Compositions Comprising Alkaline Phosphatase for Use in Treating Arthritis - Patent application

Abstract

The present invention relates to compositions for use in the treatment of arthritis. Alkaline phosphatase (AP), an ectophosphatase with an anti-inflammatory barrier-protecting mechanism of action, exhibits potent anti-rheumatoid arthritis (anti-RA) efficacy in a rat model of arthritis. In this model, RA was induced by subcutaneous immunization with a mixture of methylated bovine serum albumin (mBSA), CFA (complete Freund's adjuvant antigen), and CBP (custom pertussis antigen), followed by intra-articular injection of mBSA. Results were comparable to those obtained with MTX, a reference drug for the treatment of RA. Both knee swelling over time and the number of infiltrating macrophages were found to be reduced with AP treatment, either prophylactically or therapeutically, comparable to the effects of MTX. AP was found to be effective as both a prophylactic and therapeutic intervention. Thus, ectophosphatase intervention with AP, by combining a distinct and synergistic mode of action with MTX, fills a novel and unmet need in RA treatment.

Description

[Technical field]

[0001] The present invention relates to compositions for use in the treatment of arthritis (also referred to herein as arthritis). [Background technology]

[0002] Arthritis is a group of more than 100 types of inflammatory or degenerative diseases that damage the joints with pain and stiffness of the musculoskeletal system. Arthritis is inflammation of one or more joints. The most common types are osteoarthritis, rheumatoid arthritis (RA), gout, and ankylosing spondylitis, but it can also occur following other diseases such as infectious arthritis and systemic lupus erythematosus. Examples of arthritis (not in acute form) include osteoarthritis, rheumatoid arthritis, and gouty arthritis. The acute form is usually caused by bacterial invasion.

[0003] Although the etiology is unknown, RA is characterized by chronic inflammation in multiple joints, which develops into marginal bone and cartilage erosion, juxta-articular bone loss, and systemic loss of bone mass. It is widely accepted that inflammatory cells, especially lymphocytes and macrophages, are crucial players in the pathogenesis of RA, and that cytokines such as tumor necrosis factor alpha (TNFα), interleukin 1 (IL-1), IL-6, and IL-8 are also involved. In addition, recent findings indicate that osteoclasts, not balanced with osteoblast activity (i.e., osteoclastic activity / osteoblast activity>1), play a key role in joint destruction and osteoporosis in RA. Although RA manifests itself as a joint disease, the roots of RA may begin systemically. RA patients show increased plasma levels of IL-1, IL-6, and TNFα (during exacerbations or chronic disease episodes), which indicate a physiological condition of a pro-inflammatory state.

[0004] Rheumatoid arthritis (RA) is a major systemic autoimmune disease associated with high morbidity and mortality, with 50% of RA patients being disabled and unable to work 10 years after onset, reducing life expectancy by 3-18 years. RA is associated with significantly high medical costs and lost work time.

[0005] Although the causal factors or mechanisms that lead to RA are still unknown, a role for systemic induction of inflammation in the exacerbation or induction of RA has been proposed. For example, there seems to be a correlation between the activity and extent of intestinal disease and the severity of arthritis. Small intestinal bacterial overgrowth induces RA, and recombinant bactericidal / permeability enhancing proteins, drugs that neutralize endotoxins, and metronidazole, which is active against anaerobic bacteria, prevented the induction of arthritis. Next to gram-negative bacterial toxins (e.g., lipopolysaccharides), gram-positive bacterial toxins (e.g., lipoteichoic acid) can also induce arthritis. Next to bacterial toxins, excessive levels of ATP / ADP from bacteria in the intestinal tract can also cause a pro-inflammatory state. Also, a collagen-induced RA animal model has been described in which RA induction failed when the animals were grown under septic conditions. This indicates that bacterial components from commensal and pathogenic strains can affect local and systemic homeostasis and immune responses.

[0006] Since there is no cure for rheumatoid arthritis, the goals of pharmacological management are symptom relief, prevention of joint damage, and disease remission. The "gold standard" disease-modifying antirheumatic drug (DMARD) used in the treatment of RA is methotrexate (MTX). MTX has been shown to clearly control the inflammatory response as a first-line agent. Its long-term efficacy has been well documented, but true / complete remission is rare. Monotherapy with MTX is not often associated with sustained disease remission. The major drawbacks of MTX are its cytotoxicity and nephrotoxicity, as well as adverse effects on patients treated for long periods of time. Due to its toxicity, only 50% of patients remain on MTX after 5 years. Another disadvantage is that resistance to MTX is known to be induced, and therefore MTX is often used in combination with other drugs.

[0007] Other disease-modifying drugs used in the treatment of RA are anti-tumor necrosis factor (TNF) drugs (e.g., etanercept or infliximab), so-called TNFα blockers. These types of drugs have been shown to reduce disease activity, rapidly improve joint pain and swelling, and reduce joint damage in patients with RA. TNFα is an important cytokine that is involved in many pro-inflammatory effects and plays a major role in inflammatory diseases such as rheumatoid arthritis, and is a key element in their pathogenesis. Current anti-TNFα drugs are used to block TNFα, thereby reducing the inflammatory response and potentially preventing or reducing joint damage. Anti-TNFα drugs are used either as stand-alone therapy or in combination therapy with, for example, MTX.

[0008] However, currently available biological TNFα blockers show several side effects. One of the main effects is the destruction of immune tolerance to antibody drugs due to the non-natural nature of these drugs. This leads to the formation of inactivating antibodies, making the medication less effective. As a result, the patient no longer responds to the beneficial effects of the compound. The patient is then reintroduced to other medicines, such as corticosteroids, which have major adverse effects over a patient-specific period of time. The use of anti-TNF antibodies may also increase the risk of serious infections, especially tuberculosis. Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the above, there is a need in the art for new strategies for the treatment of arthritis. Additionally, there is a need in the art for treatments that are more effective than currently available treatments, with fewer adverse effects for the patient during treatment.

[0010] In particular, it is an object of the present invention to address the above-mentioned needs in the art. In particular, this object of the present invention is met by the present invention as outlined in the appended claims. [Means for solving the problem]

[0011] Specifically, in particular, the above objectives are met by the present invention, according to a first aspect, by ectophosphatase in combination with disease-modifying antirheumatic drugs (DMARDS) for use in treating mammals suffering from arthritis, such as osteoarthritis, rheumatoid arthritis (RA), and gouty arthritis. During pro-inflammatory injury, the disease progresses further due to an incorrect systemic response of the innate immune system. As a result, it may prove beneficial to attenuate those sudden (exacerbated) systemic inflammatory responses (e.g., cytokine storm). Alkaline phosphatase (AP) fulfills this function and can therefore be administered as a routine therapeutic compound.

