days. However, it was significantly lower than that of observed in PBS release. This slower release in cell culture medium could be due to the absorption of synovial fluid proteins onto the surface of microspheres. Non-specific protein adsorption onto the polymeric surface of microspheres limit the release of proteins and resulting in slow release profiles (CROTTS, 1997). However, this limitation was not observed in cell culture medium and the amount of biologically active anti-TNFa drug released was higher than those of determined in synovial fluids. Furthermore, amount of anti-TNFa drug released from PCL and PEG-PCL-PEG microspheres in synovial fluids of RA patients were significantly higher than that of determined in healthy synovial fluid due to the lower viscosity of synovial fluid of RA patients compared to healthy synovial fluid.

Table 3. Comparison of the total amounts of biologically active anti-TNFa drug released from 5 γ-irradiated type II PCL and PEG-PCL-PEG microspheres in PBS, cell culture medium with 5 % FBS and synovial fluids of healthy and RA patients at the end of day 7.

μg anti-TNFa drug /mg microsphere

Type II PCL Type II PEG-PCL-PEG microspheres microspheres

Release in PBS 2.27 ± 0.18 4.16 ± 0.24

Release in cell culture medium 0.55 ± 0.05 0.85 ± 0.30 containing 5 % FBS

Release in healthy synovial fluid 0.19 ± 0.02 0.23 ± 0.02

Release in synovial fluid of RA 0.22 ± 0.02 0.25 ± 0.03 patients

All groups were significantly different from each other (p < 0.05).

) Investigation of the Effects of Anti-TNFa Loaded PCL and PEG-PCL-PEG Microspheres on the Synovial Fibroblast Cells Isolated From RA Patients

Synovial fibroblast cells were isolated enzymatically from RA patients undergoing total joint replacement surgery with the approval of the local Ethics Committees. Briefly, the synovial membranes were minced and incubated with 0.4% (g/ml) Type IA collagenase (Sigma, USA) in i serum free high glucose DMEM (PAA, Austria) supplemented withlOO units/ml penicillin/streptomycin (PAA, Austria) for 1 hour in 5% C02 at 37°C (FICKERT, 2003). The synovial fibroblast cells were cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FBS; PAA, Austria) and 100 units/ml penicillin/streptomycin, in 5% C02 at 37°C in a humidified incubator (5215 Shel Lab., USA). The cell culture medium was changed every 3 days and the cells grown to confluency were subcultured in a 1:3 ratio using 0.1% trypsin-EDTA (PAA, Austria).

Anti-TNFa drug loaded PCL or PEG-PCL-PEG microspheres were incubated with the synovial fibroblast cells of RA patients for 4 weeks to examine the treatment efficacy of the delivery systems. In preferred embodiment of this invention, etanercept (Enbrel) was used as anti-TNFa drug loaded into the microspheres. However, other anti-TNFa drugs such as infliximab, adalimumab, golimumab and certolizumab pegol can be also used as the other application of this invention.

For this study, the experimental groups were as follows:

l. RA synovial fibroblast cells (Control)

