Title:  Compositions for treating autoimmune disease

United States Patent:  6,518,259

Issued:  February 11, 2003

Inventors:  Holoshitz; Joseph (Ann Arbor, MI); Shayman; James A. (Ann Arbor, MI); Tan; Shi-Yu (Ann Arbor, MI)

Assignee:  The Regents of the University of Michigan (Ann Arbor, MI)

Appl. No.:  575612

Filed:  May 22, 2000

Methods and compositions are described for treating and diagnosing autoimmune diseases, and in particular for treating and detecting rheumatoid arthritis. Treatment is described with a new class of anti-RA drug, namely compounds that inhibit proliferation and induce apoptosis.

DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for treating autoimmune diseases, and in particular for treating rheumatoid arthritis with sphingomyelin pathway inhibitors. The description of the invention that follows discusses I) Fas-mediated Apoptosis, II) Apoptosis And The Immune System, III) Reversing The Resistance to Apoptosis As A Treatment Of Autoimmune Disease. IV) Formulation of Inhibitors of the Sphingomyelin Pathway, and V) Delivery of Formulations and Intra-articular Injections.

I. Fas-Mediated Apoptosis

Homeostasis of mammalian tissues is controlled not only by proliferation and differentiation of cells, but also by cell death. There are two death processes, apoptosis and necrosis. The death of cells during embryogenesis, metamorphosis, endocrine-dependent tissue atrophy, and normal tissue turnover is called programmed cell death. Most of the programmed cell death which occurs during mammalian development proceeds by apoptosis.

Apoptosis can be distinguished morphologically and biochemically from necrosis. Necrosis occurs during pathological cell death as a result of injury, complement attack, severe hypoxia, hyperthermia, lytic viral infection, and exposure to a variety of toxins. Apoptosis is accompanied by condensation and segmentation of nuclei, loss of plasma membrane microvilli, and extensive degradation of the chromosomal DNA into nucleosome units.

It is believed that the apoptotic signal is induced by the binding, of the Fas ligand to Fas. See generally, S. Nagata. "Fas and Fas Ligand: A Death Factor and Its Receptor." Advances in Immunology 57:129 (1994). The cytoplasmic domain of Fas consists of 145 amino acids. About 70 amino acids in this region have significant similarity with a part of the cytoplasmic region of the type I (but not type II) TNF receptor. Indeed, analyses of point mutations in the Fas protein in this conserved region have shown that the domain is essential for the function of Fas.

The Fas ligand has been identified, isolated, and cloned. The amino acid sequence deduced from the nucleotide sequence of the cDNA indicates that the Fas ligand is a TNF-related type II membrane protein. However, despite the high similarity between Fas ligand and TNF (about 30% identical at the amino acid sequence level). Fas ligand does not bind to the TNF receptor.

The tissue distribution of Fas mRNA in the mouse has been examined. Fas mRNA can be detected abundantly in the thymus, heart, liver, and ovary of 8-week old mice, but not in the brain, bone marrow, and spleen. Fas is expressed in almost all populations of thymocytes. This, wide distribution of Fas is in sharp contrast to the tissue restrictive expression of Fas ligand; Fas ligand is found on the Sertoli cells of the testis, on the corneal epithelium, iris and retina, and on activated T lymphocytes.

The activation of Fas is caused by aggregation mediated by the Fas ligand (or other agonist, such as antibodies). The signal is thought to be transduced by clustering of the intracellular domain. The downstream elements of the apoptosis process are thought to be affected by the sphingomyelin signal transduction pathway. See generally,. R. N. Kolesnick et al., "The Sphingomyelin Signal Transduction Pathway Mediates Apoptosis For Tumor Necrosis Factor, Fas, and Ionizing Radiation," Biochem. Cell Biol. 72:471 (1994). This pathway is initiated by enzymatic hydrolysis of the phosphodiester bond of sphingomyelin by a specific sphingomyelin-directed phospholipase C ("sphingomyelinase"), generating ceramide and phosphocholine.

