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`
`eduction of Tissue Iron Stores and Normalization of Serum Ferritin During
`Treatment With the Oral Iron Chelator Lt in Thalassemia Iniermedia
`
`ncy F. Olivieri, Gideon Koren, Doreen Matsui, Peter P. Liu, Laurie Bien dis, Ross Cameron, Robert A. McClelland,
`and Douglas M. Templeton
`
`In patients with thalassemia lntermedia In whom hypera!J.
`sorption of iron may result in serious organ dysfunction, an
`orally effective Iron-chelating drug would have major thera(cid:173)
`peutic advantages, especially for the many patients with
`thalassemia intermedia in the Third World. We report reduc.
`tion in tissue iron stores and normalization of serum ferritin
`concentration after 9-month therapy with the oral chelator
`1,2-dimethyt-3-hydroxypyrid-4-one (L 11ina29-year-old man
`with thalassemia intermedia and clinically significant Iron
`overload (SF 2,174 !'g/l., transferrin saturation 100%; ele(cid:173)
`vated AST and ALT, abnormal cardiac radionuclide angio(cid:173)
`gram) who was enrolled in the study with L1 75 mg/kg/day
`after he refused deferoxamine therapy. L 1-lnduced 24-hour
`urinary iron excretion during the first 6 months of therapy
`was (mean :t: SD, range I 53 ::!: ao (11to109) mg (O.n mg/kg),
`declining during the last 3 months of L 1to24 :t 14 (13-40) mg
`
`(0.36 mg/kg), as serum ferritin decreased steadily to normal
`range (present value, 251 flg/LJ. Dramatic Improvement In
`signal intensity of the liver and mild improvement in that of
`the heart was shown by comparison of T,-weighted spin
`echo magnetic resonance imaging with images obtained
`Immediately before L 1 administration was observed after 9
`months of L1 therapy. Hepatic Iron concentration decreased
`from 14.6 mg/g dry weight of liver before L1 therapy to 1.9
`mg/g liver after 9 months of therapy. This constitutes the
`first report of normalization of serum fettitin concentration In
`parallel with demonstrateq reduction in tissue iron stores as
`a result of treatment with L1. Use of L1 as a therapeutic
`option in patients with thalassemia intermedia and Iron
`overload appears warranted .
`o 1992 by The American Society of Hematology.
`
`· PROGRESSIVE ORGAN dysfunction, often leading to
`mortality, is a consequence of transfusional or absorp(cid:173)
`tionaJ tissue iron loading in patients with homozygous
`~-thalassemia.1 Regular chelation therapy with deferox(cid:173)
`amine reduces hepatic iron and arrests hepatic fibrosis,2
`stabilizes iron-related cardiac disease,3 and prevents go(cid:173)
`nadal failure when started before age 10 years.• Administra(cid:173)
`tion of standard therapy with deferoxamine is problematic,
`however; the drug requires prolonged nightly subcutaneous
`(SC) infusion, frequently associated with local irritation.
`Compliance with this regimen decreases dramatically dur(cid:173)
`ing adolescence,s and many patients with cardiac iron
`overload still die.5-8 Moreover, the cost of deferoxamine
`renders it unavailable to thousands of thalassemia patients
`in the Third World. It has long been acknowledged9 that the
`added convenience of an orally available chelator would
`simplify treatment of patients with transfusion-dependent
`thalassemia, and should also simplify management of pa(cid:173)
`tients with sickle cell disease who are maintained on a
`transfusion program in an effort to prevent or arrest specific
`disease complications .
`Although transfusion-dependent anemias have focused
`attention on the worldwide need for a safe, orally active
`iron-chelating agent, disorders of increased iron absorption
`should also benefit from their development. Individuals
`with nontransfusion-dependent thalassemia (thalassemia
`"intermedia ")exhibit excessive dietary iron absorption that
`can lead to serious iron loading by the second or third
`decade of life.10The cost to these patients of neglecting iron
`loading is the development of life-threatening tissue dam(cid:173)
`age, and infrequent infusions of deferoxamine may suffice
`to prevent iron ac.cumulation and its complications.II An
`orally effective iron-chelating drug would have major thera(cid:173)
`peutic advantages for these patients, especiaHy for Third
`v.:orici patients with thalassemia intennedia.1:
`Several orally available, iron-chelating compounds have
`been developed in recent years9; few have been evaluated in
`human trials. Among those that have, l,2-dirnethyl-3-
`hydroxypyrid4-one (CP20 or LJ) has been demonstrated.to
`
`promote clinically significant urinary iron excretion in
`animal studies13·14 and in human trials in the United
`Kingdom,1s.16 Europe," India,18 and Canada.19 In a short(cid:173)
`term randomized, cross-over study, Ll's ability to induce
`urinary iron excretion was demonstrated to be comparable
`to that of SC deferoxamine and well tolerated20; recent
`studies have demonstrated its continued long-term effi(cid:173)
`cacy.16.1?