2. RA synovial fibroblast cells + free anti-TNFa drug

3. RA synovial fibroblast cells + anti-TNFa drug loaded type II PCL microspheres

4. RA synovial fibroblast cells + anti-TNFa drug loaded type II PEG-PCL-PEG microspheres

In the study of incubation RA synovial fibroblast cells were cultivated in 25 cm2 flask and cell culture medium containing anti-TNFa drugs released from PCL or PEG-PCL-PEG microspheres were added at determined time intervals to these cultivated cells. Briefly, synovial fibroblast cells were seeded into 25 cm2 flask at a density of 2.5 x 105. 10 mg of anti-TNFa drugs loaded PCL or PEG-PCL-PEG microspheres were incubated in cell culture medium containing 5% FBS in separate flasks. At the end of 3 day release, cell culture medium of synovial fibroblast cells was replaced with 2.5 ml cell culture medium containing 5% FBS and anti-TNFa drugs released from microspheres for the experiment groups 3 and 4. At the end of 4 day incubation of synovial fibroblast cells with culture medium containing 5 % FBS and anti-TNFa drugs released from microspheres, 2.5 ml culture medium containing 5 % FBS and 7 day release of anti-TNFa drug from microspheres was added to synovial fibroblast cells used for experiment groups 3 and 4. After 3 day incubation of these cells, half of cell culture medium was removed and culture medium containing 5% FBS and 10 day release of anti-TNFa drug from microspheres was added to these synovial fibroblast cells. After this time point, half of cell culture medium was replaced with new culture medium containing 5% FBS and release of anti-TNFa drug from microspheres at defined time periods. For the experiment group 2, RA synovial fibroblast cells were incubated for 4 days only once with a cell culture medium containing 5% FBS and free anti-TNFa drug equal to the amount of anti-TNFa drug loaded in 5 mg microsphere (10 pg/ml). At the end of 4 days, fresh cell culture medium containing 5 % FBS was added to these cells and cells were cultivated for 3 days. After 7 day cultivation, half of cell culture medium was replaced with fresh cell culture medium containing 5% FBS at defined time periods.

For control group (experiment group 1), RA synovial fibroblast cells were cultivated with cell culture medium containing 5% FBS and half of cell culture medium was replaced with fresh cell culture medium containing 5 % FBS at defined time periods. For experimental groups formed to examine the treatment efficacy of the delivery systems, changes in RA synovial fibroblast viability and in levels of interleukin-6 (IL-6) and matrix metalloproteinase-3 (MMP-3) released by the RA synovial fibroblast cells during inflammation were compared with the that of control group (experimental group 1) at the end of 1, 2, 3 and 4 weeks. Viability of RA synovial fibroblast cells were assessed by Alamar Blue Assay which provide continuous monitoring of cultures over time. At the end of 1, 2, 3 and 4 weeks, the culture medium was removed and fresh high glucose DMEM medium without phenol red (Biochrom, Germany) containing 10% Alamar Blue reagent (Invitrogen, USA) was added to each flask. After 4 h of incubation at 37°C in dark, media of cells were collected and their absorbance was read at 570 nm (reduced) and 600 nm (oxidize) with a microplate reader (pQuant, Biotek, USA). The percent difference in reduction of cell culture media by the RA synovial fibroblast cells that were incubated with free anti-TNFa drug or PCL or PEG-PCL-PEG microspheres loaded with anti-TNFa drug compared with that of in reduction of cell culture media by the control cells. Ratios of the percent cell viability of RA synovial fibroblasts incubated with free anti-TNFa drug or γ-irradiated anti-TNFa drug loaded type II PCL and PEG-PCL-PEG microspheres to the percent cell viability of control RA synovial fibroblast cells are given in FIG. 5. According first week results, percent cell viability of RA synovial fibroblast cells incubated with free anti-TNFa drug was significantly lower than that of RA synovial fibroblast cells incubated with type II PCL and PEG-PCL-PEG microspheres (p<0.05). However, no significant differences were observed between these groups at other time periods (p>0.05). In experimental group 2, RA synovial fibroblast cells were incubated only once with a cell culture medium containing 5% FBS and free anti-TNFa drug equal to the amount of anti-TNFo drug in 5 mg microsphere (10 pg/ml). Adding high dose of anti-TNFo drug causes a decrease at the percent cell viability at first week. However, an increase in percent cell viability of this group was observed after a reduction of amount of free anti-TNFo drug by changing cell culture medium with a fresh medium. No decrease in percent cell viability of RA synovial fibroblast cells incubated with type II PCL and PEG-PCL-PEG microspheres was observed due to slow release of anti-TNFo drug from microspheres. These results were in accordance with cytotoxicity results in which cell response depends on the applied dose used (NUZZI, 2012). Problems such as toxicity, decrease in the patient's quality of life and pain, loss of function at injection site related with a high dose drug application in clinic are observed (O'DELL, 1997,CHATZIGIANNIS, 2004). Additionally, these results showed that the potential of the use of controlled drug delivery systems are high in clinical applications.

