I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US005922761A
`5,922,761
`[11] Patent Number:
`.Jul. 13, 1999
`[45] Date of Patent:
`
`United States Patent r19J
`Lai
`
`(54] METHODS FOR IN VIVO REDUCTCON OF
`ffiO N LEVELS ANO COMPOSITCONS
`USEFUL THEREFOR
`
`[75]
`
`lnventor: C hing-Sa n Lai, Encinitas, Calif.
`
`[73] Assignee: Medinox, lnc., San Diego, Calif.
`
`[21] Appl. No.: 08/708,552
`
`[22] Filed:
`
`Sep. 6, 1996
`
`Kolaric et al., "A Phase II Trial of Cardioprotection with
`Cardioxaoe (ICRF- 187) in Patients witb Advanced Brea5t
`Cancer Receiving 5-Fluorouracel, Doxorubicin and Cyclo(cid:173)
`phosphamide" Oncology 52:251-255 (1995).
`Komarov and Lai, ·'Detection of nitric oxide production in
`mice by spin-trapping electron paramagnetic resonance
`spectroscopy" Biochim. Biopllys. Ac1a 1271:29-36 (1995).
`Kontoghiorghes, G. J., "Advances in oral iron chelation in
`man" Im. J. Hematol. 55:27-38 (1992).
`Kontoghiorghes, G. J., ''Comparative efficacy and toxici ty of
`desferrioxamine, deferiprone and otber iron and aluminium
`chelating drugs" Toxicol. letters 80:1- 18 (1995).
`Lai and Komarov, "Spin trapping of nitric oxide produced in
`vivo in septic-shock mice" FEBS Letters 345:120-124
`(1994).
`Lai and Piette, ·'Hydroxyl Radical Production Involved in
`Lipid Peroxidation of Rat Liver Microsomes" Biochem.
`Biophys. Res. Commun. 78(1) :51- 59 (1977).
`Muirden and Senator, "Iron in the Synovial Membrane in
`Rheumatoid Arthri tis and Other Joint Diseases" Ann
`Rheum. Dis. 27:3µ8 (1968).
`Myers et al., " Adriamycin: The Role of Lipid Peroxidation
`in Cardiac 1bxicity and Tumor Response" Science
`197:165-167 ( 1977).
`Pippa rd cl al., "Ferrioxamine Excretion in Iron- Loaded
`Man" Blood 60(2) :288-294 (1982).
`Proper et al., "'Reassessment of the Use of Oesferrioxamine
`B in lron Overload'. N. Engl. J. Med. 294(26) :1421-1423
`(1976).
`
`(List continued on next page.)
`
`Primary Examiner-Theodore J. Criares
`Attorney, Agent, or Firm-Gray Cary Ware & Freidenrich
`LLP; Stephen E. Reiter
`
`[57]
`
`ABSTRACT
`
`In accordance with the present inven tion, there are provided
`methods for the in vivo reduction of face iron ion levels in
`a mammalian subject. The present invention employs a
`scavenging approach whereby free iron ions are bound in
`vivo lo a suitable physiologically compatible scavenger. Tbe
`resulting complex renders tbe free iron ions harmless, and is
`evemually excreted in the urine of 1he host. Further in
`accordance with the present invention, there are provided
`compositions and formulations useful for carrying out tbe
`above-described methods. An exemplary scavenger contem(cid:173)
`plated for use in the practice of Lbe present invention is a
`ditlliocarbamate-containing compositioa. This materia l
`binds to free iron ions, forming a stable, water-soluble
`dithiocarbamate-iron complex. The present invention relates
`to methods for reducing in vivo levels of free iron ions as a
`means of treating subjecis afflicted wi1 h iron overload and
`non-iron overload diseases and/or conditions, such as
`thalassemia, anemia hereditary hemochromatosis,
`hemodia lysis, s troke and rheumatoid art hritis.
`Dithiocarbamate-contaioiog scavengers are administered to
`a bost in need of such treatment; these scavengers interact
`with in vivo forming a stable clithiocarbamate-metal
`complex, which is then filtered through the kidneys, con(cid:173)
`centrated in the urine, and eventually excreted by the
`subject, thereby reducing in vivo levels or free iron ions.
`
`40 Claims, 3 Drawing Sheets
`
`Int. C l.6
`[511
`[52] U.S. C l .
`
`........... ... ......... .... A61K 31/27; A61K 31/40
`.......................... 514/476; 514/479; 514/4SO;
`514/481; 514/4S4; 514/423
`[i'ield of Search ..................................... 514/423, 476,
`514/479, 480, 481, 484
`
`[58]
`
`[56]
`
`References C ited
`
`U.S. PATENT DOCUMENTS
`
`4,056,621
`4, 160,452
`4,173,644
`4,256,108
`4,265,874
`4,894,393
`5,380,747
`5,430,058
`
`11/1977 Brown c t al ............................ 4241273
`7 / 1979 Theeuwes ............................... 128/260
`11/1979 Brown el al ............................ 4241270
`3/ 1981 Tbeeuwes ............................... 128/260
`5/1981 Bonsen et al. ............................ 424/15
`1/1990 Nguyen et al. ......................... 514/476
`1/1995 Medford el al. ........................ 514/423
`7/ 1995 Shanzer et al. ......................... 514/575
`
`O'I1iER PUBLICKDONS
`
`Al-Refaie et al., " Efficacy and Possible Adverse Effects of
`tbe
`Oral
`Iron
`Cbelator
`1,2-Dirnethyl-3-Hydroxypyrid-4-0ne (L1 ) in Thalassemia
`Major" Blood 80(3):593-599 (1992).
`Biemond et al., ''Intraarticu.lar Ferritin-Bound lron in Rheu(cid:173)
`matoid Arthritis" Arthritis Rheum. 29(J 0): ll87- Jt93
`(1986).
`Biemond et al., " Iron Mobilization from Fcrritin by Super(cid:173)
`oxide Derived from Stimulated Polymorphonuclear Leuko(cid:173)
`cytes" J. Clin. Invest. 73:1576- 1579 (1984).
`De Sousa et al., "Iron, Iron-binding Proteins and Immune
`System Cellsn" Ann. N.Y. Acad. Sci. 526:310-323 (1988).
`De Yirgil iis et al., " Depletion of trace elements and ac11te
`ocular toxicity induced by desferrioxamine in patients wi th
`thalassaemia" Arch. Dis. Chi/. 63:250-255 (1988).
`Oizdaroglu and Bergtold, "Cbaraterization of Free Radical(cid:173)
`- Induced Base Damage in ONA at Biologically Rclcvcn t
`Levels" Anal. Biocllem. 156:182-188 (1986).
`Guyader et al., "Evaluation of Computed Tomography in lhe
`Assessment of Liver
`lron Overload" Gastroenterol.
`97:737-743 (1989).
`Haber and Weiss, "The Cataly tic Decomposition of Hydro(cid:173)
`gen Peroxide by Iron Salts" Proc. R. Soc. Ser. A.
`147:332- 351 (1934).
`Halliwell aod Gutteridge, in Halliwell and Gutteridge, Free
`Radicals in Biology and Medicine, 2nd edition. Oxford:
`Clarendon Press, 15-19 (1989).
`Harrison and Iloare, in Metals in Bioche111is1ry, Chapman
`and Hall, New York, (1980).
`Kim el al., "Loss and Degradation of Enzyme-bound Heme
`Induced by Cellular Nitric Oxide Synthesis" J. Biol. Chem.
`270(11) :5710-5713 (1995).
`
`
`1 of 14
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1008
`
`