`We report reduction in tissue iron in the liver and heart,
`as demonstrated by magnetic resonance imaging (MRI) of
`both organs, and pe.rcutaneous liver biopsy, after 9 months
`of therapy with L1 in a patient with thalassemia intermedia
`at high risk of progressive iron-induced morbidity. nus
`constitutes the first report of normalization of serum
`ferritin concentration and demonstrated reduction of tissue
`iron stores as a result of treatment with Ll.
`
`MATERIALS AND METHODS
`
`Case Report
`In 1989, a 29-year-old Italian male was evaluated for liver
`disease, presumed to be scoondary to iron overload. The son of
`
`From IM Diviswnsof Hamratology/Oncolcgyand Clinical Pharma(cid:173)
`cology, The Hospital for Sick Childnn; Divisions of Haematology,
`Canliolcgy, Gastroentuolcgy, and Pathology, The Toronto Hospital;
`and the DepartmQlls of Pediatrics, Medicine, Pathology, Chemistry,
`and Clinical Biochemistry, The University of Toronto, Toronto,
`Canada.
`Submitted November 19, 1991; accepted January 16, 1992.
`Suppot1ed by Grant No. MA 10601 from the Medical Researr;h
`Council of Canada. N.F.O. and G.K art Carter Scientists of the
`Ontario Ministry of Health.
`Address rtprint requests to Dr Nancy Oliv~ri, Division of Haematol(cid:173)
`ogy/ Oncology, The Hospital for Sick Children, Room 6'324, 555
`J
`UniversilyAve, Toronto, Canada M5G I X8.
`· Tnc publicatior. costs o,,~. tili.; a."11d~ wer~ aefraycd'\ir. par. o:~.r}iig'r-'·
`charge payment. This article must therefort be hereby maf!<t:d
`"advertisement" in accordanu with 18 U.S.C. section 1734 solely to
`indicate this fact.
`c 1992 by The American Society of Hematology .
`0006-4971192/7910-0032$3.00/0
`
`Bloocf. Vol 79, No 10 (May 15). 1992: pp 2741·2748
`
`2741
`
`~r I
`.ic I
`f I z . H
`
`
`1 of 8
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1005
`
`
`
`2742
`
`OLIVIERI El
`
`nonconsanguinous pare.nts from Calabria, he bad been diagnosed
`with homozygous P thalassemia and had undergone a splenecrorny
`in 1964 at age 3 years. In 1980, he underwent cbolcc:ystcctomy after
`repeated episodes of abdominal pain associated with radiologic
`demonstration of gallstones; RBC transfusions were administered
`before both opcratiollS. He had never re<:cived transfusions since
`then. Io 1980, observation of abnormal IC't'Cls of AST and ALT and
`an inc:rcased serum ferritin oonccntration prompted initiation of a
`program of nightly SC dcferoxamine therapy; the patient complied
`with this erratically and discontinued it after 6 months, although
`the risk of potential iron-related morbidity had been explained to
`him. He administered no iron-chelating therapy in the next 10
`years.
`In 1990, he sought medical attention because of worsening
`fatigue. Physical examination showed bim to be deeply pigmented
`and icteric, with mild muscle wasting. The liver was enlarged and
`firm. Height was at the fiftieth percentile for an adult male11;
`external genitalia were fully developed. Laboratozy hematologic
`studies were consistent with previous assessments of steady-state
`hemoglobin concentrations of 15 to 85 g/L, increased total WBC
`and platelet oounts, and a hemoglobin electrophoresis demonstrat(cid:173)
`ing 98% hemoglobin F and 2% hemoglobin Al. consistent with p0
`th!ilassemia. Fasting glucose, testosterone, TSH. thyroxine, cal(cid:173)
`cium, phosphate and PTH measurements were within normal
`limits. Serum AST and ALT levels were increased at 139 and 98
`U/L, respectively (normal for both <40 U/L); prolbrombin time,
`PlT, total protein, and albumin were within normal limits.