Ceramide serves as the second messenger of the pathway, initiating signaling for several biological agents. Ultimately, ceramide activates the death signaling pathway in a Jun kinase (JNK)-mediated pathway. See M. Verheij et al., "Requirement for ceramide-initiated SAPK/JNK signaling in stress-induced apoptosis," Nature 380:75 (1996).

Ceramide, however, can also be metabolized to sphingosine and sphingosine-1-phosphate (SPP) by the action of ceramidase and sphingosine kinase, respectively. See generally C. J. van Koppen et al., "Activation of a High Affinity G_i Protein-coupled Plasma Membrane Receptor by Sphingosine-1-phosphate," J. Biol. Chem. 217:2082 (1996). SPP has been shown to be involved in stimulating DNA synthesis and cell division, i.e., proliferation. Importantly, SPP has been recently shown to inhibit Fas-mediated cell death. See O. Cuvillier et al., "Suppression of ceramide-mediated Programmed Cell Death By Sphingosine-1-phosphate," Nature 381:800 (199).

II. Apoptosis and the Immune System

The principal physiologic function of the immune system is the elimination of infectious organisms. The effector mechanisms that are responsible for protective immunity are also capable of injuring host tissues. In some situations, specific immune responses have little or no protective value, and the harmful consequences become dominant. The best example of this is autoimmune disease caused by pathologic immune responses against self-antigents.

The immune system has evolved multiple mechanisms for controlling potentially harmful reactions. Failure of these mechanisms may lead to tissue injury and disease. Potentially harmful immune reactions may be prevented either by functionally inactivating or killing the responding lymphocytes. The primary cytolytic mechanism involved in controlling lymphocyte responses is the Fas-mediated apoptotic pathway discussed above. In this manner, the immune system actively eliminates potentially harmful cells so that the host may survive. See A. Abbas, "Die and Let Live: Eliminating Dangerous Lymphocytes." Cell 84:655 (1996).

The mechanism by which Fas-Fas Ligand interactions maintain immunological tolerance to self-antigens is yet to be completely understood. It is believed that repeated stimulation of antigen cause T cells to express high levels of Fas and Fas Ligand, thereby killing either themselves or one another. Such a homeostatic mechanism may limit the size of lymphocyte clones responding to foreign antigens. The same mechanism may be triggered by abundant and disseminated self-antigens, which are able to interact repeatedly with specific T cells.

Abnormalities in Fas-mediated cell death pathways may result in autoimmunity even in situations in which Fas and Fas Ligand are themselves normal, for example, where apoptosis is inhibited and a proliferation pathway is stimulated, activated lymphocytes may escape elimination and cause disease. The present invention contemplates reversing the resistance to apoptosis by inhibiting downstream events of the sphingomyelin pathway. In this manner, activated lymphocytes go through the apoptosis pathway and are eliminated. It is believed that such treatment of RA patients will reduce the symptoms that are characteristic of RA.

III. Reversing the Resistance to Apoptosis as a Treatment of Autoimmune Disease

It is not intended that the present invention be limited to particular points in the sphingomyelin pathway or particular inhibitors of the sphingomyelin pathway. As noted above, SPP has been recently shown to inhibit the Fas-mediated cell death pathway. While, the present invention contemplates inhibiting SPP, thereby reversing the inhibition of Fas-mediated apoptosis, the present invention also contemplates inhibiting the sphingomyelin pathway at other points.

Following Fas ligation, sphingomyelin is converted to ceramide through the activation of acid sphingomyelinase. Ceramide activates the death signaling pathway in a Jun kinase (JNK)-mediated pathway. Through its inhibitory effect on ERK 1/2, ceramide can also block proliferation, thus shifting the balance further toward apoptosis.