`

`5,922,761
`Page 2
`
`011-TER PUBLICATIONS
`
`Rowley cl al., "Lipid pcroxiclaiion in rheuma toid arthritis:
`tbiobarbiluric acid-reactive material and catalytic iron sails
`in soyovia l tluid from rheumatoid patients" Clin. Sci.
`66:691-695 (1984).
`Shinobu el al., "Sodium N-Metbyl-D-glucamine Dilbiocar(cid:173)
`bamate and Cadmium Intoxication" Ac/a P/wrmacof el
`Toxicol. 54:189- 194 (1984).
`Singal ct al., "SubcelJular Effects of Adriamycin in tbe
`Hean: A Concise Review" J. Mo!. Cell. Cardiol. l 9:8 L 7-828
`(1987).
`
`St. Louis cl al., "Comparison of oral iron chclator Ll and
`dcsferrioxamine
`in
`iron- loaded
`patients"
`Lancet
`336:1275-1279 (1990).
`Thomas et al., ''Tbe Hydrolysis Product of ICRF- 187 Pro(cid:173)
`motes Iron-Catalysed Hyd roxyl Radical Production via the
`Fenlon Reaction" Biochem. Plwrmacof. 45(10) :1967-72
`(1993).
`Yoest el al., "Iron- Chelating Agen1s in Non- Iron Overload
`Conditions" Ann. fnlern. Med. 120(6) :490-499 (1994).
`Yreugclcnbil et al., "Efficacy and Safety of Oral lron Chela(cid:173)
`lor L1 in Anaemic Rheumatoid Arlhnlis Patients" Lancet
`8:1398-1399 (1989).
`
`
`2 of 14
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1008
`
`