`Transfcrrin saturation was 95% to 100%; serum fcrritin oonceotra(cid:173)
`tioo was 2,000 JLg/L (normal < 300 µ.g/L). Analysis of DNA from
`peripheral blood lymphocytes (PBL) demonstrated homozygosity
`for the codon 39 C~T mutation respoI1S1l>le for po thalasscmia, a
`normal et-globin gene cluster, and bomozygosity for the presence of
`the C to T substitution at position 158 S' to the (Ty gene.n Resting
`electrocardiogram demonstrated atrial bigeminy; radionuclide an(cid:173)
`giogram of the heart showed a normal resting ejection fraction
`(EF), but no increase in EFwitb exercise, and mild abnormalities
`oC diastolic function.
`The evidence for and risk of progression of iron-related organ
`damage was explained in full to the patient, who nevertheless
`declined to resume SC deferoxamine or to begin a program of
`continuous intravenous (IV) defcroxamine infusions; 9 months
`later, be was offered enrollment in the Canadia~ trial of the oral
`iron ehelator Ll. At this time, serum ferritin concentration was
`increased at 2,174 JLg/L; transferrin saturation was increased at
`'100% (normal <30%); serum AST and ALT were increased at 126
`and 94 U/L, respectively; and serum triglyceride level was in·
`creased at 3.74 mmol/L (nonnal 0.34 to 1.58 mmol/L). Antinuclear
`anti~y and rheumatoid factor were negative. ACTH stimulation
`testing demoO.Strated normal baseline ACTII and cortisol, normal
`oonisol lcvcls 30 and 60 minutes after ACTII stimulation, and two
`normal urinazy free cortisol quantitations. Baseline 2~hour uri(cid:173)
`nary iron excretion was 2.9 mg. After undergoing baseline MRI of
`the liver and heart and percutaneous liver biopsy, the patient began
`therapy with Lt at 75 mg/leg body weight per day, administered in
`three divided doses at 8-hour intervals in the fasting state.
`
`Methods
`This study was approved by The Hospital for Sick Children's
`Human Subject Re& \\ ·:::Ommme~ and tile Health Protection
`Branch, Health and Welfare CanltGa (File No. 9427-Hl117-41C.
`HPB, Ottawa. Canada). Writte; informed consent was obtained
`from the patient. Lt was synthesized according lo previously
`published methodsD by the direct reaction in aqueous solution of
`methylamine and mahol, as described previously. 202'
`
`Hepo/U iron quaflliJ.ation. Pcrcutan'eous liver biopsy specirr
`were divided with a wooden splint and weighed immediate~
`determine fresh weighL One portion was then overdried (10~
`hours), reweighed and wet-ashed with hot concentrated HJ
`(Suprapur; Mercie, Dannstadt) in a sc:rew-<ap Teflon vcssc~
`analysis of total iron by Zeeman-corrected clectrothermal ato
`absorption (Varian SpectrAA·300; Techtron Ply, Ud, Malgr
`Australia). The other portion was homogenized with a pb
`pestle in a microcentrifuge tube in 0.50 mL cold 0.25 m<
`sucrose/3 mmol/L imida:zole Ha, pH 7.2 Total iron reoovere
`the homogenate, as compared with that determined in the d
`sample, was 89% to 90%. After sonication (Branson Sot
`W-350; power setting 3, 15 pulses at 20% duty cycle), a 100·
`aliquot was applied to a carboxymethylcellulose oolumn and ch
`in stepwise fashion according to the method of Selden and Pete
`Fractions eluting in the order of transferrin, ferritin, hemoprot1
`and hemosiderin were analyzed for iron by atomic absorption.
`Histok>gic t:XJtmination of the liver. Cores of liver tissue v
`fixed in 10% buffered formalin, embedded in paraffin, and stai
`witb Prussian blue for iron. Tissue was fixed in universal fua
`(2% glutaraldehyde plus 4% fonnaldehyde) and processed u
`standard methods for transmission electron microscopy usu
`Phillips 400 electron microscope.
`Magnetic resonance imaging. The patient underwent magr
`resonance imaging (MRI) in a I.ST superconducting magnet u
`a spin-echo technique.26 The patient was positioned supine ·
`simultaneous cardiac and respiratory gating. Initial coronal
`ITansverse images were talc.en to determine the Euler angle
`image the heart, liver, and peripheral muscle simultaneously a
`the cardiac short axis. Ti-weighted spin echo was perfonncd u
`cardiac gated sequence oonsisting of R-R interval/20/ 2 (TR (1
`of pulse repetition)/TE (time to ecbo)/numbcr of ~uisiti
`spanning &om the apex to the base of the heart at 1-cm centei
`center separations and 7-mm slice thiclcness.
`A midbcart slice position was then selected as the referena
`the T :-weighted spin echo acquisition. This was a gated multi1
`sequence consisting of [x R-R/20-40-60-80/2 (TR/TE)/ numb!
