WO2016102530A1

WO2016102530A1 - Cyclic phosphoric acid derivative for the treatment of chronic fatigue syndrome

Info

Publication number: WO2016102530A1
Application number: PCT/EP2015/080910
Authority: WO
Inventors: Öystein FLUGE; Olav Mella
Original assignee: Bergen Teknologioverføring As
Priority date: 2014-12-22
Filing date: 2015-12-22
Publication date: 2016-06-30

Abstract

The present invention relates in first aspect to a cyclic phosphoric acid derivative for use in the treatment of chronic fatigue syndrome,also known myalgic encephalomyelitis. In particular, the present invention relates to the use of a pharmaceutical composition containing the cyclic phosphoric acid derivative for the treatment of chronic fatigue syndrome/myalgic encephalomyelitis in a subject afflicted with said disease. In a further aspect, the present invention relates to a combination of the cyclic phosphoric acid derivative as defined herein with a B-cell depleting agent for use in the treatment of chronic fatigue syndrome. The combination may be provided in form of a kit comprising suitably effective dosages of said compounds. Further, the present invention relates to a method for treating chronic fatigue syndrome comprising the step of administering the cyclic phosphoric acid derivative to a subject afflicted therewith, optionally, in combination with a B-cell depleting agent.

Description

Cyclic phosphoric acid derivative for the treatment of chronic fatigue syndrome

The present invention relates in first aspect to a cyclic phosphoric acid derivative for use in the treatment of chronic fatigue syndrome, also known as myalgic encephalomyelitis. In particular, the present invention relates to the use of a pharmaceutical composition containing the cyclic phosphoric acid derivative for the treatment of chronic fatigue syndrome/myalgic encephalomyelitis in a subject afflicted with said disease. In a further aspect, the present invention relates to a combination of the cyclic phosphoric acid derivative as defined herein with a B- cell depleting agent for use in the treatment of chronic fatigue syndrome. The combination may be provided in form of a kit comprising suitably effective dosages of said compounds. Further, the present invention relates to a method for treating chronic fatigue syndrome comprising the step of administering the cyclic phos- phoric acid derivative to a subject afflicted therewith, optionally, in combination with a B-cell depleting agent.

Technical background

Chronic fatigue syndrome (CFS) also described as myalgic encyophalitis (ME) is a disease affecting approximately 0.2 percent of the population (Nacul et al, BMC med 201 1 , 9:91 ). It is a disease affecting women three to four times more often than men and often preceded by an infection. It is an assumed genetic predisposition for CFS (Albright et al, 201 1 , BMC neurol 1 1 :62). According to the clinical working case definition (Canadian criteria) for CFS/ME, Carruthers B.M., et al ., 2003, J . Chronic Fatigue Syndr, 1 1 :7-36, the main symptoms are post-exertional malaise, with cognitive disturbances, pain, sensory hypersensitivity, and several symptoms related to neuroendocrine and autonomic function. CFS is characterized by an unexplained, severe fatigue, persisting for at least six consecutive months, and with a substantial reduction of previous levels in occupational, social or personal activities. Although many studies have shown alterations in blood tests or radiological investigations, no consistent pattern has emerged, and no specific biomarker or diagnostic test exists. That is, the aetiology of CFS remains unclear. Various hypotheses include immunological, virological, neuroendocrinological, and psychological mechanisms. The pathogenesis of CFS is presumed to be multifactorial and to involve both host and environmental factors.

Many patients suffering from CFS have a history of an acute viral or bacterial infection preceding the development of fatigue. However, no persisting infection has been proven.

Furthermore, several gene expression studies have been performed in CFS, indicating that there are specific but complex gene alterations in accordance with the dysfunction in immune response and in defence mechanisms. For example, Kaushik N., et al., 2005, J. Clin Pathol 58:826-32 describe a microarray study showing differential expression of 16 genes in CFS suggesting T-cell activation and a disturbance of neuronal and mitochondrial function. Other microarray studies concluded that several genes affected mitochondrial function and cell cycle deregulation. Moreover, alterations in membrane transport and ion channels were described. Based on the numerous studies, the gene expression data are not conclusive, but suggest that there are disturbances in CFS representing various cellular functions.

A study in 2007 of post-infective fatigue syndrome found no differences in ex vivo cytokine production over a 12-month period, as compared to controls recovering promptly after infection (Vollmer-Conna U., et al ., 2007, Clin Infect Dis 45:732-5). It is speculated that CFS patients may have a reduced immune cell function with a low NK cell cytotoxicity and immunoglobulin deficiencies.

For example, Ogava M., et al., 1998, Eur J of Clin Invest, 28:937-943 describes decreased nitric oxide mediated natural killer cell activation in chronic fatigue syn- drome. It is identified therein, that NO donors were able to stimulate NK cell activity in healthy control subjects, but not in NK cells obtained from CFS patients and stimulated in vitro. Studies demonstrated several abnormalities in laboratory markers associated with immune functions in CFS patients. For example, a low NK cell cytotoxicity, but also an increase in CD8 + T cells, elevated numbers of CD20+ B-cells, and an increase in the B-cell subset expressing CD20 and CD5 has been described. A study comparing CFS patients and controls reported decreased expression of CD69 on T cells in NK cells after mitogenic stimulation in vitro, indicating a disorder in the early activation of cellular immunity mediated by the cells (Mihaylova I., et al ., 2007, Neuro Endocrinol Lett 28:477-83).