Since increased levels of ceramide may promote programmed cell death, in one embodiment, the present invention contemplates treatment of RA patients with inhibitors of ceramide catabolism. It is not intended that the present invention be limited by the nature of the inhibitor. In one embodiment, the present invention contemplates inhibiting ceramide catabolism by inhibiting its conversion to free sphingosine by ceramidase [e.g., by using N-oleoylethanolamine, (1S,2R)-D-erythro-2-(N-myristoylamino)-phenyl-1-propanol ("D-MAPP") or other suitable inhibitor]. In another embodiment, the present invention contemplates inhibiting ceramide catabolism by inhibiting metabolism to glucosylceramide by glucosylceramide synthase [e.g., by using D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ("PDMP") and newer homologues or by using N-butyldeoxynojirimycin or other suitable inhibitors]. In yet another embodiment, the present invention contemplates inhibiting ceramide catabolism by inhibiting metabolism to sphingomyelin by sphingomyelin synthase or by inhibiting metabolism to ceramide-1-phosphate by ceramide kinase. In still another embodiment, the present invention contemplates inhibiting ceramide catabolism by inhibiting metabolism to 1-O-acylceramide by ceramide transacylase (e.g. by using D- and L-erythro enantiomers of PDMP and related homologues).

On the other hand, as discussed above. SPP has an antagonistic effect to that of ceramide. It is produced from sphingosine by the activity of sphingosine kinase. It has a dual, Gi-protein-dependent, anti-apoptotic effect. It blocks JNK activation on the one hand, and leads to ERK 1/2 activation on the other. Thus, the net effect of SPP is inhibition of ceramide-mediated cell death.

While it is not intended that the present invention be limited to a precise understanding of the mechanism, it is believed that resistance to Fas-mediated cell death in RA is due to a shift in the ceramide/SPP rheostat. Suppressing the pathway (e.g. by suppressing SPP synthesis with a sphingosine kinase inhibitor, or blocking its Gi-mediated effects) can both restore the susceptibility of RA lymphocytes to killing by Fas ligation. Accordingly, targets for therapeutic intervention in RA include (but are not limited to) inhibition of sphingosine kinase, inhibition of Gi proteins, inhibition of MEK1, activation of JNK, or a combination of two or more of those modalities.

In one embodiment, the present invention contemplates treating RA patients with compounds that inhibit sphingosine-phosphate formation. While inhibition of sphingosine kinase (e.g., with sphingosine derivatives) has been discussed, other approaches to inhibiting sphingosine phosphate formation are contemplated, including inhibiting (1) synthesis of long chain bases (e.g. by use of .beta.-chloroalanine or L-cycloserine or other suitable inhibitor), (2) inhibition of acylation of long chain bases, a critical step in eventual sphingosine formation (e.g. using fumonisin B1 or other suitable inhibitor), and (3) stimulation of sphingosine-1-phosphate phosphatase or lyase.

IV. Formulation of Inhibitors of the Sphingomyelin Pathway

The present invention contemplates preparations comprising inhibitors of the sphingomyelin signal transduction pathway. Such formulations can be prepared either as liquid solutions or suspensions, or in solid forms. Formulations may include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and typically contain 1%-95% of active ingredient, preferably 2%-70%.

The compositions are also prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. For intra-articular injections (see below), the present invention contemplates formulations comprising one or more inhibitors of the sphingomyeline pathway along with one or more local anesthetic. It is not intended that the present invention be limited to particular anesthetics. A variety are contemplated including but not limited to procaine or lidocaine. When injecting a bursa, tendon sheath, or periarticular region, such a mixture will give immediate relief (due to the anesthetic) followed by more lasting relief (due to the inhibitor).

Where mixtures with local anesthetics are not desired, a topical anesthetic prior to injection may be used. Such topical anesthetics include but are not limited to ethyl chloride spray on the skin over the joint to be injected. Alternatively, a local anesthetic may be given first, followed by administration of one or more of the above-described inhibitors.

