United States Patent [19J
`Beutler
`
`[54] USE OF SUBSTITUTED ADENINE
`DERIVATIVES FOR TREATING MULTIPLE
`SCLEROSIS
`
`[75]
`
`Inventor: Ernest Beutler, La Jolla, Calif.
`
`[73] Assignee: The Scripps Research Institute, La
`Jolla, Calif.
`
`[21] Appl. No.: 256,931
`
`[22] PCT Filed:
`
`Feb. 18, 1993
`
`[86] PCT No.:
`
`PCT/US93/01467
`Jul. 27, 1994
`§ 371 Date:
`§ 102(e) Date: Jul. 27, 1994
`
`[87] PCT Pub. No.: WO93/16706
`
`PCT Pub. Date: Sep. 2, 1993
`
`Related U.S. Application Data
`
`[63] Continuation of Ser. No. 838,546, Feb. 19, 1992, Pat. No.
`5,310,732, which is a continuation-in-part of Ser. No. 460,
`351, Jan. 3, 1990, Pat. No. 5,106,837, which is a continu(cid:173)
`ation-in-part of Ser. No. 323,350, Mar. 14, 1989, abandoned,
`which is a continuation-in-part of Ser. No. 169,618, Mar. 16,
`1988, abandoned, which is a continuation-in-part of Ser. No.
`825,215, Feb. 3, 1986, abandoned.
`Int. CI.6
`..........•.............•............................ A61K 31/70
`[51]
`[52] U.S. CI . ................................................. 514/46; 514/45
`[58] Field of Search .......................................... 514/45, 46
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,106,837
`5,310,732
`
`4/1992 Carson et al ............................. 514/46
`5/1994 Carson et al. ............................ 514/46
`
`OTHER PUBLICATIONS
`
`Carrera et al., J. Clin. Invest., 86, 1480---1488 (1990).
`Piro, et al., "2-Chlorodeoxy Adenosine: an Effective New
`Agent for the Treatment of Chronic Lymphocitic Leuke(cid:173)
`mia", Blood, 72:1069-1073 (1988).
`Montgomery, et al., "Synthesis of Potential Anticancer
`
`I 1111111111111111 11111 111111111111111 111111111111111 lllll 111111111111111111
`US005506214A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,506,214
`Apr. 9, 1996
`
`Agents. XX. 2-Fluoropurines", J. Am. Chem. Soc.,
`82:463-468 (1960).
`Stoeckler, et al., "C (2') Substituted Purine Nucleoside
`Analogs", Biochemical Pharmacology, 31: 1723-1728
`(1982).
`Beutler, E. "Cladribine (2-Chlorodeoxy Adenosine)", The
`Lancet, 340:952-956 (1992).
`Beutler, et al., "Antileukemic and Immunosuppressive
`Activity of 2-Chloro-2'-Deoxy Adenosine", Proc. Natl.
`Acad. Sci., 81: 2232-2236 (1984).
`Carson, et al., "Deoxycytidine Kinase-Mediated Toxicity of
`Deoxy Adenosine Analogs Towards Murine L1210 Leuke(cid:173)
`mia in Vivo", Proc. Natl. Acad. Sci., 77:6865-6869 (1980).
`Liliemark, et al., "On The Bioavailability of Oral and Sub
`Cutaneous 2-Chloro-2'-Deoxy Adenosine in Humans:
`Alternative Routes of Administration," J. of Clinical Oncol(cid:173)
`ogy, 10: 1514-1518 (1992).
`Hershfield et al., "Effects of Mutational Loss of Adenosine
`Kinase and Deoxycytidine Kinase on Deoxy ATP Accumu(cid:173)
`lation and Deoxy Adenosine Toxicty in Cultured Cem
`Human T-Lymphoblastoid Cells", J. Of Biol. Chem.,
`257:6380---6386 (1982).
`Chan et al., "Deoxycytidine Excretion by Misuse Peritoneal
`Macrophages: Its Implication in Modulation of Immuno(cid:173)
`logical Functions", J. of Cellular Phys., 111:28-32 (1982).
`Juliusson et al., "Subcutaneous Injections of 2-Chloro-2'
`Deoxy Adenosine (COA) as Treatment . . ." Blood, 80:
`Suppl. 1, 1427 (1992).
`Fredrikson et al., Acta. Neural Scand., 1987, 75, 352-355.
`Huber et al., J. Exp. Med., 1984, 160, 310-316.
`Saven et al., J. Clin. Oncology 1993, 671-678 vol. 11.