`

`U.S. Patent
`
`Jul. 13, 1999
`
`Sheet 1 of 3
`
`5,922,761
`
`3.20000
`
`2. 60000
`
`1.90000
`
`1. 20000
`
`0.0
`
`c "(/) 0. 60000
`c
`Q)
`0
`ro
`.o 1.82350
`+-'
`0.
`0 1. 45880
`
`FIG. 1A
`
`FIG. 18
`
`1.09410
`
`-
`
`0.72939
`
`0.38470
`
`too
`
`300
`
`600
`500
`400
`Wavelength (nm)
`
`700
`
`800
`
`
`3 of 14
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1008
`
`

`

`U.S. Patent
`
`J uJ. 13, 1999
`
`Sheet 2 of 3
`
`5,922,761
`
`2.0
`
`-0 .
`0 -~
`
`.t:
`Cf) c
`<D 1.0
`0
`co
`() ·-.+..J
`0..
`0
`
`t
`
`440
`
`600
`500
`Wavelength (nm)
`
`700
`
`FIG. 2
`
`
`4 of 14
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1008
`
`

`

`U.S. Patent
`
`J uJ. 13, 1999
`
`Sheet 3 of 3
`
`5,922,761
`
`0.5
`
`_......
`§ 0.4
`co
`0
`LO
`........... p 0.3
`Cf) c
`Q)
`0
`ro
`0
`+:i
`0...
`0
`
`0.0
`
`0.1
`
`0. 2
`
`0.3
`
`0.7
`0.6
`0.5
`0.4
`Fe/MGD Ratio
`
`0.8
`
`0.9
`
`1.0
`
`FIG. 3
`
`
`5 of 14
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1008
`
`