However, the data on immune bioregulation in CFS are not consistent, e.g. as discussed in Brenu et al, 2012, J Trans Med 10:88.

Along with hypotheses of immune deregulation in CFS, autoimmunity to endogenous vasoactive neuropeptides has been proposed as a mechanism for the disease, Staines DR., 2005, Med Hypotheses 64:539-42, however not supported by sufficient scientific data. Other reports are discussing various autoantibodies in conjunction with CFS. However, no clear association was proved. Thus, there is no direct evidence with consistent data for the presence of pathogenic autoantibodies or for T-lymphocyte mediated autoimmunity. That is, CFS is at present not defined as an autoimmune disease. Rather, CFS is still identified as a disease with unknown aetiology.

Various hypotheses for CFS pathogenesis are discussed in the art including blood platelet dysfunction, neurological, neuroendocrine, metabolic or autonomic disturbances, ion channel dysfunction, zinc deficiency, toxin exposure or prior vac- cination, etc., However, no consistent picture has emerged for the aetiology and pathogenesis of CFS. Due to the lack of knowledge of the exact pathogenesis and with no known causal mechanism, there is no current standard specific treatment for CFS. The unknown aethiology of CFS is probably the reason for the remarkably few studies performed, evaluating therapy based upon a biological hypothesis. Studies are described in the art testing treatment with immunoglobuline, or treatment with anti-viral compositions, like valganciclovir.

The inventors of the present invention published a Case series, Fluge O., Mella O., 2009, BMC Neurol. 9:28 followed by a double-blinded and placebo controlled, randomized phase II study, Fluge O., et al., 201 1 , PLOS 6:e26359, exploring B- cell depletion using the therapeutic monoclonal anti-CD20 antibody Rituximab, showing a clinical benefit in 2/3 of CFS patients. The use of B-cell depleting agents is described in WO 2009/083602. The patterns of responses and relapses, with a time delay of 2 to 8 eight months from start of Rituximab infusion (with rapid B-cell depletion) until start of clinical responses, indicate that an antibody may be involved in the pathogenesis. Recently, a case control study performed in elderly aged more than 65 years, investigating more than 1 million cases in cancer and 100.000 healthy controls, with a prevalence of CFS diagnosis of 0.5 percent in both groups, show that elderly CFS patients had a modest but highly significant risk of B-cell lymphomas, Chang CM., Cancer, 2012, 1 18:5929-36, consistent with a chronic B-cell activation. Taken together, the data on treatment with a monoclonal anti-CD20 antibody examplified by Rituximab indicate that CFS in a subset of patients may be a post-infectious immune dysregulation, possibly a variant of autoimmune mechanisms, possibly with a genetic predisposition, in which B lympho- cytes are important for symptom maintenance.

Ifosfamide, also marketed as Ifex, and cyclophosphamide, also marketed as en- doxan, Cytoxan, neosar, procytox, refimune, and also known as cytofosfane, are nitrogen mustard alkylating agents used in the treatment of cancer.

This compounds are based on a oxazaphosphorene group. Today, cyclophosphamide is used for the treatment of numerous malignant processes and autoimmune diseases. The main effect of cyclophosphamide is due to its metabolite phosphoramide mustard. Phosphoramide mustard forms DNA crosslinks whereby these crosslinks are irreversible and lead to cell apoptosis.

It is described that cyclophosphamide induces beneficial immodulatory effects in adaptive therapy and it is speculated that it eliminates regulatory T-cells and enhances adoptive T-cell immunotherapy regimens.

For example, cyclophosphamides as a representative of nitrogen mustard alkylating agent, have been described in the 1950s and were approved by the FDA in 1959. As noted, today cyclophosphamide is used in a numerous treatment regimens against cancer and autoimmune diseases. Its main use is in combination with other chemotherapy agents in the treatment of malignant tumors and leukaemia as well as some sort of solid tumors. It is speculated that the chemotherapy drug partly works by inducing the death of certain T-cells.

As indicated above, using B-cell depletion by the monoclonal anti-CD20 antibody Rituximab in CFS, there is a significant delay from start of the treatment and the beginning of the symptoms relief. Also, in the clinical studies performed with Rituximab, approximately 2/3 of CFS patients have a clinical response.

Thus, there is an ongoing need to provide additional compounds useful in the treatment of chronic fatigue syndrome. In particular, there is an ongoing need for providing compounds which act fast in the patients without the lag period described for the B-cell depleting agents before. In addition, there is a continuous demand for compounds which may also be effective in patients not susceptible to a B-cell depleting agent treatment or with non-responsiveness to the treatment. One major problem of the treatment of CFS is the induction of tolerance against the active drug, thus, only a transient beneficial effect may be achieved. Description of the present invention

In a first aspect, the present invention relates to a cyclic phosphoric acid derivative of formula I for use in the treatment of chronic fatigue syndromes:

R

wherein Ri is selected from hydrogen, Ci - C4 alkyl optionally substituted with halogen;

X2 is selected from an ethylene imine group or a group of Formula II

formula (II)

wherein R is hydrogen or Ci - C4 alkyl optionally substituted with halogen, HAL is halogen;

Y, Z are independently from each other selected from a hydrogen, Ci - C4 alkyl optionally substituted with halogen or a hydroxy group;

m, n are independently from each other 2 or 3; or pharmaceutically acceptable salt or solvates thereof.