`Keams et al., Cancer Research 1994, 54, 1235-1239.
`Weilbach et al., Nervenarzt, 1995, 66, 299-303.
`
`Primary Examiner-Jose G. Dees
`Assistant Examiner-Barbara S. Frazier
`Attorney, Agent, or Firm-Donald G. Lewis
`
`[57]
`
`ABSTRACT
`
`Treatment of patients having multiple sclerosis with thera(cid:173)
`peutic agents containing substituted adenine derivatives
`such as 2-chloro-2'-deoxyadenosine is shown to markedly
`ameliorate the disease condition.
`
`6 Claims, No Drawings
`
`Hopewell EX1026
`
`1
`
`

`

`5,506,214
`
`1
`USE OF SUBSTITUTED ADENINE
`DERIVATIVES FOR TREATING MULTIPLE
`SCLEROSIS
`
`STATEMENT OF GOVERNMENT RIGHTS
`
`This invention was made with government support under
`FDA grant FD-R-000280 and NIH grant numbers
`NS30218 and RR00833. The government has certain
`rights in the invention.
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a national phase application of copend(cid:173)
`ing International Application PCT/US 93/01467 filed Feb.
`18, 1993, which is a continuation of copending U.S. appli(cid:173)
`cation Ser. No. 838,546, now U.S. Pat. No. 5,310,732, filed
`Feb. 19, 1992, which was a continuation-in-part of copend(cid:173)
`ing application Ser. No. 460,351, filed Jan. 3, 1990, now
`U.S. Pat. No. 5,106,837, that was a continuation-in-part of
`copending application Ser. No. 323,350 filed Mar. 14, 1989,
`now abandoned, that was a continuation-in-part of copend(cid:173)
`ing application Ser. No. 169,618, filed Mar. 16, 1988, now
`abandoned, that is a continuation-in-part of copending appli(cid:173)
`cation Ser. No. 825,215, filed Feb. 3, 1986, now abandoned.
`
`DESCRIPTION
`
`TECHNICAL FIELD
`
`This invention relates to therapeutic methods for treating
`multiple sclerosis. More particularly, this invention relates to
`the use of substituted adenine derivatives for treating mul(cid:173)
`tiple sclerosis.
`
`BACKGROUND OF THE INVENTION
`
`Multiple sclerosis (MS) is the result of demyelination in
`the brain and spinal cord (central nervous system). Symp(cid:173)
`toms resulting from this demyelination include weakness,
`visual impairment, incoordination, and paresthesia (abnor(cid:173)
`mal tingling). The course of the disease is largely unpre(cid:173)
`dictable, but often progresses through a cycle of exacerba(cid:173)
`tion of symptoms followed by remission.
`Conventional treatments presently employ therapy with
`ACTH or corticosteroids such as prednisone. Controlled
`studies suggest that such treatments induce more rapid
`clearing of acute symptoms and signs but leave the long(cid:173)
`term outcome of the disease unaffected. Long-term mainte(cid:173)
`nance therapy with ACTH or corticosteroids is contraindi(cid:173)
`cated. Evidence indicates that immunosuppressant agents
`have no long-term benefit. (Cecil, Textbook of Medicine,
`Beeson et al., eds., 15th ed., W. B. Saunders Company,
`Philadelphia, (1979) page 847)
`The etiology of multiple sclerosis is unknown but is
`linked to a variety of genetic and environmental factors.
`Both cell-mediated and humoral immune responses, trig(cid:173)
`gered by extraneous or autoantigens may contribute to the
`pathogenesis of multiple sclerosis. Certain immune response
`genes may be associated with an increased susceptibility to
`the disease. The disease may be mediated by T cells that
`recognize an as yet unidentified autoantigen. For example,
`experimental allergic encephalomyelitis (EAE), an animal
`model of demyelinating diseases such as multiple sclerosis,
`can be induced by immunizing mice with whole myelin or
`specific myelin components such as myelin basic protein.
`
`5
`
`10
`
`25
`
`2
`In humans with multiple sclerosis, exacerbations are
`correlated with high levels of neopterin in blood and cere(cid:173)
`brospinal fluid. Neopterin is a factor released from mono(cid:173)
`cytes and macrophages in the presence of activated T-cells,
`thereby implicating these cells as being involved in multiple
`sclerosis exacerbations. (Fredrickson et al. (1987), Acta
`Neurol. Scand., 75:352-355; Huber et al. (1984), J. Exp.