Changes in levels of IL-6 and MMP-3 released by the RA synovial fibroblast cells in experimental groups 2, 3 and 4 were compared with the that of control group (experimental group 1) at the end of 1, 2, 3 and 4 weeks to examine the treatment efficacy of the delivery systems developed in this invention. Levels of IL-6 and MMP-3 released by RA synovial fibroblast cells were determined by using ELISA kit (eBioscience, USA) corresponding to related cytokine or matrix metalloproteinase. Used anti-TNFo drug in preferred embodiment of this invention is Enbrel® (etanercept) which is a neutralizing biologic that does not remove TNF-a molecules from the solution (GRATTENDICK, 2008). The presence of the etanercept-bound TNF-a was still able to bind to the antibodies utilized in the ELISA and so therefore resulted in high measurements. Therefore, levels of physiologically active TNFo released by RA synovial fibroblast cells were determined by using a bioassay in which TNFo sensitive Wehi-164 varl3 cell line was used. Wehi-164 varl3 cell line was a kind gift of Prof. Dr. Atilla Isjk from Department of Virology of Faculty of Veterinary Medicine of Selguk University. The comparison of %-production levels of physiologically active TNFa produced by RA synovial fibroblast cells incubated with free anti- TNFo drug or γ-irradiated anti-TNFo drug loaded type II PCL or PEG-PCL-PEG microspheres to %-production levels of physiologically active TNFa produced by control synovial RA fibroblast cells are given in FIG. 6. After 2 weeks, amount of physiologically active TNFa released from RA synovial fibroblast cells incubated with type II PCL or PEG-PCL-PEG microspheres was found significantly lower than that of physiologically active TNFa released from RA synovial fibroblast cells incubated with free anti-TNFa drug. Amount of physiologically active TNFa released from RA synovial fibroblast cells incubated with type II PCL or PEG-PCL-PEG microspheres decreased around 35% whereas amount of physiologically active TNFa released from RA synovial fibroblast cells incubated with free anti-TNFa drug decreased around 10-25%. A decrease of TNFa levels was observed after 2 week incubation due to continuous release of anti-TNFa drug from type II PCL or PEG-PCL-PEG microspheres. However, no significant decrease was observed in active TNFa amounts in cell culture medium due to decrease of amount of free anti-TNFa drug in culture medium with each media change. FIG. 7A is a graphic representation of the comparison of %-production levels of IL-6 produced by RA synovial fibroblast cells incubated with free anti- TNFa drug or γ-irradiated anti-TNFa drug loaded type II PCL or PEG-PCL-PEG microspheres to %-production levels of IL-6 produced by control synovial RA fibroblast cells. Amount of IL-6 released from RA synovial fibroblast cells incubated with type II PCL or PEG-PCL-PEG microspheres decreased around 25-30% whereas amount of IL-6 released from RA synovial fibroblast cells incubated with free anti-TNFa drug decreased around 10-15% after two weeks. However, no difference was observed between IL-6 levels produced by RA synovial fibroblast cells incubated with anti-TNFa drug loaded type II PCL and PEG-PCL-PEG microspheres. Proinflammatory cytokines produced in RA affect the production of IL-6 (CHOY, 2001). Therefore, decrease in IL-6 levels produced by RA synovial fibroblast cells incubated with anti-TNFa drug loaded type II PCL and PEG-PCL-PEG microspheres was parallel with the decrease in physiologically active TNFa levels of RA synovial fibroblast cells incubated with microspheres. FIG. 7B shows the comparison of %-production levels of MMP-3 produced by RA synovial fibroblast cells incubated with free anti-TNFa drug or γ-irradiated anti-TNFa drug loaded type II PCL or PEG-PCL-PEG microspheres to %-production levels of MMP-3 produced by control synovial RA fibroblast cells. Amount of MMP-3 released from RA synovial fibroblast cells incubated with type II PCL or PEG-PCL-PEG microspheres was found significantly lower than that of MMP-3 released from RA synovial fibroblast cells incubated with free anti-TNFa drug at second week. At 3rd and 4th weeks, amount of MMP-3 released from RA synovial fibroblast cells incubated with free anti-TNFa drug was significantly higher than that of MMP-3 released from RA synovial fibroblast cells incubated with II PCL or PEG-PCL-PEG microspheres. Amount of MMP-3 released from RA synovial fibroblast cells incubated with type II PCL or PEG-PCL-PEG microspheres decreased around 30% after 3rd week. However, amount of MMP-3 released from RA synovial fibroblast cells incubated with free anti-TNFa drug decreased around 10%. Proinflammatory cytokines and MMP levels can be reduced by increasing the amount of microspheres incubated with cells and/or increasing the amount of anti-TNFa drug loaded into type II PCL or PEG-PCL-PEG microspheres.