That is, the present inventors have recognized that administration of the cyclic phosphoric acid derivative of formula I, for example in form of the cyclophospha- mide-Monohydrate or ifosfamide, relieve the symptoms of CFS and, thus, may be useful in the treatment of CFS accordingly. In contrast to the treatment with a B-cell depleting agent, the present inventors achieved rapid relief, e.g. within weeks from start of administration of the cyclic phosphoric acid derivative formula I with dosages similar to the dosages of said compounds used in the treatment of other diseases. This is in contrast to the so far described medication using Rituximab for a treatment of CFS. As described e.g. in WO 2009/083602 and subsequent publications of the inventors, treatment with Rituximab as a representative of a B-cell depleting agent is characterized by a remarkable lag time before clinical responses.

Surprisingly, the administration of the cyclic phosphoric acid derivatives according to the present invention allow a treatment of CFS patients for early relief of symptoms without a long delay.

In the context of the present invention, the terms "chronic fatigue syndrome", CFS and "myalgic encephalomyelitis", ME, are used synonymously.

As used herein, the term cyclic phosphoric acid derivative of formula I refer to the compound of formula I as shown herein . In this connection, the term "derivative" refers to any of the possibilities of the general formula I accordingly. That is, the present inventors recognized that treating individuals suffering from chronic fatigue syndrome with the compound of general formula I results in early relief of symptoms.

In an embodiment of the present invention, the cyclic phosphoric acid derivatives of formula I for use in the treatment of chronic fatigue syndrome or as active agent in the method of treating individuals suffering from CFS are compounds wherein Ri is hydrogen or Ci - C4 alkyl substituted with CI, Y, Z are hydrogen, m, n are independently from each other 2 or 3, X2 is Formula II with R being hydrogen or Ci - C4 alkyl substituted with CI , and HAL is CI . The term "Ci - C4 alkyl" identifies Ci , C2, C3 and C4 straight or branched alkyl groups including methyl and ethyl groups. Further, the alkyl group may be substituted with a halogen. The halogen substitution may be at any of the carbon atoms present in the alkyl group. Substitution may be with at least one halogen, but, it may also be possible that more than one, e.g. two or three halogens are present. When more than one halogen are present as substituents, said halogens may be present at one carbon atom or at least at two different carbon atoms. Preferably, substitution is at the terminal carbon atom.

The term "halogen" as used herein, also referred to as HAL, include the halogens F, CI, Br and I. It is preferred that the halogen is CI .

As used herein, the term "comprising", "comprises", "containing" or "contains" includes the embodiments of "consisting of" or "consist". In another embodiment of the present invention, the cyclic phosphoric acid derivative of formula I is for use in the treatment of CFS or administered in the method of treating CFS is a compound wherein X2 is formula II, m is 3, n is 2, z is hydrogen, y is hydrogen, R is hydrogen or C2-alkyl-CI . It is particularly preferred, that the phosphoric acid derivative is an active drug or pro-drug selected from cyclophosphamide or ifosfamide. Both, cyclophosphamide and ifosfamide are pro-drugs which are converted by oxidase enzymes in the liver to active metabolites. For cyclophosphamide, the main active metabolite is 4-hy- droxic cyclophosphamide.

The cyclic phosphoric acid derivatives according to the present invention may be used in form of its free compounds as salts thereof or in form of solvates, like hydrates. For example, cyclophosphamide are administered in form of the cyclo- phosphamide-Monohydrate. The route of administration of the cyclic phosphoric acid derivative of formula I according to the present invention depends on the formulation used. That is, the cyclic phosphoric acid derivative may be administered in form of an infusion, or in form of capsules or other suitable forms, like tablets. In addition, the cyclic phosphoric acid derivative according to the present invention may be in form of a compound of immediate release or in form of a delayed or sustained release. Furthermore, if applicable, the cyclic phosphoric acid derivative according to the present invention may be provided in powder form for oral use.

That is, the cyclic phosphoric acid derivative according to the present invention may be adapted for systemic administration, for example via the enteral or parenteral route. In another embodiment, the cyclic phosphoric acid derivative is adapted for mucosal or local administration. For example, the administration may be via the parenteral route, e. g. in form of an infusion .