[0012] According to another aspect of the present invention, the present invention relates to ectophosphatase in combination with disease-modifying antirheumatic drugs (DMARDS) for use, where the DMARDS is methotrexate (MTX). The combination therapy with alkaline phosphatase allows for an effective treatment regimen with low doses of MTX when the combination therapy is administered. MTX administered at doses for anti-RA therapy leads to the efflux / release of intracellular nucleotides such as ATP and ADP from cells, which are subsequently converted to adenosine. Adenosine has a strong anti-inflammatory effect and deactivates activated leukocytes, which are responsible for RA pathology. Thus, by inference, alkaline phosphatase is synergistic with MTX therapy by being able to convert to anti-inflammatory adenosine. It also converts nucleotides released from cells under oxidative stress, such as those in affected areas associated with RA, thereby generating an anti-inflammatory microenvironment, for example in joints.

[0013] In inflammatory conditions, AP is a potent mitigator of, for example, TNFα responses, and AP activity results in a marked attenuation of the cytokine storm, as shown in preclinical and clinical trials, for example in patients undergoing major surgery. Specifically, significant reductions were observed for proinflammatory markers such as TNFα, IL-6, and IL-8, whereas IL-10 plasma levels were not significantly affected. The latter suggests that basal proinflammatory events do not occur, for example, in macrophages and other leukocytes, as a consequence of which anti-inflammatory IL10 is not produced in the presence of sufficient AP levels. Moreover, in contrast to current anti-RA agents that target one of the main cytokine intermediates, AP is proposed to be a gatekeeper in the innate immune defense system, affecting a number of cytokines.

[0014] Alkaline phosphatase is an endogenous protein that is physiologically effective and active in healthy conditions, but has no putative adverse effects such as increased risk of infection, tolerance or resistance problems, etc. Given the fact that even high endogenous levels of AP are safe for both the mother and the developing child observed during pregnancy and are well tolerated for long time intervals, it is hypothesized that AP therapy may be of benefit to advanced rheumatoid arthritis patients.

[0015] The downstream effects of oxidative stress (e.g., ischemic injury) can result in the release of nucleotides such as ATP, ADP, and AMP from affected cells. These moieties, normally present intracellularly and involved in the intracellular energy supply, are potent pro-inflammatory factors (inducing inflammatory moieties, ITMs) when in the extracellular environment. These ITMs are detoxified by the activity of ectophosphatases such as AP, CD39, and CD73. By acting on ATP and ADP, adenosine is generated, which has an antagonistic (anti-inflammatory) effect. Thus, AP is proposed here as the sole anti-inflammatory agent, as a standalone effective anti-RA agent, or in combination therapy with further drugs. The advantage of the latter approach is that lower doses of such other pharmacological or biological anti-RA compounds are required, thereby reducing the possibility of inducing resistance or affecting tolerance.

[0016] According to yet another preferred embodiment, the present invention relates to an ectophosphatase in combination with a disease-modifying antirheumatic drug (DMARDS) for use, wherein the arthritis is rheumatoid arthritis.

[0017] According to the present invention, the mammal suffering from arthritis can be any vertebrate such as monkeys, horses, cows, rodents, humans, etc., preferably humans.

[0018] According to another preferred embodiment, the present invention relates to an ectophosphatase selected from the group consisting of alkaline phosphatase, CD39, and CD73, in combination with a disease-modifying antirheumatic drug (DMARDS) for use. The sources of such ectophosphatases can be numerous, from natural sources or recombinant techniques by expression of the ectophosphatase in unicellular organisms such as yeast, or in multicellular organisms such as plants or animals.

[0019] According to yet another preferred embodiment, the present invention relates to an ectophosphatase in combination with disease-modifying antirheumatic drugs (DMARDS) for use which is a recombinant alkaline phosphatase.

[0020] According to yet another preferred embodiment, the present invention relates to an ectophosphatase, which is a recombinant mammalian alkaline phosphatase, preferably a human alkaline phosphatase, in combination with a disease-modifying antirheumatic drug (DMARDS) for use. Preferably, the phosphatase used in the combination of the present invention corresponds to the anticipated therapeutic intervention, e.g. to aid in the treatment of a human, using a composition of the present invention comprising recombinant human alkaline phosphatase. However, other combinations can also be used, e.g. the treatment of a human, using a composition of the present invention comprising an alternative alkaline phosphatase, e.g. non-human natural or non-human recombinant, such as bovine intestinal alkaline phosphatase.

[0021] The ectophosphatase of the present invention, which is used in combination with disease-modifying antirheumatic drugs (DMARDS), can be administered either locally (e.g., orally, inhalation therapy) or parenterally, in a suitable formulation adapted for such administration route. Only after parenteral administration can the AP act directly on the target. Many treatments for chronic inflammatory diseases, such as rheumatoid arthritis, still require frequent and long-term administration, which utilizes traditional routes such as oral administration, intramuscular and intravenous injections, leading to accumulation of the drug outside the site of inflammation and sometimes also to unwanted systemic side effects. Targeting can be made specific, for example, by using nanoformulated AP.

[0022] According to another preferred embodiment, the present invention relates to an ectophosphatase in combination with disease-modifying antirheumatic drugs (DMARDS) for use, which is further combined with nanoparticles. By incorporating the ectophosphatase of the present invention in nanoparticles or combining it with DMARDS that show themselves to be effective against RA, an effective treatment modality can be developed. The use of nanoparticles for the delivery of the composition of the present invention allows the drug to be specifically released at the site of inflammation in a controlled or sustained manner, resulting in reduced undesired effects and improved patient compliance.

[0023] According to a preferred embodiment, the present invention relates to an ectophosphatase in combination with a disease-modifying antirheumatic drug (DMARDS) for use, wherein the nanoparticles consist of a substance selected from the group consisting of fullerenes, liposomes, gold, poly-lactic-co-glycolic acid (PLGA) and poly-L-lactic acid (PLA). The nanoparticles that can be used to encapsulate the composition of the present invention preferably consist of gold, poly-lactic-co-glycolic acid (PLGA) or poly-L-lactic acid (PLA), more preferably PLGA or PLA, most preferably PLGA.

[0024] A suitable AP is preferably a recombinant human AP with a long plasma residence time, which can thus be positioned as a routinely used alternative compound to current drugs administered in the treatment of RA.

[0025] In addition, the use of recombinant human APs circumvents putative immune responses that may follow long-term use of non-human, such as (glyco)proteins such as bovine APs administered in cardiac surgery for short-term use only. Typical for glycoproteins with guaranteed long plasma residence times are fully complex glycosylated oligosaccharide chains. Many potential sources of APs do not have sufficient complex glycans attached, and as a result, show plasma residence times that are a fraction of the favorable therapeutic agents. We believe that alternative non-human APs such as bovine APs are not very effective when used chronically, even though they have significant TNFα blocker activity, even when their residence times are short. However, they can be used as "resting drugs (off-period drug use)" in RA patients during drug-free periods from therapy with currently administered TNFα blockers, with short-term dosing regimens. These APs are therefore proposed herein to act as "resting drugs". For primary use, several sources of APs can be used as "resting drugs". Sources for which identified and biologically relevant AP activity has been established include natural and recombinant APs expressed in, for example, yeast, plants, moss, and mammalian expression models.