`

`5,922,761
`
`1
`METHODS FOR TN vrvo REDUCTION OF
`ffiON LEVELS ANO COMPOSITIONS
`USEFUL THEREFOR
`
`FIELD OF THE 1NVEN110N
`
`The present invention relates to methods for reducing iron
`levels in mammals. lo a particular aspect, the present
`invention relates 10 methods fo r reducing free iron ion levels
`in mammals by administration of ditbiocarbamates as scav(cid:173)
`engers of free iron ions in hosts undergoing anthracycline
`chemotherapy, as wcU as hosts suffering from iron overload
`or non-iron overload diseases and/or conditions, such as
`thalassemia, anemia, hereditary hemochromatosis,
`hcmodialysis, stroke aod rheumatoid arthritis. Jo a further
`aspect, the present invention relates 10 compositions and
`formulations useful in the methods disclosed herein.
`
`BACKGROUND OF THE INVENTION
`
`Iron is crucial for maintaining normal structure aod func- 20
`lion of virtually all mammalian cells (see, for example, Voest
`ct al., in Ann. Intern. Med. 120:490-499 (1994) and
`Kontoghiorghes, G. J., in 1bxicof. Lerre1:s 80:1-18 (1995))
`Adult humans contain 3-5 g of iron, mainly in the form of
`hemoglobin (58%), ferritin/hemosiderin (30%),. myoglobin
`(9%) and other heme or nonheme enzyme proteins (Harrison
`and Hoare, in Metals in Biochemistry, Chapman and Hall,
`New York, 1980).
`Total iron levels in 1be body are regulated mainly through
`absorption from the intestine and the erythropoietie activity
`of the bone marrow. Upon absorption, iron is transported to
`various tissues and organs by the serum protein transferrin.
`Onc.-e 1ransported to the target tissue or organ, iron is
`transported and stored intracellularly io tbe form of ferritio/
`hcmosiderin. Under normal conditions, transferrin is about
`30% sa1uratcd wi1h iron in healthy individuals, and an
`equilibrium is maiotaioed between the sites of iron
`absorption, storage and utilization. The presence of these
`homeostatic controls ensures the maimenance of physiologi(cid:173)
`cal levels of 001 only iron, but also other essential metal ions
`such as copper, zinc and cobalt.
`Breakdown of 1hese controls could resull in me1al imbal(cid:173)
`ance and metal overload, causing iron overloading toxicity
`and possibly death in maoy groups of patients, especially
`those with idiopathic bemocbromalosis (see, for example,
`Guyader et al., io Gastroenterof. 97:737-743 (1989)).
`Among its toxic effects, iron is known to mediate a reper(cid:173)
`toire of oxygen related free radica 1 reactions (sec, for
`example, llalliwell and Gu tteridge, in llalliwell and
`Gutteridge, Pree Radicals io Biology and Medicine, 2od
`edi1ion. Oxford: Clarendon Press, 15- 19 (1989)). For
`example, iron, particularly io the form of free iron ions, can
`promote the generation of reactive oxygen species through
`Lbe iron-catalyzed Haber-Weiss reaction (see, for example,
`Haber and Weis.5, in Proc. R. Soc. Ser. A. 147:332 (1934)) as
`follows:
`
`2
`acids (see, for example, Lai and Pielle, in Biochem. Biophys.
`Res. Commun .. 78:51-59 (1977); and Dizdaroglu and
`Berg1old, in Anal. Biochem., 156:182 (1986)).
`The occurrence of iroo imbalance resulting io excessive io
`s vivo iron levels can be categorized into two conditions,
`namely iron-overload and non-iron overload conditions (see,
`for example, Yoest e1 al., supra; Kootoghiorghes, supra).
`lron-overload conditions are common in such patients as
`those suffering from tbalassemia, sickle cell anemia,
`10 repeated blood transfusion and hereditary bemocbromatosis.
`lo such patients, transferrin is fully sarurated with iron, and
`excess low-molecular-weigh t iron appears in 1be serum. Tbis
`low-molecular-weight iron appears to originate from the
`iron released mainly from the liver and spleen, aod from the
`15 breakdown of effete red cells. Other iron overload diseases
`and conditions include hereditary spherocytosis,
`hemodialysis, die tary or Iatrogenic iron intake, intramuscu(cid:173)
`lar iron dextra n and hemolytic disease of the newborn (see,
`for example, Voest el al., supra; Kontogbiorghes, supra).
`Non-iron overload condi1ions relate to situations where
`elevated iron levels are the result of 1herapeutic in1erven1ion,
`such as, for example, anthracycliae anti-cancer therapy or
`inflammatory diseases such as rheumatoid arthritis. While
`antbracycliaes such as adriamycin (doxorubicin) are effec-
`25 live in the trea tmen t of a number of neoplastic diseases,
`these compounds have limited clinical utility due to the high
`incidence of cardiomyopa th y (see, for example, Singal et al.,
`in J. Mof. Cell. Cardiof. 30 19:817-828 (1987)).
`The molecular mechanism of cardiomyopathy is now
`JO attributed to the adriamycin-induced release of iron from
`iotraccllular iroo-cootaining proteins, resu !ting in the for(cid:173)
`mation of an ad riamycin-iron complex, which generates
`reactive oxygen species causing the scission and condensa(cid:173)
`tion of DNA, peroxidation of phospbolipid membranes,
`35 depletioo of cellular reducing equivalents, interference with
`mitochondrial respira tion, and disrup tion of cell calcium
`homeostasis (see, for example, Myers et al., Science
`197:165-167 (1977); and Gianni et a.I., in Rev. Bioche111.
`1bxicof. 50: 1-82 ( 1983)). On the other band, several clinical
`40 studies have shown that patients with rheumatoid arthri tis
`exhibit eleva1cd low-molecular weigb1 iron species and
`ferritin-bound iron levels in synovial fluid. Iron, presumably
`via its mediation of oxygen free radical pathways, exerts its
`proioflammatory effects in rheumatoid arthritis (see, for
`45 example, Muirden aod Senator, in Ann. Rheum. Dis.
`27:38-48 (1968); and Biemond el al., in Arthritis Rheum.
`29:1187-1193 (1986)).
`Iron also plays an important role in many aspects of
`immune and nonimmune host response (see, for example,
`so De Sousa et al., in Ann. N. Y. A cad. Sci. 526:310- 323
`(1988)). It is known that increased concentrations of iron arc
`deleterious to tbe immune system tbrougb the initiation or
`maint.enance of inflammatory reactions (see, for example,
`Biemond et al., in J. Cfin. Jnves1. 73:1576-9 (1984); and
`55 Row ley el al., in Clin. Sci. 66:691-5 (1984)). Other non-iron
`overload diseases and conditions include reperfusion injury,
`solid tumors (e.g., neuroblastoma), hema tologic cancers
`(e.g., acu te myeloid leukemia), malaria, renal failure, Afzhc(cid:173)
`imer's disease, Parkinson's disease, inflammation, bear!
`60 disease, AJOS, liver disease (e.g., chronic hepatitis C),
`microbia!Jparasitic infections, myelofibrosis, drug-induced
`lung injury (e.g., paraguat), graft-versus-host disease and
`transplant rejection and preservation.
`Hence, not surprisingly, there bas been a tremendous
`interest in the therapeutic use of cbelators in the treatme nt of
`both iron-overload and non-iron overload diseases and con(cid:173)
`ditions. A chelator (Greek, cbcle-claw of a crab) is a mol-
`
`The oet resul t of these reactions is as follows:
`
`The Haber-Weiss reac1ion is seen 10 produce the hydroxyl 65
`radical (.OH), a highly potent oxidant which is capable of
`causing oxidative damage to lipids, proteins and nucleic
`
`
`6 of 14
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1008
`
`