`Med., 160:310-316). At the microscopic level, monocytes,
`microglial cells (macrophages of the central nervous sys-
`tern), and activated T-cells are found within the demyeli(cid:173)
`nated regions of the nerve cells during multiple sclerosis
`exacerbations. (Cecil, Textbook of Medicine (1979), Beeson
`et al. (eds.), W. B. Saunders Co., Philadelphia, Pa.).
`Various conventional treatment methodologies have been
`15 employed to ameliorate the symptoms of multiple sclerosis.
`Many of these are directed to use of palliative, anti-inflam(cid:173)
`matory agents. No treatment to date has had any consistent
`positive effect on the course of the disease.
`Recently, the art has described the use of specific deox-
`20 yribosides as anti-inflammatory agents. For instance, U.S.
`Pat No. 4,481,197 (Rideout et al.) relates to the use of
`unsubstituted 3-deaza-2'-deoxyadenosine derivatives in the
`treatment of inflammation. U.S. Pat. No. 4,381,344 (Rideout
`et al.) relates to a process for the synthesis of deoxyribosides
`that utilizes a bacterial phosphorylase.
`A deoxyriboside derivative, 2-chloro-2'-deoxyadenosine
`(CdA), has been found to be an effective agent for the
`treatment of chronic lymphocytic leukemia and some T cell
`malignancies. (Carson et al. (1984) Proc. Natl. Acad. Sci.
`30 U.S.A., 81:2232-2236; Piro et al.
`(1988), Blood
`72: 1069-1073) The pharmacokinetics of orally and subcu(cid:173)
`taneously administered 2-chloro-2'-deoxyadenosine in the
`treatment of chronic lymphocytic leukemia have been
`described and compared. (Liliemark et al. (1992) Journal of
`35 Clinical Oncology, 10, (10): 1514-1518; Juliusson et al.
`(1992) Blood, 80 (Suppl. 1): 1427) Chronic lymphocytic
`leukemia is a malignancy of B lymphocytes that bear the
`Leu-1 surface antigen.
`The Leu-1 B cells represent a minor proportion of the
`40 normal pool of B lymphocytes, usually less than 20 percent.
`The Leu-1 B cells express surface markers that are typically
`found on monocytes (Mac-I antigen) and T-lymphocytes
`(Leu-1 antigen). Approximately 10 percent of patients with
`chronic
`lymphocytic
`leukemia exhibit accompanying
`·
`autoimmunity, and recently, Leu-1 B cells have been impli-
`cated in the pathogenesis of autoimmune diseases.
`Phase I clinical trials on human patients with chronic
`lymphocytic leukemia indicate that infusion of increasing
`50 doses of 2-chloro-2'-deoxyadenosine [0.1-0.5 milligrams
`per kilogram of body weight per day (mg/kg/day)] yielded
`increasing plasma concentrations of the drug [10-50 nano(cid:173)
`molar (nM)]. Those infusions indicated that the drug was
`well tolerated and did not induce nausea, vomiting or fever.
`The dose-limiting toxicity was bone marrow suppression,
`which usually occurred at doses greater than about 0.2
`mg/kg/day or at plasma levels of greater than about 20 nM.
`Other studies, Montgomery et al. (1959) J. Am. Chem.
`Soc., 82:463-468, indicated that 2-fluoroadenosine exhibits
`60 a relatively high degree of cytotoxicity. Those workers
`reported that C57 black mice implanted with Adenocarci(cid:173)
`noma 755 (Ad755) could tolerate only about 1 milligram per
`kilogram of body weight. 2-Fluoroadenosine was found to
`be inactive at that level against Ad755 as well as leukemia
`65 L1210 and the Erlich ascites tumor.
`U.S. Pat. No. 4,751,221 and its division No. 4,918,179 to
`Watanabe et al. describe the synthesis and use of several
`
`45
`
`55
`
`2
`
`

`

`5,506,214
`
`4
`2-Chloro-2'-deoxyadenosine is phosphorylated by non(cid:173)
`dividing (normal) human peripheral blood lymphocytes and
`is converted to the 5'-triphosphate. This adenine derivative
`is not catabolized significantly by intact human cells or cell
`5 extracts, and is phosphorylated efficiently by T lymphocytes.
`(Carson et al.
`(1980) Proc. Natl. Acad. Sci. USA,
`77: 6865-6869)
`As discussed before, high levels of adenosine kinase have
`been found in murine peritoneal macrophages and in human
`monocytes. Adenosine kinase can phosphorylate 2'-deoxy(cid:173)
`adenosine derivatives, but does so less efficiently than
`deoxycytidine kinase. (Hershfield et al. (1982) J. Biol.