In another embodiment, the cyclic phosphoric acid derivative according to the present invention is adapted for the administration to a subject in a single therapeutically effective daily dosage thereof or in form of multiple therapeutically active daily dosages thereof. The skilled person is well aware of the effective dose to be administered. Typically, the daily dose is similar to the daily doses administered in the treatment of other diseases treated with the cyclic phosphoric acid derivative as described herein. For example, for cyclophosphamide, the dosage administered by infusion presently tested in three pilot patients is an intravenous pulse given 4 weeks apart to a total of 6 doses (six months total treatment time). The single doses were 500 mg/nn2, increasing to 700mg/nn2 given intravenously. Alternatively, oral continuous cyclophosphamide may be used . The dosage regimen of ifosfamide can be at about 1 -2g/m2 every 3-4 weeks as an intravenous infusion That is, in another aspect, the present invention relates to a pharmaceutical composition comprising the cyclic phosphoric acid derivative or formula I as defined herein and a pharmaceutically acceptable diluent or carrier for use in the treatment of chronic fatigue syndrome. Pharmaceutical compositions can be obtained by processing the cyclic phosphoric acid derivative of the formula I as defined herein with pharmaceutically acceptable inorganic or organic carriers. Lactose, corn starch or derivatives thereof, steric acid or salts and the like can be used for example as such carriers for tablets, coated tablets, dragees and hard gelatine capsules. Suitable carriers for soft gelatine capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols and the like. Depending on the nature of the active substance, no carriers are, however, usually required in the case of soft gelatine capsules. Suitable carriers, excipients or diluents, for the reduction of solutions and syrups are for example water, polyols, glycerol, vegetable oil and the like.

The pharmaceutical compositions can, moreover, contain preservatives, solubil- izers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.

Moreover, the present invention relates to a composition containing a combination of cyclic phosphoric acid derivative of formula I as defined herein and a B-cell depleting agent. In addition, as used herein, the composition or pharmaceutical composition may be in form of one composition or maybe formulated into two separate compositions, e. g. to separate pharmaceutical composition.

As used herein, the terms "subject", "individuals" and "patient", are used herein interchangeably.

In a further embodiment, the composition of the present invention comprising a combination of cyclic phosphoric acid derivative of formula I and a B-cell depleting agent, are in form of a pharmaceutical composition for use in the treatment of chronic fatigue syndrome, in particular for use in the treatment of chronic fatigue syndrome wherein the compounds are administered simultaneously, separately or sequentially.

In an embodiment, the pharmaceutical composition is designed to allow admin- istration of the cyclic phosphoric acid derivate in a pharmaceutically effective dosage over a time range of the first eight to sixteen weeks, for example, for the first twelve weeks of treatment, like over a time range of the first eight weeks of the treatment, likewise in the three months or four months from the beginning of the treatment. Of course, the treatment regimen depends on the drug administered as well as on the way of administration. The skilled artisan is well aware of suitable dosages and treatment regimen depending on the drug.

In addition, the pharmaceutical composition is designed that the B-cell depleting agent is adapted for administration 1 or 2 infusions twice within the first two weeks and, thereafter, administering the B-cell depleting agent once every two or three months for maintaining a beneficial effect.

As used herein, the term "B-cell depletion" or "B-cell depleting activity" or "B-cell depleting agent" refers to the ability of the entity, either a chemical or biological entity, e. g. an antibody, to reduce circulating B-cells levels in a subject. B-cell depletion may be achieved e. g. by inducing cell death, inducing apoptosis, or reducing proliferation.

The CD20 molecule, also called human B-lymphocyte-restricted differentiation antigen or Bp35, is a hydrophobic transmembrane protein located on pre-B and ma- ture B lymphocytes that has been described extensively in the art.

CD20 is expressed on greater than 90 % of B-cells from peripheral blood or lymphoid organs in humans. CD20 is present on both normal B-cells as well as pathological B-cells, but is not expressed on stem cells. The term CD20 includes the equivalent molecules of other species than human. Of note, recently low level expression of CD20 on a substitute of T-cells and in K-cells have been reported.

A "B-cell" is a lymphocyte that matures within the bone marrow, and includes a naive B cell, memory B cell, or effector B cell (plasma cells).

In a broader sense, the present invention relates not only to the use of antibodies or fragments thereof for the treatment of CFS, but to the use of antagonists of the CD20 molecule in general having a B-cell depleting activity for the treatment of CFS.

An "antagonist" or "B-cell depleting agent" which is used herein interchangeably is a molecule which, e. g. upon binding to a B cell surface marker, like CD20 on B cells, destroys or depletes B cells in a mammal and/or interferes with one or more B cell functions, e.g. by reducing or preventing a humoral response elicited by the B cell. The antagonist or B-cell depleting agent according to the present invention is able to deplete B cells (i.e. reduce circulating B cell levels) in a mammal treated therewith. Such depletion may be achieved via various mechanisms such antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC), inhibition of B cell proliferation and/or induction of B cell death (e.g. via apoptosis). Antagonists included within the scope of the present invention include antibodies, synthetic or native sequence peptides and small molecule antagonists which bind to the B cell surface marker, optionally conjugated with or fused to a cytotoxic agent. A preferred antagonist is a CD20 antibody or CD20-binding antibody fragment. Furthermore, small molecule antagonists are preferred, like the known B-cell depleting agent Methotrexat.