[0026] According to a preferred embodiment, the present invention relates to an ectophosphatase in combination with a disease-modifying antirheumatic drug (DMARDS) for use, wherein the treatment comprises parenteral or oral administration of said composition. The therapeutic intervention can be administered either locally (e.g., orally, inhalation therapy) or parenterally, in suitable formulations adapted for such administration routes.

[0027] According to another preferred embodiment, the present invention relates to ectophosphatase in combination with disease-modifying antirheumatic drugs (DMARDS) for use, where the treatment includes prevention of arthritis, or delay of onset, or attenuation of progression.Accordingly, the ectophosphatase in combination with disease-modifying antirheumatic drugs (DMARDS) for use of the present invention can be used as a prophylaxis to prevent or reduce inflammatory response during proinflammatory injury, which leads to attenuation of further progression of the disease.AP can also be used therapeutically for the treatment of mammals suffering from arthritis.

[0028] According to another preferred embodiment, the present invention relates to an ectophosphatase in combination with a disease-modifying antirheumatic drug (DMARDS) for use, wherein said treatment comprises attenuation of the inflammatory response in a mammal suffering from arthritis. Attenuation of the inflammatory response may also comprise treatment of exacerbations in a patient suffering from arthritis.

[0029] According to yet another preferred embodiment, the present invention relates to an ectophosphatase which is a tissue specific ectophosphatase in combination with a disease modifying antirheumatic drug (DMARDS) for use, wherein the treatment is the treatment of a chronic arthritic disease.

[0030] According to a preferred embodiment, the present invention relates to an ectophosphatase in combination with a disease-modifying antirheumatic drug (DMARDS) for use, wherein the tissue-specific ectophosphatase is selected from the group consisting of intestinal AP (IAP), placental ALP (PALP) and hepatic AP (LAP), preferably placental ALP (PALP). Although both tissue non-specific alkaline phosphatase (TNSALP) and tissue-specific AP such as intestinal AP (IAP) cq placental ALP (PALP) share activity towards nucleotides, as shown by both in vitro and in vivo studies carried out in the laboratory, significant differences are found in the molecular structure between TNSALP on the one hand and IAP or PALP on the other hand. The so-called crown domain, which in TNSALP isozymes contains the RGD binding site for bone-type AP, is missing in tissue-specific IAP and PLAP. Herein, we propose that this crown domain is the homing moiety for TNSALP, which may explain why, for example, intestinal ALP is relatively increased in RA patients, but not bone formation, which may partially compensate for nucleotide toxicity. Preferably, placental ALP can be used instead in RA patients. Since plasma levels are elevated during pregnancy up to 30 times normal levels, the safety of long-term administration of placental AP is guaranteed. Increased plasma levels further correlate with a reduction in clinical RA symptoms in pregnant RA patients, possibly because the need for endogenous tissue-specific (bone, liver, kidney AP) supplementation is reduced. It is known that in pregnant women, placental ALP is significantly upregulated during the second and third trimester of pregnancy and is cleared from the circulation with a half-life (T1 / 2) of 7 days. Administration of human placental ALP is advantageous as a treatment for chronic disease in RA patients. It has also been described that the clinical phenotypes of many autoimmune diseases improve during these pregnancy periods, but reappear after pregnancy with the same kinetics as the clearance of placental AP. Human placental ALP is an enzyme that is fully glycosylated and exposes terminal sialic acids on the oligosaccharide side chains, and therefore is not rapidly cleared from the circulation by the hepatic asialoglycoprotein receptor.

[0031] In principle, the intestinal AP isoenzyme could also be used due to its close similarity to the placental alkaline phosphatase, but we suggest using the placental form rather than the intestinal form due to its favorable plasma residence time of about 6-7 days, thus corresponding to an acceptable treatment regimen for the treatment of chronic diseases. Compared to the placental isoenzyme, the intestinal isoenzyme has a shorter plasma residence time, making the intestinal enzyme suitable for administration in certain acute therapeutic interventions, such as major surgery to combat complications mediated by ischemic injury.

[0032] According to another preferred embodiment, the present invention relates to an ectophosphatase in combination with a disease-modifying antirheumatic drug (DMARDS) for use, wherein the tissue specific ectophosphatase is selected from the group consisting of intestinal AP (IAP), placental ALP (PALP) and hepatic AP (LAP), preferably an IAP, more preferably a PALP or a combination thereof.

[0033] According to yet another preferred embodiment, the present invention relates to a composition, wherein the ectophosphatase is a tissue non-specific ectophosphatase and said treatment is the treatment of a non-chronic arthritic disease.

[0034] According to a preferred embodiment, the present invention relates to an ectophosphatase in combination with a disease-modifying antirheumatic drug (DMARDS) for use, which is administered at least once a month, preferably at least twice a month, more preferably at least three times a month, even more preferably at least four times a month, and most preferably at least five times a month. Furthermore, the present invention relates to a composition, in which the combination is administered at least once a week, more preferably twice a month, preferably at least three times a week, more preferably at least once a month, and most preferably for a period of more than a monthly cycle. Furthermore, the combination of the present invention can be administered at least once every two months, preferably at least once every three months, more preferably at least once every four months, and most preferably at least once every five months. The combination of the present invention can be administered at least once a week.

[0035] As mentioned above, the advantage of placental AP (e.g., native placental AP or recombinant human alkaline phosphatase (hRESCAP)) over replacement of both intestinal and bone, liver or kidney isoenzymes is its extreme plasma residence time of 6-7 days. This T1 / 2 allows for acceptable parenteral administration twice a month, whereas for example intestinal AP must be administered daily, which is undesirable for chronic disease dosing schedules.

[0036] The invention is further described in the following examples, which refer to the figures. [Brief description of the drawings]