V. Delivery of Formulations and Intra-articular Injections

It is not intended that the present invention be limited to the particular route of administration. The sphingomyelin pathway inhibitors can be given orally or injected (including but not limited to intravenous injection). The present invention specifically contemplates intra-articular injections in RA patients.

To perform an arthrocentesis, the specific area of the joint to be injected is palpated and is then marked, e.g., with firm pressure by a ballpoint pen that has the inked portion retracted. This will leave an impression that will last 10 to 30 minutes. (The ballpoint pen technique can also be used with soft tissue injection.) The area to be aspirated and/or injected should be carefully cleansed with a good antiseptic, such as one of the iodinated compounds. Then the needle can be inserted through the ballpoint pen impression.

Helpful equipment includes the following items: alcohol sponges; iodinated solution and surgical soap; gauze dressings (2.times.2); sterile disposable 3-, 10- and 20-ml syringes; 18- and 20-gauge, 11/2-inch needles; 20-gauge spinal needles; 25-gauge, 5/8-inch needles; plain test tubes; heparinized tubes; clean microscope slides and coverslips; heparin to add to heparinized tubes if a large amount of inflammatory fluid is to be placed in the tube; fingernail polish to seal wet preparation; chocolate agar plates or Thayer-Martin medium; tryptic soy broth for most bacteria; anaerobic transport medium (replace periodically to keep culture media from becoming outdated); tubes with fluoride for glucose; plastic adhesive bandages; ethyl chloride; hemostat; tourniquet for drawing of simultaneous blood samples; and 1 percent lidocaine.

Knee

The knee is the easiest joint to inject. The patient should be in a supine position with the knee fully extended. The puncture mark is made just posterior to the medial portion of the patella, and an 18- to 20-gauge, 11/2-inch needle directed slightly posteriorly and slightly inferiorly. The joint space should be entered readily. On occasion thickened synovium or villous projections may occlude the opening of the needle, and it may be necessary to rotate the needle to facilitate aspiration of the knee when using the medial approach. An infrapatellar plica, a vestigal structure that is also called the ligamentum mucosum, may prevent adequate aspiration of the knee when the medial approach is used. However, the plica should not adversely affect injections or aspirations from the lateral aspect.

Shoulder

Injections in the shoulder are most easily accomplished with the patient sitting and the shoulder externally rotated. A mark is made just medial to the head of the humerus and slightly inferiorly and laterally to the coracoid process. A 20- to 22-gauge, 11/2-inch needle is directed posteriorly and slightly superiorly and laterally. One should be able to feel the needle enter the joint space. If bone is hit, the operator should pull back and redirect the needle at a slightly different angle.

The acromioclavicular joint mast be palpated as a groove at the lateral end of the clavicle just medial to the shoulder. A mark is made, and a 22- to 25-gauge. 5/8 to 1-inch needle is carefully directed inferiorly. Rarely is synovial fluid obtained.

The sternoclavicular joint is most easily entered from a point directly anterior to the joint. Caution is necessary to avoid a pneumotharax. The space is fibrocartilaginous, and rarely can fluid be aspirated.

Ankle Joint

For injections of the inhibitors of the present invention in the ankle joints, the patient should be supine and the leg-foot angle at 90 degrees. A mark is made just medical to the tibialis anterior tendon and lateral to the medial malleolus. A 2- to 22-gauge, 11/2-inch needle is directed posteriorly and should enter the joint space easily without striking bone.

Subtalar Ankle Joint

Again, the patient is supine and the leg-foot angle at 90 degrees. A mark is made just inferior to the tip of the lateral mallcolus. A 20- to 22-gauge, 11/2-inch needle is directed perpendicular to the mark. With this joint the needle may not enter the first time, and another attempt or two may be necessary. Because of this and the associated pain, local anesthesia may be helpful.