Insofar that other cells than B-cells express the CD20 antigen like a subset of T- cells or NK-cells, these cells are also depleted with the B-cells depleting agent being an agent acting via CD20. Antagonists which "induce apoptosis" are those which induce programmed cell death, e.g. of a B cell, as determined by standard apoptosis assays, such as binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies {e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

In a preferred embodiment, the antibody useful for the treatment of CFS is a B- cell depleting CD20-binding antibody fragment.

"CD20-binding antibody fragments" comprise a portion of an intact antibody which comprises the antigen binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single- chain antibody molecules; and multispecific antibodies formed from antibody fragments. For the purposes herein, an "intact antibody" is one comprising heavy and light variable domains as well as an Fc region.

Moreover, it is assumed that other B-cell depleting agents, in particular, anti-CD22 antibodies, like Epratuzumab or anti-CD19 humanized antibodies, like MDX-1342, can be used for the treatment of CFS.

The terms "rituximab" or "RITUXAN®" or "mabthera" herein refer to the genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen and designated "C2B8" in US Patent No. 5,736,137, expressly incorporated herein by reference, including fragments thereof which retain the ability to bind CD20. Purely for the purposes herein and unless indicated otherwise, "humanized 2H7" refers to a humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody is effective to deplete primate B cells in vivo.

The expression "effective amount" of the B-cell depleting agent or antagonist, in particular of the anti-CD20 antibody or CD20-binding antibody fragment thereof, refers to an amount of the B-cell depleting agent or antagonist which is effective for treating CFS. For example, the anti-CD20 antibody for the treatment of chronic fatigue syndrome/myalgic encephalomyelitis is administered in the range of 10 mg to 5000 mg per dosage. For example, the dosage may be in the range of from 100 to 1000 mg/m2, in particular, 500 mg/m2 as a single infusion for Rituximab. Typically, the dosage for Methotrexate is in the range of 5 mg to 30 mg per week.

In one preferred embodiment, the B-cell depleting agent is a chemical entity, e.g. a small molecule. A variety of B-cell depleting agents are known in the art for example known B-cell depleting agents are BAFF-antagonists. Furthermore, known B-cell depleting agents include antagonists of BR3, agonists of alpha-4- integrins etc. For example, Methotrexate is an analogue of folic acid displaying B- cell depleting activity. Other useful B-cell depleting agents are small modular im- munopharmaceuticals (SMIP) against CD20. For example, SMIP acting as B-cell depleting agents are TRU-015 or SBI-087 of Trubion Pharmaceuticals. Also, SMIP can be single chain polypeptides, smaller than antibodies, having a potent B-cell depletion activity. In a preferred embodiment, a combination of an anti CD20 antibody and representing a biological entity of a B-cell depleting agent and Methotrexat, representing a chemical entity of a B-cell depleting agent, are used for treating chronic fatigue syndrome of myalgic encephalomyelitis. Administration of these entities may be effected simultaneously, separately or sequentially. For example, in a first regimen either the antibody or Methotrexat is administered to the subject while in a second regimen the other agent is administered. The composition comprising the B-cell depleting agent, the antagonist, in particular, the anti CD20 antibody or the CD20-binding antibody fragment thereof, will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the stage of the particular disease or disorder being treated, the particular mammal being treated, the clinical condition of the individual subject, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The effective amount of the B-cell depleting agent, like an antibody or antibody fragment to be administered will be governed by such considerations. As a general proposition, the effective amount of the antagonist administered parenterally per dose will be in the range of about 20mg/m² to about 10,000mg/m² of subject body, by one or more dosages. Exemplary dosage regimens for intact antibodies include 375 mg/m2 weekly x 4; IOOO mg x 2 (e.g. on days 1 and 1 5); or 1 gram x 3. The antibody for the administration to a subject in a single therapeutically effective dosage of said antibody is of 50 to 2000 mg/m² or multiple of therapeutically effective dosages of said antibody or CD20-binding antibody fragment thereof of 50 to 2000 mg/m2. As noted above, however, these suggested amounts of antibody are subject to a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and scheduling is the result obtained, as indicated above. The B-cell depleting agent antagonist, like the anti- body, is administered by any suitable means, including parenteral, topical, subcu- taneous, intraperitoneal, intrapulmonary, intranasal, and/or intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Intrathecal administration is also contemplated. In addition, the B-cell depleting agent antagonist, like the antibody may suitably be administered by pulse infusion, e.g., with declining doses of the antagonist. Preferably the dosing is given by intravenous injections.

The terms "CD20" and "CD20 antigen" are used interchangeably herein, and include any variants, isoforms and species homologs of human CD20 which are naturally expressed by cells or are expressed on cells transfected with the CD20 gene. The term "anti-CD20 antibody" according to the invention is an antibody that binds specifically to CD20 antigen. As identified, the B-cell depleting agent is a B-cell depleting anti-CD20 antibody or CD20 binding antibody fragment thereof, preferably, a monoclonal anti-CD20 binding antibody or a fragment thereof, in particular, a humanized antibody or antibody fragment thereof. Alternatively, the antibody may be a chimeric antibody.

The term "humanized antibody" refers to antibodies in which the framework or complementarity determining regions (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the humanized antibody.