[0037] [Figure 1] Figure 1 shows the effect of prophylactic AP treatment (2xAP before intra-articular antibody injection) on ED1+ synovial macrophages in arthritic rats. Massive infiltration of activated macrophages (ED1+) is observed in the inflamed knees of rats ("rheumatoid arthritis") after immunization with methylated BSA (mBSA) and intra-articular boost. The effect is observed in both the lining and sublining, at a depth of 10 (Figure 1A) and 20 (Figure 1B) cell layers, respectively. Note that control knees show only mild macrophage infiltration. A similar mild infiltration is observed after AP treatment ("hRESCAP prophylactic treatment"), with little reaction in the control knees of each rat. [Diagram 2]Figure 2 shows the effect of AP therapeutic treatment (4xAP after the first ia and during the boost) on ED1+ synovial macrophages in arthritic rats. AP therapeutic treatment is compared to MTX treatment. Rats show a positive effect by showing an increase in ED1+ macrophages after mBSA stimulation of the right knee, while AP-treated rats do not show this activity. The effect is observed in both the lining and sublining, at a depth of 10 (Figure 2A) and 20 (Figure 2B) cell layers, respectively. It is noted that AP treatment reduces ED1+ infiltration obtained after intra-articular mBSA injection. However, MTX treatment results in an even greater decrease in the count of ED1+ macrophages, although the count in the negative control knee appears to be higher than in AP-treated rats. [Diagram 3] Figure 3 shows the effect of AP treatment (2xAP before intra-articular antibody injection) on ED2+ synovial macrophages in arthritic rats. After mBSA stimulation of the right knee, rats showing a positive effect by showing an increase in ED1+ macrophages also show a response of ED2+ activity, whereas AP-treated rats do not show this activity. This is consistent with the general concept of inducing an anti-inflammatory response only after a pro-inflammatory event. The effect is observed both in the lining and in the sublining, at a depth of 10 (Figure 3A) and 20 (Figure 3B) cell layers, respectively. It is clear from this that the absence of a pro-inflammatory event even upon AP treatment does not lead to an anti-inflammatory response, as seen by the immunohistochemical readout of reduced ED2+ macrophages. [Figure 4]Figure 4 shows the effect of AP treatment (4xAP after the first ia and during the boost) on ED2+ synovial macrophages in arthritic rats. After stimulation of the right knee with mBSA, rats showing a positive control effect by showing an increase in ED1+ macrophages also show a response of ED2+ activity, while AP-treated rats do not show this activity. This is consistent with the general concept of inducing an anti-inflammatory response only after a pro-inflammatory event. From this it is clear that during therapeutic treatment with AP, even after intra-articular injection with mBSA, the absence of a pro-inflammatory event does not lead to an anti-inflammatory response, as seen by the immunohistochemical readout of reduced ED2+ macrophages. It is noted that with AP treatment, even after intra-articular injection of mBSA, the effect is very reduced and very similar to that obtained with MTX treatment. The effect is observed both in the lining and in the sublining, at a depth of 10 (Figure 4A) and 20 (Figure 4B) cell layers, respectively. [Diagram 5] Figure 5 shows swelling of rat knees (millimeters (mm)): A study of a group of rats treated therapeutically with four intra-articular injections. The black line through the open triangle indicates the positive control. Swelling of the untreated left knee of these rats is mild (Figure 5A). Swelling of the treated right knee is evident (Figure 5B). Reduction in swelling is observed after both prophylactic and therapeutic AP or MTX treatment upon intra-articular injection of mBSA into the right knee. Note that the beneficial effect of both AP and MTX in the untreated left knee is also evident from the reduced swelling when compared to the positive control treatment. [Figure 6] 1 shows immunohistochemical localization and quantification of ED1+ macrophages in synovial tissue of rheumatoid knees from groups of rats that were either untreated, prophylactically or therapeutically treated with AP (i.e., hRESCAP), or therapeutically treated with MTX. Untreated rheumatoid knees contain numerous ED1+ positive macrophages, which are almost absent in control knees. The amount of ED1+ macrophages in arthritic knees is very significantly reduced after both prophylactic and therapeutic AP or MTX treatment. [Figure 7]Figure 1 shows the pharmacokinetics of AP plasma in healthy and arthritic rats. (A) Treatment schedule and time of pharmacokinetic measurements are shown. Healthy and arthritic rats were administered 700 U / kg human recombinant AP, and arthritic rats (as control) received injections of PBS. (B) Blood samples were drawn at baseline and at 15, 60, 120, and 240 min after AP injection, after which plasma samples were processed for AP enzyme activity analysis. Results are expressed as mean ± SD for two rats per group, and assays were performed in duplicate. [Figure 8] Representative images and quantification of ED1 and ED2 positive macrophages in sections of rat knees receiving AP (2x) as prophylactic treatment. (A) Timeline of induction of arthritis in rats and prophylactic intervention with alkaline phosphatase. The first and second immunizations with mBSA (Im1 and Im2) were performed on days 7 and 14, and intra-articular (ia) injections were given on day 21. Prophylactic treatment with AP or PBS was given on days 16 and 19. (B) ED1 images of arthritic and contralateral knees of untreated and 2xAP-treated rats. (C) Quantification of ED1+ macrophages of untreated and 2xAP-treated rats. (D) ED2 images of arthritic and contralateral knees of untreated and 2xAP-treated rats. (E) Quantification of ED2+ macrophages of arthritic and contralateral knees of untreated and 2xAP-treated rats. Error bars indicate SD. [Figure 9]Representative images and quantification of ED1-positive macrophages in sections of knees from arthritic rats receiving AP, MTX, and AP / MTX combination therapy. (A) Treatment schedule. The first and second immunizations with mBSA (Im1 and Im2) were performed on days 7 and 14, followed by intra-articular (ia) injections of mBSA on days 21, 26, 30, and 34. Interventions were performed with AP (700 U / kg), MTX (0.3 mg / kg or 1.0 mg / kg), or the AP / MTX combination. As a control, untreated arthritic rats received injections of PBS. (B) ED1 images of healthy, arthritic, and contralateral knees from rats treated with PBS, MTX (1.0 mg / kg), AP (700 U / kg), and AP+MTX (1). (C) Quantification of ED1+ macrophages in healthy, arthritic and contralateral knees of rats treated with PBS, MTX, AP and AP+MTX. MTX was tested at two doses, namely 0.3mg / kg and 1.0mg / kg. Both doses had the same effect on the number of ED1 macrophages in the arthritic knees, so the results of these two experimental groups were grouped. Group sizes: PBS: n=4, MTX: n=8, AP: n=4 and MTX+AP: n=8. The combined effect of AP and MTX was significantly different from the MTX or AP alone groups (**p<0.01). [Figure 10] Representative coronal PET-CT scans of [18F]fluoro-PEG-folate in arthritic knees from (B) PBS and (C) 4xAP treated rats. [18F]fluoro-PEG-folate is a PET tracer that binds relatively specifically to macrophages. (A) Treatment schedule. (D, E) Standard uptake value (SUV) scale bar, min 0 to max 1, represents tracer uptake. Time activity curves of [18F]fluoro-PEG-folate uptake are expressed as SUV in (D) untreated (PBS only) and (E) 4xAP treated rat arthritic and contralateral knees. PET scans were made on day 40 of the experiment. [Figure 11]Representative immunohistochemical (HE) images of ED1+ and ED2+ macrophages in liver sections from healthy, PBS-treated (control) and AP-treated arthritic rats. (A, B) ED1+ macrophages in livers from PBS-treated and AP-treated arthritic rats, respectively. (C, D) Isotype control stained liver sections from PBS-treated and AP-treated arthritic rats, respectively. (E, F) ED2+ macrophages in livers from PBS-treated and AP-treated arthritic rats, respectively. (G, H) Isotype control stained liver sections from PBS-treated and AP-treated rats, respectively. (I, J) Quantification of ED1+ and ED2+ macrophages in livers from healthy, PBS-treated and AP-treated arthritic rats. Values ​​indicate the mean number of macrophages counted in predefined areas of the liver. Error bars indicate SD. **: p<0.01, ***: p<0.001. [Figure 12] Representative immunohistochemical (HE) images of ED1+ and ED2+ macrophages in spleen sections from healthy, PBS-treated (control) arthritic, and AP-treated arthritic rats. (A, B) ED1+ macrophages in spleen sections from PBS-treated and AP-treated rats, respectively. (C, D) Isotype control stained spleen sections from PBS-treated and AP-treated rats, respectively. (E, F) ED2+ macrophages in spleen sections from PBS-treated and AP-treated rats, respectively. (G, H) Isotype control stained spleen sections from PBS-treated and AP-treated rats, respectively. (I, J) Quantification of ED1+ and ED2+ macrophages in spleens from healthy, saline-treated, and AP-treated rats. Values ​​indicate the mean number of macrophages counted in predefined areas of the spleen. Error bars indicate SD. ***: p<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0038] Rheumatoid Arthritis Knee Effect of hRESCAP (AP treatment) on synovial macrophage infiltration in arthritic rats Experiments were performed using a rat model for RA as published by Chandruputla et al. (BioMed Int, 2015) to show the effect of recombinant human alkaline phosphatase (hRESCAP) in RA. hRESCAP was compared to MTX, used as a positive control. The antigen-induced rat model uses a mixture of methylated bovine serum albumin (mBSA), CFA (complete Freund's adjuvant antigen), and CBP (custom Bordetella pertussis antigen), as well as two subcutaneous immunizations with an intra-articular injection of mBSA in one knee and saline (negative control) in the contralateral knee. Immunohistochemistry (IHC) analysis specifically focuses on infiltration with macrophages to follow knee swelling over time after therapeutic treatment with MTX or hRESCAP.