`acquisitions]. Finally, the slice was angled so that it also trave
`both left and right lobes of the liver and the shoulder !"usdes.
`Image analysis. The signal intensities from the cardiac scp1
`posterior wall, liver, and peripheral muscle were calculated I
`identically positioned slices of the T 1-weighted spin echo
`gradient echo images. A 0.25-cmZ of the region of interest
`applied over these organs, and ai;i average signal intensity
`obtained. The ratio of signal intensities between. the heart
`peripheral muscle and the liver and peripheral muscle were 1
`subsequently for comparison. Image-derived Tz values were
`tained by fining the signal intensities from each echo of
`multiecho sequence to an exponential function: SI = SO · e- 1
`where SI is signal intensity, SO is nondecayed signal intensity,
`lE is time to echo. The signal intensities were first correcte<
`variation in image field site and receiver attenuation when app
`ble.n
`
`RESULTS
`
`Efficacy of LI
`Urinary iron excretion. Urinary iron excretion durinf
`first 6 mo nths of LI thera py varied becween 113 and l~
`iron excreted in 24 hours, with a m ean excretio n of 53.3
`equal to a mean urinary iron of 0. 77 mg/ leg body weight
`day. During the next 6 months of Ll administration,
`patient's urinary iron excretion varied be tween 4.6 and
`mg in a 24-hour period, with a mean excretion of
`
`
`2 of 8
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1005
`
`
`
`'·
`
`OAAl IRON CHELATION IN THAl.ASSEMIA INTcRMEDIA
`
`2743
`
`mg/day, or 0.27 mg/kg/day. The decrease in· urinary iron
`excretion is shown in Fig lA
`Serum Jerri/in concetµradon. Serum ferritin concentra·
`tion decreased steadily during 6 months of LI therapy, from
`a prechelation level of 2,174 to 251 µ.g/L after 6 months of
`Ll therapy. The decreases in urinary iron excretfon and
`serum ferritin concentration are shown in Fig lB.
`
`MRI of Cardiac and Hepatic Tron
`Image inLerpreJati.on. On the MRI before the start of LI
`therapy (Fig 2A), the initial Ti-weighted spin echo image in
`the coronal view identified an enlarged liver, with a venical
`span of 18 cm in the right midclavicular line. The most
`notable feature was complete absence of signal from the
`liver, compatible with significant field inhomogeneity cre(cid:173)
`ated by iron deposition. This corresponds to heavy iron
`deposition in the liver. Similarly, on the cardiac shon axis
`view (Fig 3A) before initiation of chelation therapy, the
`liver signal was severely depressed. The myocardial signal
`also was depressed in both views, corresponding to iron
`deposition in the cardiac septum and posterior wall of the
`bean.
`The MR image after 9 months of Ll therapy demon(cid:173)
`strated a distinct improvement in signal intensity from the
`liver region by Trwcighted spin echo imaging, observed in
`both the coronal (Fig 2B) and cardiac short axis (Fig 3B)
`views. Cardiac muscle signal intensity also improved. All
`these arc indications that the iron content in the organs
`imaged was reduced as compared. with that of the previous
`study.
`Quantila.tive MR image-derived parameters. The signal
`intensity ratio between the liver and the peripheral muscle
`was less than 0.01 (normal > 1.0) before therapy with Ll,
`owing to the very low-to-absent signal intensity in the liver.
`After L1 therapy, the same signal ratio improved to 0. 72.
`Before therapy, image-derived T 2 relaxation time for the
`liver was also very short at 9.2 ms (normal 32 to 36 ms).
`After completion of 9 months of Ll therapy, the T2
`relaxation values increased to 17.4 ms. This near doubling
`of relaxation time can be attributed to a reduction of liver
`
`iron content, owing to a fixed relationship between iron
`concentration and l/T2.
`The myocardial/peripheral muscle ratio was 0.90 before
`Ll therapy (normal > 1.3). This improved to nearly nonnal
`values of 0.96 after 9 months of LI therapy. Myocardial T2
`values before therapy was 33 ms, improving to 35 ms after 9
`months of therapy. The less severe iron deposition in the
`myocardium before chelation therapy rendered the improve(cid:173)
`ment in the signal from this organ less dram;lfic than that
`observed in the liver.