Wrist

This is a complex joint, but fortunately most of the intercarpal spaces communicate. A mark is made just distal to the radius and just ulnar to the so-called anatomic snuff box. Usually a 24- to 26-gauge, 5/8 to 1-inch needle is adequate, and the injection is made perpendicular to the mark. If bone is hit, the needle should be pulled back and slightly redirected toward the thumb.

First Carpometacarpal Joint

Degenerative arthritis often involves this joint. Frequently the joint space is quite narrowed, and injections may be difficult and painful. A few simple maneuvers may make the injection fairly easy, however. The thumb is flexed across the palm toward the tip of the fifth finger. A mark is made at the base of the first metacarpal bone away from the border of the snuff box. A 22- to 26-gauge, 5/8 to 1-inch needle is inserted at the mark and directed toward the proximal end of the fourth metacarpal. This approach avoids hitting the radial artery.

Metacarpophalalangeal Joints and Finger Interphalangral Joints

Synovitis in these joints usually causes the synovium to bulge dorsally, and a 24- to 26-gauge. 1/2 to 5/8-inch needle can be inserted on the either side just under the extensor tendon mechanism. It is not necessary for the needle to be interposed between the articular surfaces. Some prefer having the fingers slightly flexed when injecting the metacarpophalangeal joints. It is unusual to obtain synovial fluid. When injecting, a mix of the inhibitors of the present invention with a small amount of local anesthetic is preferred.

Metatarsophalangeal Joints and Toe Interphalangeal Joints

The techniques are quite similar to those of the metacapophalangeal and finger interphalangeal joints, but many prefer to inject more dorsally and laterally to the extensor tendons. Marking the area(s) to be injected is helpful as is gentle traction on the toe of each joint that is injected.

Elbow

A technique preferred by many is to have the elbow flexed at 90 degrees. The joint capsule will bulge if there is inflammation. A mark is made just below the lateral epicondyle of the humerus. A 22-gauge, 1 to 11/2-inch is inserted at the mark and directed parallel to the shaft of the radius or directed perpendicular to the skin.

Hip

This is a very difficult joint to inject even when using a fluoroscope as a guide. Rarely is the physician quite sure that the joint has been entered; synovial fluid is rarely obtained. Two approaches can be used, anterior or lateral. A 20-gauge, 31/2-inch spinal needle should be used for both approaches.

For the anterior approach, the patient is supine and the extremity fully extended and externally rotated. A mark should be made about 2 to 3 cm below the anterior superior iliac spine and 2 to 3 cm lateral to the femoral pulse. The needle is inserted at a 60 degree angle to the skin and directed posteriorly and medially until bone is hit. The needle is withdrawn slightly, and possibly a drop or two of synovial fluid can be obtained, indicating entry into the joint space.

Many prefer the lateral approach because the needle can "follow" the femoral neck into the joint. The patient is supine, and the hips should be internally rotated the knees apart and toes touching. A mark is made just anterior to the greater trochanter, and the needle is inserted and directed medially and sightly cephalad toward a point slightly below the middle of the inguinal ligament. One may feel the tip of the needle slide into the joint.

Temporomandibular Joint

For injections, the tempormandibular joint is palpated as a depression just below the zygomatic arch and 1 to 2 cm anterior to the tragus. The depression is more easily palpated by having the patient open and close the mouth. A mark is made and, with the patient's mouth open, a 22-gauge, 1/2 to 1-inch needle is inserted perpendicular to the skin and directed slightly posteriorly and superiorly.

Claim 1 of 5 Claims

What is claimed is:

1. A composition, comprising at least one inhibitor of the sphingomyelin signal transduction pathway, selected from the group consisting of methylsphingosine, dimethylsphingosine, (1S,2R)-D-erythro-2-(N-myristoylamino)-phenyl-1-propanol, N-butyldeoxynojirimycin and 2-(2'-amino-3'-methoxyphenyl-oxanaphthalen-4-one) in combination with an anesthetic.
 

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