[0039] In the rat model, RA was induced in the right knee (RA knee) and the contralateral left knee served as an internal control (control knee). The model allows different options for therapeutic intervention, either before the intra-articular injection or during the boost antibody injection with mBSA. Dosing of hRESCAP (700 U / kg, ip) was administered on different schedules as shown in the figure. Two hours before AP administration, rats received a dose of levamisole (50 mg / kg, sc). For comparison, arthritic rats were treated with methotrexate (1 mg / kg, ip).

[0040] At the end of the experiment, rats were sacrificed and knees were decalcified and processed for IHC analysis of synovial macrophage infiltration, including two rat macrophage antibodies, ED1 (homolog of human CD68) and ED2 (homolog of human CD163, a proposed marker for human M2 anti-inflammatory macrophages). IHC analysis of ED1 and ED2 macrophages was performed in multiple quadrants of the synovial tissue as described by Chandruputla et al. (BioMed Int, 2015). Macrophage counts included the synovial lining layer (SL1-10) and the synovial sublining layer (SL1-20).

[0041] animal Groups of 3-6 Wistar rats (male, 150-200 grams, Charles River International Inc, Sulzfeld, Germany) were provided with standard diet, water (ad libitum) and conditions. The animal experiments performed met the standards of the European Communities Council Directive 2010 / 63 / EU for institutional animal care and the Dutch Act on animal experimentation. The experimental protocol was validated and approved by the local committee for animal experimentation of the VU University Medical Center (DEC PET13-07).

[0042] Induction of arthritis and therapeutic intervention All rats (except healthy) were immunized and arthritis was induced via 1x or 4x intra-articular (ia) mBSA injections, 4 or 5 days apart, in the arthritic (right) knee and the contralateral (left, non-arthritic) knee as control knee. For therapeutic interventions, AP (human recombinant placental AP, TNO, Zeist, The Netherlands) was administered subcutaneously (sc) at a dose of 700 U / kg (≈200 μg) and MTX (pharmacy of VU University Medical Center) was administered intraperitoneally (ip) at two doses: 0.3 mg / kg (low dose) and 1.0 mg / kg (high dose). Rats were divided into 8 groups based on different treatments and treatment schedules. In the prophylactic setting, 2 rats received AP twice before intra-articular (ia) arthritis induction and 4 rats received AP twice before the intra-articular and 4x PBS between the ia injections. In the treatment groups, arthritic rats were administered AP twice or 4x after arthritis induction, either as stand-alone therapy or in combination with low or high doses of MTX. Control rats received 500 μL PBS (ip). Healthy rats did not receive arthritis induction or any treatment. At the end of the study, all rats were sacrificed and tissues were excised for further processing and various analyses.

[0043] Alkaline phosphatase activity Using enzyme assays, the plasma concentrations of alkaline phosphatase before and 0 - 4 hours after administration of AP at a dose of 700 U / kg were determined in healthy rats and rats with arthritis (Figure 7). At the time points of 0 minutes, 15 minutes, 60 minutes, 120 minutes, and 240 minutes after AP administration, blood samples were withdrawn from the tail vein of the rats and transferred to 1 ml Eppendorf microtubes containing heparin (454081, Greiner bio-one, Charlotte, USA) as an anticoagulant. As a control, blood was withdrawn at the same time points from arthritic rats injected with 500 μl of PBS at t = 0. The Eppendorf tubes were centrifuged at 3,000×g for 5 minutes at 40°C, and then the plasma was collected and stored at -80°C until use.

[0044] The enzyme assay for AP was based on the conversion of the substrate paranitrophenyl phosphate (PNP, 104105, Sigma-Aldrich, Zwijdrecht, the Netherlands) to paranitrophenol, which was spectrophotometrically measured at 405 nm and 25°C. To a 3 ml reaction cuvette, 2.9 ml of substrate solution (containing a final concentration of 25 mM glycine, 10 mM MgCl2, 3 mM PNP, adjusted to pH 9.6 with NaOH) was added. The reaction was initiated by adding 30 μl of plasma sample diluted 1:1 in enzyme dilution buffer (25 mM glycine, 1 mM MgCl2, 0.1 mM ZnCl2, 10% (v / v) glycerol, adjusted to pH 9.6 with NaOH). In parallel, a reference cuvette was assayed without substrate. The reaction was continued online for 5 minutes at 25°C while continuously monitoring the increase in absorbance at 405 nm using a (10037 - 434, VWR, Radnor, PA <USA) spectrophotometer. From the linear phase of the A405 increase, the AP activity in the plasma sample (U / L) was calculated from a standard curve of serial dilutions of human recombinant placental AP stock solution. One unit of activity was defined as the amount of enzyme that degrades 1 μmol of PNP per minute at 25°C.