`Chem., 257:6380- 6386)
`Chemotherapeutic agents are described hereinafter that
`15 may be employed as therapeutic agents in the treatment of
`multiple sclerosis.
`
`3
`2-substituted-2'-deoxy-2' -fluoroarabino-furanosy 1 nucleo(cid:173)
`sides including adenine derivatives. Those compounds were
`said to have anti-tumor and antitrypanosomal biological
`activities. Cytotoxicity data showing anti-tumor activity of
`2-amino- 6-thiopurine, guanine and thiopurine derivatives
`against murine and human cell lines were reported.
`U.S. Pat. No. 5,034,518 to Montgomery et al. teaches the
`synthesis of 2-substituted-2'-deoxy-2'-fluoroaraadenosines.
`Those compounds were said to have anticancer activity, and
`data for prolongation of life of mice transplanted with P388 10
`leukemia cells were provided.
`The biochemical activity of 2-CdA in cells has been
`reviewed by Ernest Beutler. (The Lancet (1992), 340:
`952-956-incorporated herein by reference)
`The 2',3'-dideoxynucleosides are phosphorylated at the
`5'-position in T cells to form the 5'-nucleotide triphosphate
`derivatives. Those derivatives are well known to be sub(cid:173)
`strates for reverse transcriptase molecules. (Ono et al. (1986)
`Biochem. Biophys. Res. Comm., 2:498-507)
`Those 2',3'-dideoxynucleoside 5'-triphosphates are also 20
`utilized by mammalian DNA polymerases beta and gamma.
`(Waquar et al. (1984) J. Cell. Physiol., 121:402-408) They
`are, however, poor substrates for DNA polymerase-alpha,
`the main enzyme responsible for both repair and replicative
`DNA synthesis in human lymphocytes. In part, these prop(cid:173)
`erties may explain the selective anti-HIV activity of the
`2' ,3 'dideoxynucleosides.
`Chan et al. (1982) J. Cell Physiol., 111:28-32 studied the
`pathways of pyrimidine nucleotide metabolism in murine
`peritoneal macrophages and monocytes, and reported unde(cid:173)
`tectable levels of deoxycytidine kinase or thymidine kinase
`in these cells. High levels of adenosine kinase were found,
`however.
`Similar high levels of adenosine kinase have been found 35
`in human monocytes and human monocyte-derived mac(cid:173)
`rophages (MDM). MDM were found to exhibit about one(cid:173)
`tenth to about one-fourth the nucleoside kinase activity of
`GEM T lymphoblasts (e.g. ATCC CCL 119) toward uridine,
`deoxycytidine and thymidine, and about two-thirds the 40
`adenosine kinase activity of GEM cells. In addition, that
`adenosine kinase activity of MDM cells was at least about
`10-fold higher than any of the other kinase activities. Those
`studies also indicated relatively low levels of nucleoside
`phosphorylation using AZT, dideoxycytidine (ddC) and 45
`2',3'-dideoxyadenosine (ddA) in intact GEM T lymphoblasts
`and still lower levels with the MDM.
`Several 2-substituted adenosine derivatives have been
`reported not to be dearninated by adenosine deaminase. For
`example, Coddington (1965) Biochim. Biophys. Acta,
`99:442-451 reported that deoxyadenosine-1-N-oxide, as
`well as 2-hydroxy-, 2-methyl-, 2-chloro-, 2-acetarnido-, and
`2-methylthioadenosines were neither substrates nor inhibi(cid:173)
`tors for adenosine dearninase. Montgomery, in Nucleosides,
`Nucleotides, and Their Biological Applications, Rideout et 55
`al. eds., Academic Press, New York, page 19 (1983) pro(cid:173)
`vides a table of comparative Km and V max data for the
`deamination of adenosine, 2-haloadenosines 2-halo-deoxy(cid:173)
`adenosines and 2-fluoroarabinoadenosine that also indicates
`that those 2-halo adenine derivatives are poor substrates for 60
`the enzyme relative to adenine itself. Stoeckler et al. (1982)
`Biochem. Pharm., 31:1723-1728 reported that the 2'-deoxy-
`2' -azidoribosy 1 and 2' -deoxy-2' -azidoarabinosy I-adenine
`derivatives were substrates for human erythrocytic adenos(cid:173)
`ine deaminase, whereas work of others indicated 2-fluoro- 65
`adenosine to have negligible activity with adenosine dearni-
`nase.