In addition, the antibody may be a human antibody. As used herein, the term "hu- man antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.

Typically, the humanized antibodies and human antibodies are recombinant antibodies. The antibodies and the antagonistic B-cell depleting agents in general are characterized in binding specifically a binding partner on the cells, like to CD20. As used herein, the term "binding" or "specifically binding" refers to the binding of the antibody to an epitope of the antigen, in particular, the CD20 antigen, in an in vitro assay. The affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody/antigen complex), kd (dissociation constant), and KD (kd/ka). Binding or specifically binding means a binding affinity (KD) of 10^"8 mol/litre or less, preferably 10^"9 M to 10^"13 mol/litre. The pharmaceutical composition or the composition according to the present invention may be in form of at least two different components whereby each component may be separately administered. For example, while one component of the combination according to the present invention may be provided systemically, at least one other component may be adapted for local administration or mucosal administration.

Pharmaceutical Formulations

Therapeutic formulations of the cyclic phosphoric acid derivatives and, optionally, the B-cell depleting agents, like antibodies or other antagonists used in accord- ance with the present invention are prepared for storage by mixing an antibody or a fragment thereof having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers {Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabene; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Exemplary anti-CD20 antibody formulations which may form the bases of the compositions according to the present invention are described in WO98/56418, expressly incorporated herein by reference. This publication describes a liquid mul- tidose formulation comprising 40 mg/mL rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0 that has a minimum shelf life of two years storage at 2-8°C. Another anti-CD20 formulation of interest comprises lOmg/mL rituximab in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7mg/mL polysorbate 80, and Sterile Water for Injection, pH 6.5. Lyophilized formulations adapted for subcutaneous administration are described in US Pat No. 6,267,958 (Andya et ah). Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the mammal to be treated herein. Crystalized forms of the antibody or antagonist are also contemplated. See, for example, US 2002/0136719AI.

The active ingredients may also be entrapped in microcapsules prepared, for ex- ample, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocap- sules) or in macroemulsions. Such techniques are disclosed in Remington's Phar- maceutical Sciences 16th edition, Osol, A. Ed. (1980). Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylac- tides (U .S. Pat. No. 3,773,919), non-degradable ethylene- vinyl acetate, degrada- ble lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Finally, the present invention relates to a method for treating chronic fatigue syndrome comprising administering to a patient in need thereof a therapeutically effective amount of the cyclic phosphoric acid derivative of formula I as described herein for example, administration in the method according to the present invention may be effected by infusion or injection of the cyclic phosphoric acid derivatives.

It is possible that the method for treating CFS with the cyclic phosphoric acid derivative comprises administration of a suboptimal or low dosage at the beginning. In particular, the starting dosage may be reduced to avoid any undesired side effects. All the time, the dosage may be increased to dosages as administered in other diseases. Furthermore, the dosage may be an immediate release or sustained dosage depending on the way of administration.

In a preferred embodiment, the method for treating CFS according to the present invention is a method comprising step of administering the cyclic phosphoric acid derivatives of formula I as defined herein to a subject four infusions four weeks apart, for example, within initial twelve weeks. Moreover, in another embodiment, the method for treating chronic fatigue syndrome according to the present invention comprises a step of administering the cyclic phosphoric acid derivative of formula I as defined herein and the B-cell de- pleting agent as defined herein whereby the administration is simultaneously, separately or sequentially to the subject suffering from chronic fatigue syndrome. That is, the cyclic phosphoric acid may be administered in the initial weeks of treatment or for the whole treatment period while the B-cell depleting agent may be administered initially once a week over two weeks and thereafter, in a free determined time of schedule, e. g. in every second or every third month. In particular, cyclic phosphoric acid derivatives may be administered in the initial eight to sixteen weeks, like the first eight to twelve weeks of treatment, to allow rapid relief of the symptoms of CFS while the administration of the B-cell depleting agent as defined herein allows relief to the symptoms of the subject suffering from CFS over a long time period due to the delayed effectiveness thereof.

The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Examples

Initial observations

Two patients with long-standing CFS got breast cancer. One had suffered from CFS for nearly 40 years; the other had suffered moderate CFS for ten years. Both received adjuvant chemotherapeutic treatment for breast cancer using a combination of 5-fluorouracil, epirubicin and cyclophosphamide. Both patients demonstrated a clear relief of the CFS symptoms after chemotherapy, which lasted at least 18 months after the end of treatment (at present still without relapse of CFS symptoms). The third patient had suffered from CFS for 7 years. She then got Hodgkin lymphoma and during a relapse treatment for lymphoma she received treatment with ifosfamide, as part of a chemotherapeutic regimen consisting of ifosfamide, etopo- side, methotrexate and methyl-Gag (the latter no longer in use), showing major improvement of the CFS status lasting until five months after the last chemotherapy course, followed by gradual relapse of CFS symptoms.