`

`5,922,761
`
`3
`4
`a dithiocarbamate-based fo rmulation. Dithiocarbamates
`ecule forming a cyclic ring with a metal as the closing
`according to the invention bind to Cree iron ions, form ing a
`member. Hundreds of chelating agents have been designed
`stable, water-soluble dithiocarbamate-iroo complex. Dithio-
`aod developed for animal and bumao studies. Amoog them,
`carbamates are a class of low molecular-weight sulphur-
`at least fifteen different cbelators bave been used io buroaos,
`iocludiog dcsfcrrioxamine (OF), ethyleoediamioetetraacetic 5 containing compounds tbat are effective cbclators (see, for
`example, Sbioobu ct al., io Acrn Plrnrmacol et Toxicol.
`acid (EDTA), dietbyleoetriaroioe peotaacetic acid (DTPA)
`pyridoxalisooicotinoylbydrazone (PIH), l,2-dimethyl-3-
`54:189-194 (1984)). For example, diel.byldithiocarbaroate
`hydroxypy rid-4-one (LI) and [ +] 1,2-bis-(3,5-
`(DETC) is used clinically for I.be treaLroent of nickel poi-
`dioxopiperazioe-1-yl) propane (ICRF-187).
`soning.
`For 1he past 30 years, OF (i.e., desfe rrioxamine) has been 10
`Dithiocarbamates, such as N-methyl-D-glucamine dithio-
`tbe roost commonly used chelating drug for the treatment of
`carbamate (MGD), chelate wi th ferrous or ferric iron 10 fo rm
`a stable and water-soluble two-to-one [(MGD)2 -Fe2+] or
`transfusional iron overload (see, for example, Pippard et al.,
`[(MGD)z-Fe3+] complex (sec, for example, Lai and
`Komarov, in FEBS Leffers 345:120-124 (1994)). However,
`in Blood 60:288-294 (1982); Proper et al., in N EnglJ. Med.
`294:1421- 1423 (1976); and St. Louis ct al, in Lancet
`MGD administrated into normal rats did oot chelate eodog-
`336:1275-1279 (1990)). Patieotssufferiog from tbalassernia 15 enous iron 10 form the [(MGD)
`-Fc] complex, suggesting
`2
`that MGD does not remove iron from either hemoglobin or
`lived longer with the DF trealmeot. However, major draw-
`backs in the use of OF include the cost thereof (-$7,000/
`other iron containing enzymes or proteins. Oo the other
`pa1ieL11/year), which can be affordable only by a very small
`hand, administration of MGD into endotoxin-1rea1ed rats
`perceniage of thalassemia patients worldwide. Another
`resulted in the formation of the [(MGD)2 -Fe] complex
`drawback to the use of OF includes the toxici ty thereof, 20 wbich could be detected io body fluids such as blood plasma
`and urine.
`including ophihalmic and auditory toxicities as well as
`It is known tbat endotoxin cballengc induces the release
`induction of pulmonary aod renal damage.
`Unlike OF, L1 (i.e., 1,2-dimcthyl-3-hydroxypyrid-4-one)
`of cellular iron from tissues (see, for example, Kim et al., in
`J. Biol. C!te111. 270:5710-5713 (1995)). Thus, dithiocarbam-
`and related compounds are orally available iron cbelators,
`showing promise in improving the quali ty of life in patients 25 ates such as MGD are capable of removi ng free iron in vivo,
`with thalassemia (see, for example, Olivieri et al., in Drugs
`particularly during the infectious and inflammatory condi-
`Today 28(Suppl. A): 123-132 (1992)) and rheumatoid arthri-
`tions where intracellular iron loss is common, therefore
`1is (see, for example, Vreugden hil et al., in Lancet 2:1398--9
`preventing iron-induced oxidative damage to the tissues.
`(1989)). However, the major side effects of Ll therapy
`Additionally, MGD is safe inasmucb as injections of up to
`include myelosuppression, fatigue, and maternal, embryo JO l % of the body wcighi in rats did no t produce any ill-effects
`(see, for example, Komarov and Lai, in Biochi111. Biophys.
`and teratogenic toxicity, whicb severely limits tbe potential
`Acta 1272:29-36 (1995)).
`clinica l appl ications t he reof (see, for example,
`Kontoghiorgbes, io Int. J. Hemntol. 55:27-38 (1992)).