`
`SUMMARY OF THE INVENTION
`
`The present invention contemplates a method for treating
`multiple sclerosis. In this method, a patient having multiple
`sclerosis is treated with a composition having a pharmaco(cid:173)
`logically acceptable carrier and a substituted adenine deriva(cid:173)
`tive dissolved or dispersed therein. The substituted adenine
`25 derivative is present in the pharmacologically acceptable
`carrier in an amount sufficient to provide a therapeutically
`effective dose over the course of treatment.
`Preferred substituted adenine derivatives useful for treat(cid:173)
`ing multiple sclerosis may be represented by Formula I
`30 having a structural formula corresponding to:
`
`wherein Z is o- or absent,
`Y is hydrogen or a substituent containing one to about 20
`atoms that is free from net ionic charge at physiological pH
`values, provides a soluble adenine derivative and whose
`presence on the adenine moiety inhibits deamination of the
`adenine derivative by adenosine deaminase; and
`X is hydrogen or fluoro, with the proviso that Y is
`50 hydrogen only when Z is present.
`Particularly preferred compounds of Formula I are free of
`the Z group; i.e, Z is absent, and contain a halo group at the
`2-position. The most preferred compounds are 2-chloro-2'(cid:173)
`deoxyadenosine and 2-chloro-2'-deoxy-2'-arafluoroadenos(cid:173)
`ine.
`Methods for synthesizing all of the above compounds are
`indicated in U.S. Pat. No. 5,106,837 (Carson et al., Apr. 21,
`1992, incorporated herein by reference).
`The invention teaches that the disease condition of a
`patient having multiple sclerosis may be ameliorated by
`administration of an amount of the above-described com(cid:173)
`position having a sufficient quantity of the compound of
`Formula I to provide a therapeutically effective dose. Exem(cid:173)
`plary dosages range from about 0.04 to about 1.0 mg/kg/day,
`with dosages of about 0.04 to about 0.2 mg/kg/day being
`more preferred. Typically, the amount is sufficient to provide
`a concentration in the patient's plasma of about 0.5 nano-
`
`3
`
`

`

`5,506,214
`
`5
`
`5
`molar (nM) to about 50 nM, more preferably of about 1 nM
`to about 10 nM.
`Preferably, the agent contemplated for use in the present
`invention is a 2-halo-2'-deoxyadenosine (2-halo-2'-deoxy-9,
`l '-beta-ribofuranosyladenine) or a 2-halo-2'-deoxy-2'-
`arafluoroadenosine, and most preferably the halo group is
`chloro.
`A further aspect contemplated by the present invention
`comprises the use of subcutaneous injection for administer(cid:173)
`ing an effective amount of the active ingredient (agent) of 10
`the invention for treating multiple sclerosis.
`An alternative aspect contemplated by the present inven(cid:173)
`tion comprises the peroral administration of an effective
`amount of the active ingredient (agent) of the invention in a
`method of treating disease. Preferred compounds of Formula 15
`I for oral administration include compounds in which X is
`fluoro.
`In each of the before-described methods, the substituted
`2'-deoxyadenosine derivative is administered in a therapeu(cid:173)
`tically effective amount. The effect of a compound of 20
`Formula I is dependent upon the route of administration and
`upon the time and dosage. As a consequence, one can tailor
`the dosage and duration for which a particular compound is
`administered to the stage of the disease and the condition of
`the patient being treated. Where the stage of multiple 25
`sclerosis is advanced or life-threatening, treatment may be
`more aggressive, and a therapeutically effective amount is an
`amount that is sufficient to kill at least 50 percent of the
`monocytes present but is less than that which substantially
`impairs bone marrow function as determined by usual 30
`procedures when administration is in vivo. The monocyte
`killing amount of a compound of Formula I is another
`measure of a therapeutically effective dose and monocyte
`death is measured at a time seven days after the initial
`administration.
`
`6
`particularly preferred embodiments, when Y is chloro, X is
`fluorine.
`The preferred compound included in Formula I is
`2-chloro-9, 1, '-beta-D-2' -deoxyribosy !adenine,
`otherwise
`known as 2-chlorodeoxyadenosine or CdA.
`Of the compounds of Formula I, those where Xis fluoro
`are among the preferred compounds for use by oral admin(cid:173)
`istration.
`Other illustrative compounds included in Formula I are:
`2-bromo-9, 1 '-beta-D-2'-deoxyribosy !adenine;
`2-methyl-9, l '-beta-D-2' -deoxyribosyladenine;
`2-fluoro-9,1 '-beta-D-2'-deoxyribosyladenine;
`2-acetoamido-9, l '-beta-D-2' -deoxyribosy I adenine;
`2-methylthio-9,1'-beta-D-2'-deoxyribosyladenine; ....