[0045] Histopathology and immunohistochemistry Arthritic and contralateral knees from all rats were dissected in whole and fixed in 10% fresh paraformaldehyde with 2% sucrose in PBS (pH=7.3) at 4°C for 7 days, then decalcified in Osteosoft (101728, Merck, Germany) at room temperature for approximately 2.5 weeks. Knees were then embedded in paraffin. 5 μm sections were cut longitudinally at the center of the joint and stained with hematoxylin and eosin (HE) to evaluate the degree of inflammation in the synovial tissue. Liver and spleen sections from all rats were dissected and fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin. 5 μm sections were cut and stained first with HE and then for macrophages. Cells were stained with ED1 (homologous to human CD68) and ED2 (homologous to human CD163, a marker for M2 anti-inflammatory macrophages) for macrophages or with an isotype control antibody. Images were taken using a Leica 4000B microscope and a Leica digital camera DC500 (Microsystems BV, Rijswijk, The Netherlands).

[0046] FRβ immunofluorescence and microscopy Liver and spleen tissues collected at the end of the study were flash frozen in liquid nitrogen and stored at -80°C. Tissues were embedded in the appropriate medium (OCT; SKU4583, Tissue-Tek, Netherlands), cut using a cryotome cryostat (-20°C) (Leica, The Netherlands), and placed on Superfrost (4951PLUS4, ThermoFisher, The Netherlands) slides for immunofluorescence (IF) staining. 8 μm sections were cut and stained with hematoxylin and eosin. Immunostaining of FRβ was performed with mouse anti-rat FRβ antibody or isotype control antibody. Specifically, liver and spleen tissue sections were first brought to room temperature (RT) for 30 min, fixed in acetone (439126, Sigma-Aldrich, The Netherlands) for 10 min (-20°C), and air-dried for 10 min (RT). Sections were marked using a DAKO pen (S2002, DAKO, Santa Carla, CA, USA), which was then washed three times with PBS on a shaker. Sections were then incubated with 100% fetal bovine serum (FBS) for 30 min (RT) to block non-specific binding and washed again with PBS (3 x 5 min). Sections were then incubated with anti-rat FRβ (1:50) in 10% FBS / PBS or with 10% FBS / PBS for 24 h at 4°C. After washing (3 x 5 min in PBS on a shaker), sections were incubated with secondary antibody goat anti-mouse Alexa 488 (1:500) (R37120, ThermoFisher Scientific, Netherlands) in 10% FBS / PBS for 1 h at RT, air-dried and mounted with 2 μl MOWIOL mounting medium (81381, Merck, Zwijndrecht, The Netherlands). 2D IF slides were photographed with a Zeiss Axiovert 200M Marianas™ inverted microscope (40x oil immersion lens).The microscope, camera, and data processing were controlled by SlideBook™ software (SlideBook™ version 6 (Intelligent Imaging Innovations, Denver, Colo.)).

[0047] Quantifying macrophages in knee sections, liver, and spleen All stained slides were blinded and counted by two independent observers for ED1 and ED2 positive synovial macrophages. For this, knee sections were divided into four quadrants (Q1-Q4), each representing the joint capsule with synovial tissue lining on either the proximal or distal side of the bone. Macrophages were counted in the synovial lining and sublining (layers 1-10) in 2-3 areas of each quadrant under a microscope (Leica) at 400x magnification. The mean number of macrophages per area from all four quadrants was combined and presented as the total number (±SD) of ED1 or ED2 macrophages.

[0048] Stained slides of liver and spleen sections from arthritic and AP-treated arthritic rats were blindly counted by two independent observers for FRβ, ED1, and ED2 positive synovial macrophages. For quantification, representative areas of liver and spleen sections were divided into four regions, each representing the central pulp and vein, respectively. FRβ, ED1, and ED2 positive macrophages were counted at 400x magnification as described above. The average number of macrophages per region from all four regions was combined and presented as the total number of FRβ, ED1, or ED2 macrophages. As a reference, liver and spleen sections from healthy rats were analyzed as controls.

[0049] [ 18 F]fluoro-PEG-folate and PET-CT It is a macrophage PET tracer with a radiochemical purity of >96.5% and a mean specific activity of 27.6 ± 3.5 GBq / μmol. 18[F]fluoro-PEG-folate was synthesized. Untreated and 4×AP-treated arthritic rats were anesthetized using inhalation anesthesia (isoflurane 2–2.5% and oxygen 0.45% by volume). The tail vein was cannulated with a polyurethane 3-French cannula (0.7 mm × 19 mm, BD Angiocath, Breda, The Netherlands). During PET-CT (Mediso nanoPET-CT, Budapest, Hungary), rats were placed on an integrated heated bed (approximately 35 °C) while respiratory function was monitored. A computed tomography (CT) scan was performed for 5 min, after which the tracer was administered (10.7 ± 1.8 MBq) at the start of a 60-min dynamic PET scan. PET data were normalized and corrected for scatter, randomness, attenuation, decay, and dead time. List-mode PET data were rebinned in 19 consecutive frames (4x5, 4x10, 2x30, 3x60, 2x300, 3x600, and 1x900 sec) and reconstructed using a 3D Poisson ordered subset iterative expectation-maximization algorithm with 4 iterations and 6 subsets. The resulting images have a matrix size of 225x225x236 voxels with dimensions of 0.4x0.4x0.46 mm3. Images were analyzed using AMIDE software (A Medical Image Data Examiner, version 0.9.2) and expressed as standard uptake values ​​(SUVs). CT and PET images were overlaid to delineate regions of interest (ROIs). Using the final frame with a fixed size, an elliptical shaped ROI (dimensions: 7x4x8 mm3) was hand-drawn over the area of ​​both the arthritic and contralateral knees. Time activity curves (TACs) were extracted by projecting the ROIs onto the video sequences and were expressed as standard uptake values ​​(SUVs), i.e., the mean ROI radioactivity concentration normalized to the injected dose and body weight.

[0050] Ex vivo tissue distribution study At the end of the treatment period, arthritic rats treated with 4×AP, 4×AP / low-dose MTX, 4×AP / high-dose MTX, and untreated rats were [ 18F]fluoro-PEG-folate tracer was administered. 60 min after tracer administration, the rats were sacrificed. Low- and high-dose MTX-treated arthritic rats were sacrificed without tracer administration. At the time of sacrifice, all rats were excised, and knees, blood, and various internal organs were collected, rinsed, immersion-dried, weighed, and the amount of radioactivity was determined using an LKB 1282 Compugamma CS gamma counter (LKB, Wallac, Turku, Finland). Tracer uptake results in various tissues were expressed as percentage of injected dose per gram of tissue (%ID / g).

[0051] statistical analysis Statistical analysis was performed using SPSS for Windows (version 15) (SPSS Inc., Chicago, IL, USA). Wilcoxon signed rank (exact) test was used to determine differences in paired observations such as macrophage infiltration in arthritic knees vs. contralateral knees. Mann-Whitney (exact) test was performed to analyze differences in macrophage infiltration in arthritic knees vs. PBS-treated knees. A p value of less than 0.05 was considered statistically significant. All results were expressed as mean ± standard deviation (SD).