`Aootber major complication in tbe therapeutic use of
`Recently, l CRF-187 has been demonstrated to be effective
`chelators is the propensity of chelators to affect not only the
`io removing iroo from the anthracycline-iron complex, 35 desired metal but also many other essential metals, their
`therefore preventing the cardiac toxicity in cancer patients
`associated metabolic pathways and other proces.5es. Thus,
`for example, the treatment with OF aod LI requires zioc
`receiving adriamycin chemotherapy (see, for example,
`Kolaric et al., in Oncology 52:251-5 (1995)). However,
`supplementation to prevent the occurreoce of zinc deficiency
`diseases (see, for example, De Virgil is e t al., Arc/1. Dis. Chi!.
`when chelated with iron, the iron-ICRF-187 complex per se
`is also very effective in the promotion of hydroxyl radical 40 63:250-255 (1988); and Al- Refai et a l., in Blood
`geocration via the Fenton reaction, causiog oxidative dam-
`80:593-599 (1992)).
`age to tissues (see, for example, T homas et al., in Bioche111.
`The low-molecular-weigh t iron pool in serum is though t
`P!tar111ncol. 45:1967-72 (1993)). Io addition, since ICRF-
`to be tbe most labile iron source during chelation therapy.
`Cbelators that remove I.bis low-molecular-weight iron with
`187 is a strong chelator (having a structure similar to
`EDTA), it chelates not only low-molecular-weight iroo, but 45 only a minimal effect on other essential metal contents in the
`body are highly desirable, particularly for the treatment of
`also chelates iron from iransferrin and ferritin, as well as
`copper from ceruloplasmin, thus potentially affecting nor-
`transfusion-induced iron overload, as well as iron overload
`induced by antbracycline anti-cancer agents, inOammatory
`ma! cellular iron metabolism.
`Therefore, there is still a need in the an for a new class of
`diseases such as rhemoatoid arthritis and multiple sclerosis,
`iron chelaiors that are capable of removi ng free iron ions so and 1he like.
`from body fluids, without affecting 1be normal cellular iron
`BRTEF DESCRIPTION OF THE FIGURES
`metabolism.
`FIG. 1 provides UY-visible spectra of N-mei byl-D(cid:173)
`glucarnioe dithiocarbamate (MGD) and [MGD-Fe] com-
`55 plexes in aqueous solution.
`FIG. l A provides a spectrum of MGD alone. Ao aliquo t
`(10 p l) of MGD (100 mM) in wa ter was added lo 2 ml o(
`water. Water was used as the control The spectrum was
`recorded from 800 nm to 200 om. Note that MGD showed
`60 ao intensive absorption in the 200-300 om range.
`FIG. 1B provides a spectru m of the [MGD-Fe] complex.
`Ao aliquot (4-01t1) of ferrous sulfate (10 mM) was added to
`2 ml of a 0.5 mM MGD solution in wa ter. An MGD solution
`(0.5 mM) without ferrous sulfate was used as ihe control.
`65 Note the appearance of a prominent cl1arge transfer band at
`508 nm, indicative of the formation or an iron-chela tor
`complex.
`
`BRlEF DESCRu>'rION OF 11-lE INVENTION
`In accordance wi th the present invention, methods have
`been developed for the in vivo reduction of free iron ion
`levels in a subject. The present invention employs a scav(cid:173)
`enging approach whereby free iron ions are bound in vivo to
`a suitable physiologically compatible scavenger, i.e., a com(cid:173)
`pound capable of binding free iron ions. The resulting
`complex readers the free iron ions harmles.s, and is eventu(cid:173)
`ally excreted in the urine of the host. Further in accordance
`with lhe present invention, there bave been developed com(cid:173)
`positions and formulations useful for carrying out the above(cid:173)
`described me thods.
`An exemplary physiologically compatible scavenger con(cid:173)
`templated for use in tbe practice of the present ioveatioa is
`
`
`7 of 14
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1008
`
`