`2-chloro-9, l 'beta-2' -deoxy-2' -fluoro-D-arabinofuranosy !(cid:173)
`adenine;
`2-bromo-9, l '-beta-2 '-deoxy-2' -fluoro-D-arabinofuranosyl(cid:173)
`adenine;
`2-(N-acetamido )-9, 1 '-beta-2'-deoxy-2'-fluoro-D-arabino(cid:173)
`furanosy !adenine;
`2-methylthio-9,1'-beta-2'-deoxy-2'-fluoro-D-arabinofurano(cid:173)
`syladenine.
`Further illustrative of compounds of Formula I include the
`following arabinofuranosyl derivatives of adenine:
`2-methyl-9,1 '-beta-2'-deoxy-2'-fluoro-D-arabinofuranosyl(cid:173)
`adenine;
`2-isopropy 1-9, l '-beta-2' -deoxy-2'-fluoro-D-arabinofurano(cid:173)
`syl-adenine;
`2-hydroxy-9, l '-beta-2'-deoxy-2'-fluoro-D-arabinofuranosyl(cid:173)
`adenine;
`2-chloro-9, 1 '-beta-2' -deoxy-2'-fluoro-D-arabinofuranosy(cid:173)
`ladenine-1-N-oxide;
`2-fluoro-9,1'-beta-2'-deoxy-2'-fluoro-D-arabinofuranosy(cid:173)
`ladenine-1-N-oxide;
`35 2-bromo-9,1'-beta-2'-deoxy-2'-fluoro-D-arabinofuranosy(cid:173)
`ladenine-l-N-oxide;
`2-methyl-9,1 '-beta-2'-deoxy-2'-fluoro-D-arabinofuranosy(cid:173)
`ladenine-1-N-oxide;
`2-(N-acetamido )-9, 1 '-beta-2'-deoxy-2' -fluoro-D-arabino(cid:173)
`furanosy ladenine-1-N-oxide;
`2-hydroxy-9,1'-beta-2'-deoxy-2'-fluoro-D-arabinofuranosy(cid:173)
`ladenine-1-N-oxide;
`2-(2-methylbutyl)-9,l '-beta-2'-deoxy-2'-fluoro-D-arabino(cid:173)
`furanosy ladenine-1-N-oxide;
`45 2-fluoro-9,1 '-beta-D-2'-deoxyadenosine-1-oxide; and
`2-chloro-9,1 '-beta-D-2'-deoxyadenosine-1-oxide.
`It is noted that when X is hydrogen the sugar ring can be
`named as a 2'-deoxyribosyl or 2'-deoxyarabinofuranosyl
`radical. Both nomenclatures are utilized herein. When the
`50 class of compounds embraced by Formula I is discussed, all
`of the compounds are considered herein as derivatives of
`arabinose. However, when specific compounds of the sub(cid:173)
`class where X=H are discussed, the more familiar deoxyri(cid:173)
`bose nomenclature is used, such as in deoxyadenosine.
`55 These compounds are also referred to herein more simply as
`adenine derivatives.
`In the above formulas, and in all other formulas shown
`herein, hydrogen atoms on the purine and furanosidyl rings
`that are not needed to show conformation about a particular
`60 bond are not shown. Thus, the 8-position adenine hydrogen
`is not shown.
`It is also to be understood that the D isomers of com(cid:173)
`pounds of the formulas are the isomers contemplated. It is
`further to be noted that the designation "halo" used herein is
`65 meant to include fluorine, chlorine and bromine derivatives,
`and to exclude iodine derivatives, which are unstable and
`decompose, and astatine derivatives that are radioactive.
`
`40
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`A. Compounds
`The present invention contemplates the use of substituted
`adenine derivatives, i.e. substituted- 2'-deoxy-arabinofura(cid:173)
`nosyladenine, for treating multiple sclerosis. Preferred sub(cid:173)
`stituted adenine derivatives have a structure represented by
`the following formula, viz. Formula I:
`
`wherein z is an oxide radical co-) or is absent;
`Y is hydrogen or a radical containing one to about twenty
`atoms that is free from net ionic charge at physiological pH
`values, provides a soluble adenine derivative, and whose
`presence on the adenine moiety inhibits deamination of the
`adenine derivative by adenosine deaminase; and
`X is hydrogen or fluorine, with the proviso that Y is
`hydrogen only when Z is present.