Initial treatment of pilot patients suffering from CFS

To demonstrate possible benefit of treatment with cyclophosphamide (or the related drug ifosfamide), three pilot CFS patients were treated with cyclophosphamide as intravenous infusions, given four weeks apart, six infusions in total . Patient 1

The first patient was a 35-year-old woman with CFS for almost 19 years, who had suffered severe CFS symptoms and been mostly bedridden the last three years. Before intervention, she was only able to walk 300 steps per day, assessed using an electronic armband (Sensewear activity registration for seven consecutive days). She received infusions of cyclophosphamide at four weeks intervals, dose 600 mg/m² at first infusion, thereafter 700 mg/m² for the further five infusions. Improvements were observed five weeks after the first infusion. However, the improvements were slight and variable during the first months. After the last infusion, from seven months after the start of treatment, she experienced a major relief of CFS symptoms; the patient was then able to walk an average 13.000 steps per day with a maximum of 17.000 steps per day, according to activity registration for seven consecutive days. Her function level varies to some degree, but she is still able to walk 8-10 km almost every day, and until December 2015 she has no relapse (i.e. 18 months after the last cyclophosphamide infusion). Her self-re- ported function level, according to a form with examples, was 7% at baseline. At 24 months after the first infusion, she now reports a function level varying between 30% and 70%.

Patient 2:

Patient two is a 59-year-old man with a 6-year disease history of CFS. He had received treatment with Rituximab previously, with a major clinical response after Rituximab treatment lasting until 12 months after the last infusion. Thereafter, he had a gradual relapse of CFS symptoms over the following year. After relapse, this patient received six infusions of cyclophosphamide. He had cyclophosphamide infusions at four-week intervals, dose 500 mg/m² at each infusion. The relief of ME/CFS symptoms started from four weeks after the first infusion, but a stable clinical response was first obtained approx. 5 months after the first cyclophosphamide infusion. The symptom relief improved during the next months and was similar to the relief observed after Rituximab treatment. However, initial signs of relief started earlier with cyclophosphamide than with Rituximab. He had a sustained moderate response for approximately 12 months after the last infusion .

Patient 3:

Patient three is a 35-year-old woman with a 3-year history of moderate CFS. The patient received six infusions of cyclophosphamide. At first infusion she received 500 mg/m², increasing to 700 mg/m² for the following infusions. She experienced some relief of the CFS symptoms. However, the effect was less consistent over time, and not sustained. Five months after her last infusion there was only a slight residual effect on the CFS symptoms and no clear response. All patients experienced side effects commonly described for cyclophosphamide treatment, such as nausea and general discomfort. No serious side effects were observed.

To conclude, cyclophosphamide as well as ifosfamide demonstrate usefulness in the treatment of CFS. Cyclophosphamide:

Cyclophosphamide infusions were given four weeks apart. A moderate dose (500 mg/m² to 700 mg/m²) of cyclophosphamide was solubilized in 250 ml 0.9 % sodium chloride and administered over fifteen minutes.

Further studies

After observing the three pilot CFS patientsexperiencing signs of clinical response after cyclophosphamide infusions, an open-label phase-ll study aiming to include 40 CFS patients (at least 25 drug treatment-na^'fve and up to 15 who were previously given rituximab in clinical studies) has been initiated.

Patients with a mild degree of CFS, or patients with very severe CFS (completely bedridden with need for help for all daily tasks), are not included.

Based on the pilot observations described above, in this study patients are given cyclophosphamide 600 mg/m² at first infusion, and 700 mg/m² for the further five infusions, each given four weeks apart. Thereafter the patients are observed until 12 months after the start of intervention, with regular visits for assessment of CFS disease symptoms, physical activity registration using Sensewear armbands (at baseline, at 8 months, and at 12 months), and blood samples for the research biobank.

Already, 21 patients have received the planned 6 cyclophosphamide infusions, and a further 10 patients have received the first three infusions. A preliminary analysis shows that several patients already fulfil the predefined response criteria (sustained moderate improvement in fatigue symptoms for at least 6 weeks). Out of patients not given previously rituximab treatment, approximately half the patients describe improvement in fatigue symptoms, cognitive function, pain or sleep disturbances. Some patients describe a clear improvement in fatigue and malaise after exertion, improvement in sleep, less pain, and a better level of daily functioning. Even though treatment with cyclophosphamide may cause some discomfort (nausea, transient increases in CFS symptoms the first days after infusions) to the patients during the first half year of the study, there have been no cases of hae- matological toxicity.

Two patients (A and B) included in the study, illustrating clinical response after cyclophosphamide infusions, will be described in more detail .