`

`5,922,761
`
`5
`FIG. 2 illustrates the time dependent changes of visible
`spectra of [MGD-Fe] complex. Thus, an MGD solution (25
`ruM) in wa ter was purged witb a stream of nitrogen gas for
`15 min prior to addition of an aliquot of oilrogen-salurated
`ferrous sulfate solutioo in water to a fina l concentration of 5 5
`mM. The superimposed spectra were obtained by repetitive
`scanning using a three-min scan time. Other spectrometer
`settings included scan speed 100 nm/min and chart speed 25
`nm/cm. Note that the charge transfer peak at 508 nm
`increased with time, indicating the autoxidation of ferrous 10
`iron to ferric iron in the [MGD-Fe] complex.
`FIG. 3 presents the resu lts of titration experiments on the
`complexation between MGD and Fe3
`+. Serial ti tration
`experiments were performed 10 determine the binding sto(cid:173)
`ichiometry between MGD and Fe3
`+. The MGD solution in
`aerated wa ter was kept at a constant concentration of 0.5
`mM to which was added various amounts of ferrous sulfate
`(Fe2 •) from 0.05 mM to 0.5 mM with a tenth increment The
`mixtures of MGD and Fe2
`• were incubated at 22° C. for 10
`rain to allow the time required for reaching an equilibriun1 20
`between the [MGD-Fe2
`• ] and [MGD-Fe3+] complexes. The
`0.D. at 508 nm was plotted against the Fe/MGD ratios.
`
`15
`
`30
`
`35
`
`6
`alkynyl, wherein the substituents are selected from
`carboxyl, -C(O)H, oxyacyl, phenol, phenoxy,
`pyridinyl, pyrrolidinyl, amino, amido, hydroxy, oitro or
`sulfuryl, and
`M=H+ or Na ....
`Especially preferred compounds having the above(cid:173)
`described generic s tructure are tbose wherein:
`R1=a C2 up to C8 al kyl or substituted al1'.')'l, wherein the
`substitue nts are selected from carboxyl, acetyl,
`pyridinyl, pyrrolidinyl, ami no, am ido, hydroxy or oitro,
`R2 is selected from a C1 up to C6 alkyl or substituted
`alkyl, or R2 can cooperate with R1 to form a 5-, 6- or
`7-mernbered ring including N, R2 and R1, and MmI-J+ or
`Na+.
`Tbe presently most preferred compounds having the
`above-described generic structure are those wherein:
`R1 =a C2 up to C8 alkyl or substituted alkyl, wherein the
`substituents are selected from carboxyl, acetyl, amido
`or hydroxy,
`R2 =a C1 up to C.1 alkyl or substituted alkyl, aod
`M=H+ or Na ....
`When R1 and R2 cooperate to form a 5-, 6- or7-membered
`ring, the combination of R1 and Rz can be a variety of
`saturated or unsaturated 4, 5 or 6 atom bridging species
`selected from alkenylene or -0-, -S-, -C(O)- and/
`or - N(R)-containing alkylene moieties, wherein R is
`hydrogen or a lower alkyl moiety.
`Monovalent cations contemplated for incorporation into
`the above compounds include H+, Na+, NH.1+, tetraalkyl
`ammonium, and the like.
`As employed herein, "substituted alkyl" comprises alkyl
`groups further bearing one or more substituents selected
`from hydroxy, alkoxy (of a lower alkyl group), mercapto (of
`a lower al.h.')'1 group), cycloal.h.')'I, substituted cycloalkyl,
`beterocyclic, substituted heterocyclic, aryl, substituted aryl,
`heteroaryl, substituted he teroary l, aryloxy, substituted
`aryloxy, halogen, trifiuoromethyl, cyano, oitro, nitrone,
`amino, amido, -C(O)H, acyl, oxyacyl, carboxyl,
`carbamate, sulfonyl, sulfonamide, sulfuryl, and the like.
`As employed herein, "cycloalkyl" refers to cyclic ring(cid:173)
`containing groups contai ning in the range of about 3 up to
`8 carbon atoms, and "substituted cycloalkyl" refers to
`cycloalkyl groups further bearing one or more substituents
`as set forth above
`As employed herein, ''alkenyl" refers to straigbt or
`branched chain hydrocarbyl groups baving at least one
`carbon-carbon double bond, and havi ng in the range of
`about 2 up to 12 carbon atoms, and "substituted alkenyl"
`refers to alkenyl groups further bearing one or more sub(cid:173)
`stituents as set fo rth above.
`As employed herein, "alkynyl" refers to s traight or
`branched chain bydrocarbyl groups b aving at least one
`carbon-carbon triple bond, and having in the range of about
`2 up to 12 carbon atoms, and "substituted alkynyl" refers to
`alkynyl groups further bearing one or more substituents as
`55 set forth above.
`As employed herein, " aryl" refers to aromatic groups
`havi ng in the range of 6 up to 14 carbon atoms and
`"substituted aryl" refers to aryl groups further bearing ooe or
`more substituents as set forth above.
`As employed herein, " alkylaryl" refers to alkyl(cid:173)
`substituted aryl groups and ··substituted alkylaryl" refers to
`alkylaryl groups further bearing one or more substitueots as
`set forth above.
`As employed herein, "arylalkyl" refers to aryl-substituted
`65 alkyl groups and "substituted arylalkyJ" refers to arylalkyl
`groups further bearing one or more substituents as set fo rth
`above.
`
`45
`
`DETAJLED DESCRIPTION OF THE
`INVENTION
`ln accordance with the present invention, there are pro- 25
`vided methods for the in vivo reduction of free iron ion
`levels in a subject. Invention methods comp rise:
`administering lo a subject an effective amount of at least
`one pbysiologicaliy compatible compound capable of
`binding free iron ions.
`Exemplary physiologically compatible compounds con(cid:173)
`templated for use in the practice of the present invention are
`ditbiocarbamates. These materials are said to be "physi(cid:173)
`ologically compatible" because they do not induce any
`significant side effects. In other words, the main effect
`exerted by these compounds is to bind free iron ions.
`As used herein, the phrase "free iron ions" refers to
`transient iron species which are not stably incorporated into
`a biological complex (e.g., hemoglobi n, ferritin, and the 40
`like). Scavengers contemplated for use he rein are higbly
`selective for " free iron ions", relative to other forms of iron
`present in a physiological system.
`Dithiocarbamate compounds contemplated for use in the
`practice oft be present invention include any pbysiologlcally
`compatible derivative of the ditbiocarbamate moiety (i.e.,
`(R)2N-C(S)-SH). Such compounds can be described wi th
`reference to the following generic structure:
`
`(I)
`
`50
`
`wherein:
`eacb of R1 and R2 is independently selected from a C1 up
`to C18 alkyl, substituted alkyl, cycloalkyl, substitu ted
`cycloalkyl, beterocyclic, substitu led he terocyclic,
`alkenyl, substituted alkenyl, alkynyl, substituted
`alkynyl, aryl, substituted aryl, heteroaryl, substituted
`heleroaryl, alkylary l, substituted alkylaryl, arylalkyl,
`s ubsti tuted ary la lkyl, aryla lkeny l, substituted
`arylalkenyl, arylalkynyl, substituted arylalkyoyl, aroyl,
`substituted aroyl, acyl, substituted acyl or R1 and R2 60
`can cooperate to form a 5-, 6- or 7-membered ring
`including N, R1 and R2 , and
`Mis a monovalen t cation.
`Presently preferred compounds having the above(cid:173)
`described generic structure are those whe rein:
`each of R1 and R2=a C1 up to C12 alkyl, substituted alkyl,
`alkenyl, substituted alkenyl, alkyoyl or substituted
`
`
`8 of 14
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1008
`
`