`Preferably, Y is chloro. Other Y substituents may be
`selected from the group consisting of lower alkyl, lower
`alkanoylamido, lower alkylthio and hydroxyl radicals. In
`
`4
`
`

`

`5,506,214
`
`15
`
`35
`
`7
`Where specific halogen derivatives are intended, those com(cid:173)
`pounds are named specifically.
`As used herein, "a substituent free from net ionic charge"
`includes both charged and uncharged radicals, wherein when
`the substituent radical is charged, an internal zwitterionic 5
`charge pair is present that results in the absence of a net ionic
`charge for the molecule at physiologic pH values. N-oxide
`compounds are exemplary of such substituents.
`As used herein, a "soluble adenine derivative" is an
`adenine derivative which is able to dissolve and remain
`soluble in a body fluid such as blood at a therapeutically
`effective dose as is discussed hereinafter.
`As used herein, a "substituent whose presence on the
`adenine moiety inhibits deamination of an adenine deriva(cid:173)
`tive by adenosine dearninase" is one that, when 100 micro(cid:173)
`liters of a I millimolar solution of the substituted adenine
`derivative is incubated for three hours at room temperature
`with 25 units of calf spleen adenosine deaminase (1 unit
`catalyzes the dearnination of I micromole of adenosine per
`minute), produces a single UV-absorbing spot upon cellu(cid:173)
`lose-thin layer chromatography of the reaction mixture
`whose Rf value is the same as that of the substituted adenine
`derivative used.
`The metabolism of a compound by adenosine dearninase
`can be investigated by the following procedure. The indi(cid:173)
`vidual nucleosides, at concentrations from 5-200 µMin 10
`mM sodium phosphate, pH 7.5, are incubated at 18-20
`degrees C. with 0.01 EU/ml calf intestinal adenosine dearni(cid:173)
`nase. The change in the optical density at 265 nm and 250
`nm is monitored spectrophotometrically. The Km and V max
`values are determined by the Lineweaver-Burke method
`utilizing the ~M 265 between adenosine and inosine.
`The ratio V ~Km also provides a measure of relative
`efficiency of deamination by the enzyme. A substituent that
`provides a V mdKm ratio that is about 1 percent or less than
`that for the ratio obtained using 2'-deoxyadenosine is also a
`"substituent whose presence on the adenine moiety inhibits
`deamination of an adenine derivative by adenosine dearni(cid:173)
`nase."
`As used herein, lower alkyl radicals include C1-C6
`straight chain, branched and cyclic alkyl groups, for
`example, methyl, ethyl, n-butyl, t-butyl, n-hexyl, 1-ethylbu(cid:173)
`tyl, cyclopentyl, cyclohexyl and the like. Lower alkanoyla(cid:173)
`mido radicals include C1-C6 radicals, for example, forma(cid:173)
`mido, acetylarnido, propionamido, hexamoylarnido and the
`like. Lower alkylthio radicals include C1-C6 straight chain,
`branched and cyclic alkyl groups as discussed above linked
`to a thio radical.
`The pharmacologically acceptable salts of a compound of
`Formula I are also utilized. The phrase "pharmacologically
`acceptable salts," as used herein, refers to non-toxic acid
`addition salts that are generally prepared by reacting a
`compound with a suitable organic or inorganic acid. Rep(cid:173)
`resentative salts include the hydrochloride, hydrobromide,
`sulfate, phosphate, citrate, acetate, maleate and the like.
`B. Compositions
`A compound of Formula I dissolved or dispersed in or
`together with a pharmacologically acceptable carrier con(cid:173)
`stitutes a composition of this invention.
`A compound of Formula I and its pharmacologically
`acceptable salts are useful in both short and long term
`treatment. For instance, a 2-substituted-9,1 '-beta-2'-deoxy-
`2'-fluoro-D-arabinofuranosyladenine is administered to the
`injection,
`subcutaneously by
`patient
`internally, e.g.,
`parenterally, orally, or rectally as a suppository, in an effec(cid:173)
`tive amount.
`
`8
`Although a compound of Formula I and its pharmaco(cid:173)
`logically acceptable salts can be administered as the pure
`chemical, it is preferred that it be administered as a phar(cid:173)
`maceutical composition. In either event, it is administered in
`an amount sufficient to provide a therapeutically effective
`dose as is discussed hereinafter.