[0052] result Induction of arthritis and AP / MTX therapeutic intervention Induction of arthritis in rats was associated with macroscopic hypertrophy of the arthritic knees compared with the contralateral control knees (data not shown). Therapeutic intervention with AP, MTX, or their combination was well tolerated and was not associated with any adverse effects, and no significant changes in body weight were observed.

[0053] Alkaline phosphatase pharmacokinetics Plasma AP pharmacokinetics was evaluated in healthy and arthritic rats after ip injection of 700 U / kg human recombinant placental AP, and the amount of AP used for therapeutic intervention is shown in Figure 7A and Figure 7B. As a control, plasma AP levels were determined in arthritic rats injected with PBS. The baseline plasma levels of AP were slightly higher in healthy rats (0.27 ± 0.01 U / ml) than in arthritic rats (0.21 ± 0.02 U / ml) (Figure 7B). After AP administration, plasma AP levels increased over 1 h, reaching a maximum of 1.5-1.7 times higher than baseline levels in healthy and arthritic rats, respectively. Notably, the increase in plasma levels of AP levels in healthy rats continued for up to 4 h, while in arthritic rats, AP plasma levels reliably returned to baseline levels. AP levels in PBS-injected arthritic rats did not change over the 4 h sampling time frame (Figure 7B).

[0054] Effects of AP, MTX, and AP / MTX combination therapy on synovial macrophage infiltration The ability of AP, MTX, and AP / MTX to suppress synovial macrophage infiltration in the knee joints of arthritic rats was used as the primary endpoint for evaluating therapeutic efficacy. To this end, macrophage numbers were quantified in sections of arthritic knees versus sections of contralateral knees of arthritic rats by immunohistochemical assessment of the abundance of total ED1- and ED2-positive macrophages, the latter being a representative marker of anti-inflammatory macrophages. Representative images and quantification of ED1- and ED2-positive macrophages in sections of arthritic and contralateral knees before and after therapeutic intervention are shown in Figures 8 and 9. First, two doses of AP administered prior to intra-articular mBSA injection elicited potential preventive activity by suppressing the induction of arthritis, as shown by a significant reduction in the infiltration of both ED1+ (Figure 8B) and ED2+ (Figure 8D) macrophages in the arthritic knees. This was confirmed by quantification of 4-fold (p<0.01) and 8-fold (p<0.01) ED1+ macrophages (FIG. 8C) and 8-fold (p<0.01) ED2+ macrophages (FIG. 8E) in the arthritic knees of AP-pretreated arthritic rats compared to untreated rats (1×ia). These reduced levels are comparable to the macrophage numbers in the contralateral knees. 2×AP treatment (followed by 4×PBS) reduced synovial ED1+ and ED2+ macrophage infiltration by 2-fold (p<0.01) and 3-fold (p<0.01) in the arthritic knees of rats. In a therapeutic setting, 4×AP treatment further reduced the number of these macrophages in the arthritic knees by another approximately 8-fold (<0.001). Next, the effect of AP treatment in combination with MTX on synovial macrophage infiltration was examined. For this, we tested either an effective dose of 1 mg / kg MTX or a lower dose of 0.3 mg / kg MTX. The AP / MTX combination was well tolerated and a slightly increased further reduction in synovial macrophage numbers was observed with the AP / MTX combination when compared to standalone MTX or AP treatment (Figure 9).

[0055] [ 18 PET imaging of [F]fluoro-PEG-folate macrophages To test whether the AP treatment caused a decrease in synovial macrophage infiltration in arthritic rats could also be monitored by PET imaging, PET scans were performed on one of the 4 × AP-treated rats with a macrophage tracer [ 18 F]fluoro-PEG-folate and compared to untreated (PBS only) arthritic rats (Figure 10). Coronal PET-CT images visualize higher tracer uptake in untreated arthritic rats (Figure 10A) compared to 4xAP-treated rats (Figure 10B). 18 Standard uptake values ​​(SUV) of [F]fluoro-PEG-folate were quantified in synovial regions of interest (filled ovals) showing increased (1.5-fold) tracer uptake in arthritic knees from untreated rats (FIG. 10C) compared to arthritic knees from 4×AP-treated rats (FIG. 10D).

[0056] Ex vivo tissue distribution study In other organizations 18 The effect of AP treatment alone or in combination with MTX on [F]fluoro-PEG-folate tracer uptake is shown in Table 1. In all treatment groups, 18 F]fluoro-PEG-folate was rapidly cleared from plasma (Table 1). Note that AP and AP / MTX treatment also increased [ 18 F]fluoro-PEG-folate uptake was decreased in the kidney and intestine (Table 1). Consistent with the high expression of folate receptor α in these organs, tracer uptake was high in these organs but was not affected by AP and AP / MTX treatment.

[0057] [Table 1]

[0058] Effect of AP on systemic inflammation in arthritic rats To test whether the reduced tracer uptake in liver and spleen of AP-treated arthritic rats was associated with reduced macrophage infiltration in these organs, ED1 and ED2 immunohistochemistry was performed on sections of liver (Figure 11) and spleen (Figure 12) of saline-treated vs. arthritic rats, along with liver and spleen tissues of healthy rats as references. Representative images of ED1- and ED2-positive macrophages in liver and spleen sections are shown in (Figures 11A-H) and (Figures 12A-H), respectively. Quantification of ED1- and ED2-positive macrophages in liver (Figures 11I / J) and spleen (Figures 12I / J) showed significantly higher (approximately 4-5 fold, p<0.01) levels in arthritic rat organs compared to those of healthy rats, pointing to a systemic inflammatory component. Following AP treatment, a significant and dramatic reduction (approximately 50%, p<0.001) of both ED1 and ED2 positive infiltrating macrophages was observed in the liver of arthritic rats (FIG. 11I / FIG. 11J). Similarly, in the spleen of arthritic rats, AP treatment also resulted in a significant reduction (approximately 30%, p<0.001) of both ED1 and ED2 positive infiltrating macrophages was observed in the liver of arthritic rats (FIG. 11I / FIG. 11J). Antibody control stained liver and spleen sections were clearly negative for both ED1 and ED2 positive macrophages (FIG. 11C, FIG. 11D, FIG. 11G, FIG. 11H, FIG. 12C, FIG. 12D, FIG. 12G, FIG. 12H). These results highlight that AP establishes a systemic anti-inflammatory effect by reducing macrophage infiltration in the liver and spleen of arthritic rats.

[0059] conclusion Herein, we show that intervention with alkaline phosphatase (AP) induced preventive anti-arthritic activity in rats by suppressing the induction of arthritis after intra-articular antigen injection. Furthermore, in a therapeutic setting, i.e., after induction of arthritis, AP intervention also conveyed a local anti-arthritic effect, represented by a significant reduction in synovial macrophage infiltration in arthritic rats, and a systemic anti-arthritic effect, represented by lower macrophage infiltration in the liver and spleen of arthritic rats. Finally, AP maintains activity in combination treatment with MTX.