`

`5,922,761
`
`fl uoride,
`
`7
`As employed herein, "'a rylalkenyl" refers lo aryl(cid:173)
`substi1u 1ed alkenyl groups and "substiiuted arylalkenyl"
`refers 10 arylalkenyl groups further bearing one or more
`substi1uents as set forlh above.
`As employed berein, '"arylalkynyl" refers to aryl(cid:173)
`substituted alkynyl groups and "substituted arylalkynyl"
`refers 10 arylalkynyl groups fortber bearing one or more
`subs1i1uents as set forth above.
`As employed herein, "aroyl" refers 10 aryl-carbonyl spe(cid:173)
`cies such as benzoyl and "subs1i1uted aroyl" refers to aroyl
`groups further bearing one or more substiluents as sel fo rth
`above.
`As employed berein, " belerocyclic" refers to cyclic (i .e.,
`ring-containing) groups containing one or more hetcroatoms
`(e.g., N, 0, S, or tbe like) as part of the ring slruclure, and
`baving in 1he range of 3 up to 14 carbon atoms and
`"substituted heterocyclic" refers to beterocyclic groups fur(cid:173)
`ther bearing one or more substiluems as set forth above.
`As employed herein, "acyl" refers to alh.')'l-carbonyl spe(cid:173)
`cies.
`As employed herein, "halogen" refers 10
`chloride, bromide or iodide atoms.
`lo accordance wilh another embodiment of the present
`invention, there are provided methods for treating subjects
`having elevated circulating levels of free iron ions. inven(cid:173)
`tion methods comprise:
`administering to a subject an effective amount of at leas!
`one physiologically compatible compound capable of
`binding free iron ions.
`To accordance with ye t another embodiment of the present
`invention, !here are provided methods for treating overpro(cid:173)
`duction of free iron ions io a subject. Invcn1ion methods
`comprise:
`administering to a subject an effective amounl of at least
`one physiologically compatible compound capable of
`binding free iron ions.
`lbe presence of elevated iron levels in a subject is
`associa ted with a wide range of disease stales and/or
`indications, such as, for example, thalassemia, sickle cell
`anemia, repealed blood transfusions, hereditary hemocbro- 40
`malosise hereditary spberocytosise hemodialysis, dietary
`iron uptake, Iatroge nic iron uptake, imramuscular iron
`dextran, hemolytic disease of the newborn, and lhc like.
`Additional indications associated with elevated levels of
`free iron ions include anthracycline anti-cancer therapy, 45
`inflammation, septic shock, toxic shock syndrome, rheuma(cid:173)
`toid arth ritis, ulcerative colitis, inflammatory bowel disease,
`gas1ri1is, adult respiratory distress syndrome, asthma,
`cachexia, transplant rejection, myocarditis, multiple
`sclerosis, diabetes mellims, autoimmune disorders, eczema, so
`psoriasis, glomerulonephritis, heart failure, heart disease,
`atherosclerosis, Crobn's disease, dermatitis, urticaria, cere(cid:173)
`bral ischemia, systemic lupus erythema tosis, AIDS, AIDS
`dementia, chronic neurodegenerat