`Accordingly, the present invention utilizes a pharmaceu(cid:173)
`tical composition comprising a therapeutically effective
`dose of a compound of Formula I or a pharmacologically
`10 acceptable salt thereof, hereinafter referred to as the "active
`ingredient" or "agent," dissolved or dispersed in a pharma(cid:173)
`cologically acceptable carrier or diluent.
`A pharmaceutical composition is prepared by any of the
`methods well known in the art of pharmacy all of which
`involve bringing into association the active compound and
`the carrier therefor. For therapeutic use, a compound utilized
`in the present invention can be administered in the form of
`conventional pharmaceutical compositions. Such composi(cid:173)
`tions can be formulated so as to be suitable for oral,
`20 subcutaneous, or parenteral administration, or as supposito(cid:173)
`ries. In these compositions, the agent is typically dissolved
`or dispersed in a physiologically tolerable carrier.
`A carrier or diluent is a material useful for administering
`the active compound and must be "pharmacologically
`25 acceptable" in the sense of being compatible with the other
`ingredients of the composition and not deleterious to the
`recipient thereof. Thus, as used herein, the phrases "physi(cid:173)
`ologically tolerable" and "pharmacologically acceptable"
`are used interchangeably and refer to molecular entities and
`30 compositions that do not produce an allergic or similar
`untoward reaction, such as gastric upset, dizziness and the
`like, when administered to a mammal. The physiologically
`tolerable carrier can take a wide variety of forms depending
`upon the preparation desired for administration and the
`intended route of administration.
`As an example of a useful composition, a compound of
`Formula I can be utilized in liquid compositions such as
`sterile suspensions or solutions, or as isotonic preparations
`containing suitable preservatives. Particularly well-suited
`40 for the present purposes are injectable media constituted by
`aqueous injectable isotonic and sterile saline or glucose
`solutions. Additional liquid forms in which these com(cid:173)
`pounds can be incorporated for administration include fla(cid:173)
`vored emulsions with edible oils such as cottonseed oil,
`45 sesame oil, coconut oil, peanut oil, and the like, as well as
`elixirs and similar pharmaceutical vehicles.
`The agents can also be administered in the form of
`liposomes. As is known in the art, liposomes are generally
`derived from phospholipids or other lipid substances. Lipo-
`50 somes are formed by mono- or multilamellar hydrated liquid
`crystals that are dispersed in an aqueous medium. Any
`non-toxic, physiologically acceptable and metabolizable
`lipid capable of forming liposomes can be used. The present
`compositions in liposome form can contain stabilizers, pre-
`55 servatives, excipients, and the like in addition to the agent.
`The preferred lipids are the phospholipids and the phos(cid:173)
`phatidyl cholines (lecithins), both natural and synthetic.
`Methods to form liposomes are known in the art. See, for
`example, Prescott, Ed., Methods in Cell Biology, Volume
`60 XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.
`An agent of Formula I can also be used in compositions
`such as tablets or pills, preferably containing a unit dose of
`the compound. To this end, the agent (active ingredient) is
`mixed with conventional tableting ingredients such as com
`65 starch, lactose, sucrose, sorbitol, talc, stearic acid, magne(cid:173)
`sium stearate, dicalcium phosphate, gums, or similar mate(cid:173)
`rials as non-toxic, physiologically tolerable carriers. The
`
`5
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`5,506,214
`
`9
`tablets or pills can be laminated or otherwise compounded to
`provide unit dosage forms affording prolonged or delayed
`action.
`It should be understood that in addition to the aforemen(cid:173)
`tioned carrier ingredients the pharmaceutical formulation 5
`described herein can include, as appropriate, one or more
`additional carrier ingredients such as diluents, buffers, fla(cid:173)
`voring agents, binders, surface active agents, thickeners,
`lubricants, preservatives (including antioxidants) and the
`like, and substances included for the purpose of rendering
`the formulation isotonic with the blood of the intended 10
`recipient.
`The tablets or pills can also be provided with an enteric
`layer in the form of an envelope that serves to resist
`disintegration in the stomach and permits the active ingre(cid:173)
`dient to pass intact into the duodenum or to be delayed in 15
`release. A variety of materials can be used for such enteric
`layers or coatings, including polymeric acids or mixtures of
`such acids with such materials as shellac, shellac and cetyl
`alcohol, cellulose acetate phthalate, and the like. A particu(cid:173)
`larly suitable enteric coating comprises a styrene-maleic 20
`acid copolymer together with known materials that contrib(cid:173)
`ute to the enteric properties of the coating. Methods for
`producing enteric coated tablets are described in U.S. Pat.
`No.