Patient A is a 42-year-old woman who has suffered CFS for more than 10 years. The last years before inclusion her CFS disease was varying between severe (mostly bedridden) and moderate (housebound). At baseline she reported a function level 10% (scale 0-100%, in which 100% denotes completely healthy like before acquiring the disease). She started to note small positive changes already during the first months after the start of infusions. She has received the planned six cyclophosphamide infusions. She reports a clear response of CFS symptoms, and compared to baseline she expresses that the change has a major significance for her daily life. She has the last months been able to go for walks up to 3 kilometres long several times each week, and has been swimming regularly. She describes a major relief of muscle and joint pain, and a clear benefit on fatigue with less malaise after exertion. Before intervention, when she had engaged in too much activity, she would be bedridden with major CFS symptoms for weeks before recovery. Now, her activity tolerance is much higher, and she also recovers from exertion much faster. Before intervention she rested almost all day (perhaps with slight activity 2 hours per day), while now she is not at rest in bed or coach at all during daytime. Her sleep is improved and she feels that sleep results in restoration and relief, which was lacking before. At present, she reports a level of functioning of 30-40 % (and improving) compared to 10% at baseline. Her cognitive function is also improved, and she has less hypersensitivity to noise or light. Patient B included in the study is a 50-year-old woman with CFS the last 9 years. Before inclusion in the cyclophosphamide study, she had a moderate CFS severity, being housebound and partly bedridden. From 3 months after first infusion, she reported gradual improvement of CFS symptoms, with less fatigue and less malaise after exertion, less need for rest, less cognitive dysfunction and less brain "fog", less pain in muscles and joints, and improvement in sleep quality with better recovery. At baseline she reported a function level 14% (scale 0-100%). At seven months after inclusion, after the planned six cyclophosphamide infusions, she has reached a 35% function level, with a number of steps per day varying from 5000 up to 17000 at maximum. She is able to do some housework, go for walks and do shopping, with less sensitivity for light and noise, and generally reports a clear improvement in her quality of life.

Claims

Claims:

1 . A cyclic phosphoric acid derivative of formula I for use in the treatment of chronic fatigue syndrome:

D Pt-|

formula (I) wherein Ri is selected from hydrogen, Ci - C4 alkyl optionally substituted with halogen;

X2 is selected from an ethylene imine group or a group of Formula II

formula (II)

2. The cyclic phosphoric acid derivative of formula I for use according to claim 1 wherein Ri is hydrogen or Ci - C4 alkyl substituted with CI,

Y, Z are hydrogen,

m, n are independently from each other 2 or 3, X2 is Formula I I with

R being hydrogen or Ci - C4 alkyl substituted with CI, and

HAL is CI.

The cyclic phosphoric acid derivative of Formula I according to any one of claims 1 to 2 wherein X2 is formula I I, m is 3, n is 2, Z is hydrogen, Y is hydrogen, R is hydrogen or C2-alkyl-CI.

The phosphoric acid derivative of Formula I for use according to any one of the preceding claims being selected from cyclophosphamide or ifosfamide, or a salt or solvate thereof.

The cyclic phosphoric acid derivative of Formula I for use according claim 4 being cyclophosphamide-monohydrate or ifosfamide.

The cyclic phosphoric acid derivative of the Formula I for use according to any one of the preceding claims wherein these cyclic phosphoric acid derivatives are adapted for systemic administration.

The cyclic phosphoric acid derivative of the Formula I for use according to any one of the preceding claims adapted for the administration to the subject in a single therapeutically effective daily dosage thereof or as multiple therapeutically effective daily dosages thereof.

Pharmaceutical composition comprising a cyclic phosphoric acid derivative of Formula I as defined in any one of claims 1 to 6 and a pharmaceutically acceptable diluent, excipient or carrier for use in the treatment of chronic fatigue syndrome.

9. A composition containing a combination of a cyclic phosphoric acid derivative of Formula I as defined in any one of claims 1 to 6 and a B-cell depleting agent.

The composition of claim 9 in form of a pharmaceutical composition for use in the treatment of chronic fatigue syndrome, in particular, for use in the treatment of chronic fatigue syndrome wherein the compounds are administered simultaneously, separately, or sequentially.

The pharmaceutical composition of the cyclic phosphoric acid derivative of Formula I and the B-cell depleting agent according to any one of claims 8 to 10, wherein the cyclic phosphoric acid derivative of Formula I is administered in a pharmaceutically effective dosage over a time range of the initial 4 to 12 weeks of treatment.

12. The pharmaceutical composition of the cyclic phosphoric acid derivative of the Formula I and the B-cell depleting agent according to any one of claims 9 to 1 1 wherein the B-cell depleting agent is adapted for administration of one or two infusions twice within two weeks.

13. The pharmaceutical composition of a cyclic phosphoric acid derivative of the Formula I and a B-cell depleting agent according to any one of claims 9 to 12 wherein the B-cell depleting agent is a B-cell depleting anti-CD20 antibody or CD20 binding antibody fragment thereof, preferably, a monoclonal anti- CD20 binding antibody or a fragment thereof, in particular, a humanized antibody or antibody fragment thereof.

14. A method for treating chronic fatigue syndrome comprising administering to a patient in need thereof a therapeutically effective amount of the cyclic phosphoric acid derivative of Formula I as defined in any one of claims 1 to

6 or a pharmaceutical composition according to any one of claims 8 to 13.

15. The method for treating chronic fatigue syndrome according to claim 14 comprising the step of administering the cyclic phosphoric acid derivative of Formula I as defined in any one of claims 1 to 6 to a subject afflicted therewith daily within initial 4 to 12 weeks.

16. The method for treating chronic fatigue syndrome according to any one of claims 14 or 15 wherein the composition of the cyclic phosphoric acid derivative of Formula I and the B-cell depleting agent is administered simultaneously, separately or sequentially to the subject suffering from chronic fatigue syndrome.