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Claims

1. An intra-articularly injectable polymeric microcarrier system providing long-term controlled release, for the treatment of inflammatory rheumatic disease, comprising anti-TNFa drug as active ingredient encapsulated in biocompatible and biodegradable polymeric microspheres wherein active ingredient encapsulation efficiency is between 50-75 %, and particle size of microspheres is between 4.9-5.3 μιτι.
2. An intra-articularly injectable polymeric microcarrier system according to claim 1 where anti- TNFa drug is selected from the group of infliximab, etanercept, adalimumab, golimumab and certolizumab pegol.
3. An intra-articularly injectable polymeric microcarrier system according to claim 2 where anti- TNFa drug is etanercept.
4. An intra-articularly injectable polymeric microcarrier system according to claim 1 where microspheres are produced from biocompatible polymers selected from the group consisting of polyesters and multiblock copolymers, based on caprolactone and poly(ethylene glycol) (PEG).
5. An intra-articularly injectable polymeric microcarrier system according to claim 4 wherein the polymer is polycaprolactone (PCL).
6. An intra-articularly injectable polymeric microcarrier system according to claim 4 where multiblock copolymers based on poly(ethylene glycol) (PEG) -polycaprolactone (PCL) is triblock copolymer.
7. An intra-articularly injectable polymeric microcarrier system, according to claim 1, wherein the microcarrier system is sterilized by y-irradiation.
8. An intra-articularly injectable polymeric microcarrier system, according to claim 1 for local treatment approach to suppress the inflammation at defined joints.
9. An intra-articularly injectable polymeric microcarrier system, according to claim 1 for treatment of adult and juvenile Rheumatoid arthritis.
10. An intra-articularly injectable polymeric microcarrier system, according to claim 1 for treatment of adult and juvenile Ankylosing spondylitis.
11. An intra-articularly injectable polymeric microcarrier system according to claim 1 for treatment of adult and juvenile Psoriatic arthritis.
12. An intra-articularly injectable polymeric microcarrier system according to claim 1 for treatment of Juvenile idiopathic arthritis.
13. An intra-articularly injectable polymeric microcarrier system according to claim 1 for treatment for oligoarticular Juvenile idiopathic arthritis.
14. An intra-articularly injectable polymeric microcarrier system according to claim 1 for treatment of inflammatory rheumatic disease in pediatric patients.
15. A method for the treatment of inflammatory rheumatic diseases, wherein the intra- articularly injectable polymeric microcarrier system according to claim 1, is administered with at least 3 months interval or longer .
PCT/TR2012/000148 2012-09-19 2012-09-19 CONTROLED DRUG DELIVERY SYSTEMS FOR ANTI-TNF-α WO2014046631A1 (en)

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