[0060] Multiple interventions with human recombinant placental AP (hRESCAP) every 4 days are well tolerated by arthritic rats. The once every 4 days schedule was designed taking into account the half-life of hRESCAP in rats, approximately 3 days. Monitoring AP plasma pharmacokinetics after a single ip dose of 700 U / kg AP in healthy and arthritic rats showed peak plasma levels at 1 h, 50-70% above baseline (Figure 7). Except in healthy rats, supplemented hRESCAP remained stable above control levels in plasma over 4 h, while plasma levels in arthritic rats dropped to basal levels within 4 h. These results confirm previous data on the consumption of available AP during conditions of oxidative stress such as RA and confirm the results reported in human clinical trials. During its action, AP is consumed and eliminated by Kupffer cells as proposed by conjugating with ITM substrates. Consistently, this mode of action may also be involved in the preventive activity of AP in the induction of wet antigen-induced arthritis in rats. Acting as an anti-inflammatory protein, the net effect of AP is to prevent pro-inflammatory cytokines such as TNFα and IL6 from being produced by activated immune cells, thus preventing downstream effects in the inflammatory cascade. The same mode of action of AP may contribute to its therapeutic activity in rats with established arthritis, reflected in reduced macrophage infiltration of the synovium. Removal of ITM inhibits the production of pro-inflammatory cytokines and reduces chemotaxis to attack monocyte / macrophage cells. In addition, systemic and local inflammation leads to increased vascular permeability and extravasation of leukocytes. In this case, AP has also been associated with improving barrier dysfunction by restoring tight junctions between polarized cells, thereby attenuating cell migration.

[0061] Inflamed RA synovium is characterized by the presence of polarized macrophages covering a spectrum of pro-inflammatory (so-called M1 type) and anti-inflammatory macrophages (so-called M2 type). AP intervention influenced both ED1 and ED2 positive macrophage infiltration in synovial tissue. ED2 represents the rat homologue of human CD163, which has been assigned as a marker for M2 type macrophages. This classification may not be strict, since M2 macrophages in the microenvironment of arthritic synovium with ACPA antibodies and complex IgG autoantibodies were found to produce pro-inflammatory cytokines. Thus, AP may affect synovial infiltration of polarized inflammatory macrophages.

[0062] As many cDMARD and bDMARD treatments in RA are combined with MTX, we tested the efficacy of AP and MTX in arthritic rats. The AP / MTX combination was well tolerated and more effective in reducing synovial macrophage infiltration. This was shown above for a dose of 1.0 mg / kg MTX, but also at a lower dose of 0.3 mg / kg MTX, indicating that the MTX dose can be further reduced to identify a schedule for optimal efficacy in combination with AP. Alternatively, AP is the mode of action of MTX by complementing the extracellular conversion of pro-inflammatory AMP, ADP, and ATP to anti-inflammatory adenosine by the action of ectophosphatases CD39 and CD73 on immunocompetent cells.

[0063] Macrophage PET tracer [ 18Ex vivo tissue distribution studies with [F]fluoro-PEG-folate indicate that AP and the AP / MTX combination have a systemic effect beyond the reduction of synovial macrophage infiltration. Systemic inflammation, as indicated by increased macrophage infiltration in the liver and spleen, has been reported in rats with adjuvant-induced arthritis. In the present study, the liver and spleen of arthritic rats were also characterized by increased macrophage infiltration, which was significantly reduced upon AP treatment (Figure 11, Figure 12). The reduction in macrophages included FRβ-positive macrophages, which constitute a marker for activated macrophages. The lower number of FRβ-positive macrophages after AP treatment could explain the reduced tracer uptake in the liver and spleen in the ex vivo tissue distribution studies (Table 1). These results point to a systemic anti-arthritic effect induced by AP, which highlights the systemic activity of AP observed in other animal models for inflammatory diseases.

[0064] Thus, ectophosphatase intervention with AP fills a novel, unique and unsolved gap in RA treatment by combining a different and even synergistic mode of action with MTX and other cDMARDs and bDMARDs. As an anti-inflammatory protein, AP is positioned for the "drug off" period by discontinuing either cDMARDs (due to the development of resistance or toxicity) or biologic therapy (due to tolerization). Given the overall different mode of action, AP can be administered as a stand-alone therapy or combined with other treatment modalities, thus establishing a significant impact in the treatment window. Being an endogenous protein, AP lacks resistance-forming or tolerizing effects. Finally, the potential of AP is further supported by an extremely wide "safety window of use", proving the safety of recombinant human AP in human safety studies.

[0065] AP, both as a preventative and therapeutic intervention, has demonstrated favorable joint and systemic anti-arthritic efficacy in rat models of arthritis. These studies warrant further preclinical and clinical evaluation as a novel putative therapeutic entity for arthritis.

Claims

1. 1. A composition comprising an ectophosphatase in combination with a disease-modifying antirheumatic drug (DMARDS) for use in treating a mammal suffering from arthritis, comprising 11. The composition of claim 1, wherein the arthritis is rheumatoid arthritis, the DMARDS is methotrexate (MTX), and the ectophosphatase is an ectophosphatase selected from the group consisting of intestinal alkaline phosphatase (IAP) and placental alkaline phosphatase (PALP).

2. The composition of claim 1, wherein the ectophosphatase is placental alkaline phosphatase (PALP).

3. The composition of claim 1 or 2, wherein the ectophosphatase is a recombinant alkaline phosphatase.

4. 4. The composition of claim 1, wherein the ectophosphatase is a recombinant mammalian alkaline phosphatase.

5. The composition of claim 4, wherein the ectophosphatase is human alkaline phosphatase.

6. 6. The composition of any one of claims 1 to 5, wherein the ectophosphatase in combination with a disease-modifying antirheumatic drug (DMARDS) is further combined with nanoparticles, the nanoparticles being comprised of a material selected from the group consisting of fullerenes, liposomes, gold, poly-lactic-co-glycolic acid (PLGA), and poly-L-lactic acid (PLA).

7. The composition of any one of claims 1 to 6, wherein the treatment comprises parenteral or oral administration.

8. 8. The composition of any one of claims 1 to 7, wherein the treatment is treatment of a chronic arthritic disease.

9. 9. The composition of any one of claims 1 to 8, wherein the treatment comprises preventing, or delaying the onset, or attenuating the progression of, arthritis.

10. 10. The composition of any one of claims 1 to 9, wherein the treatment comprises attenuation of the inflammatory response in a mammal suffering from arthritis.

11. 11. The composition of any one of claims 1 to 10, wherein the ectophosphatase is administered at least once a month.

12. 12. The composition of claim 11, wherein the ectophosphatase is administered at least twice a month.

13. 13. The composition of claim 11 or 12, wherein the ectophosphatase is administered at least three times a month.

14. 14. The composition of any one of claims 11 to 13, wherein the ectophosphatase is administered at least four times a month.

15. 15. The composition of any one of claims 11 to 14, wherein the ectophosphatase is administered at least five times per month.

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