`
`Hepatic Iron Stain and Histology
`Histologic examination of the liver biopsy specimen
`taken before the start of treatment with Ll (Figs 4 and S)
`showed more than 90% of hepatocytes strongly positive for
`cytoplasmic iron by Prussian blue staining, with iron also
`abundant in portal macrophages. The liver architecture was
`altered with moderate portal fibrosis. Light microscopic
`examination of the liver biopsy specimen taken after 9
`months of Ll therapy showed approximately 30% of hepa(cid:173)
`tocytes staining positively for iron, with a marked reduction
`in the amount of iron within each hepatocyte (Figs 4 and
`SB).
`
`Hepatic Iron Concentration of Liver Bi.opsy Specimens
`Hepatic iron concentfation after 9 months of Ll therapy
`was 1.9 mg/g dry weight of liver tissue, substantially
`reduced from the value of 14.6 mg/g dry weight of liver
`immediately before initiation of chelation therapy. The
`pattern of iron loading was unchanged, however. Before
`therapy, 83% of hepatic iron was associated with hemo(cid:173)
`siderin, 16% was ferritin, and a very small portion was
`observed in other fractions. After 9 months of chelation,
`85% of hepatic iron was still associated with hemosiderin
`and most of the remainder was associated with ferritin.
`
`Cardiac FWlction
`Radionuclide angiography before Ll therapy showed an
`abnormal resting electrocardiogram with atrial bigeminy,
`
`· :
`,I
`·~
`
`>
`)
`
`I : .
`
`A 120
`c :s
`~ 80
`g
`z
`0 ex:
`....
`ll:l z ....
`
`0
`,Q
`
`100
`
`60
`
`40
`
`B 2soo
`~ 2000
`.:,
`~ 0:
`1500
`~ 1000
`~ 0:
`
`500
`
`Czl
`Cl)
`
`~
`;:i
`
`20
`
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`
`5
`
`MONTH
`
`I
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`: 5
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`
`3
`
`6
`MONTII
`
`13
`
`(A) Urinary iron excretion during 9 months of L 1 therapy in• patient with thalassemla lntennedia, approximately0.77 mg/kg/ day at the
`Ag 1.
`start of L 1 therapy, decreased during the last months as serum ferritln normalized. (Bl Decrease In serum ferTitln concentration during 9 mon~hs of
`L 1 therapy, from a prechelation level of 2, 174 to 251 i<g/L. ls shown.
`
`
`3 of 8
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1005
`
`
`
`2744
`
`OLIVIERI ET A
`
`Fig 2. MAI of hepatic iron IA) before l 1 therapy.
`Initial r,.welghted spin echo MRI in the coronal view
`Identified an enlarged liver with• vertical span of 18
`cm In the right mldclavlcufar Une. Complete absence
`of signal lrorn the liver i$ compatible w ith significant
`field inhomogeneity crHted by Iron deposition. (Bl
`Aller 9-month t 1 therapy. T1-welghted spin echo MRI
`In the coronal view demonstrated improvement In
`signal intensity from the liv1n. Indicating lhal the Iron
`content was reduced 1s compued with that In th•
`pr~vious study.
`
`rcvcrti111: In 1111rmal :;inus rhvthnl tlurinl! cXt:r<"i~e·. The· ldl
`,·c111ri,·uJar EF (l.VEF) wa~ i, l '~i· a[ rcsl.·~·ilh 1111 im:n.::1se· a1
`pe·~k c~i.:rci~c. Mild righ l v\1nlrirnlar dila1a1i11n. and mild
`;1iinormali11i.:s in tlia-;1niic fu11~·1 i',;ri (pl:·ai.. lilii11g r:1ll". time· to
`pl·ak lillinc,. and alri;il con1rihutio111 \\l"t~·°';ih~L:rvi.:d.
`l\f1cr ') monlhs of I.I therapy. Iii.- n:~tin!! 1.VEF re(cid:173)
`mained normal I 57~"i. ). with a n1>rmal incr.:;1\c ( 111 <>.;~·; 1
`
`11m,· ohse·1-v1:d :11 pi.: :1~ ncffi'". Thi.: a h1111r111:di1 ;,., 111' ri!tlll
`v.:111ricul:1r dilat:ition :rntl ahnum1alit il·' in dia,111li<: luni.:·
`li<IU did 111>1 r fl :1ll [!L" during ! .I lhrrapy. 1.:~<"Cpl fm :1 ' li!!hl
`i111pn11·i.:111cn1 in atrial l·11111rihll1ion ( 1-l '·; :1fln I.I the·r;1py.
`:is wmparl·d wilh 17"; hcfurl" 1hc1-.1py ).
`Sc(/i-ty cf I.I. The· pa1iL·nt v..;is rc\'icw.:d in th.: i.:linii.:
`wi.:ddy. I k tuli.:rakd I .I wdl. and rqwrwd 1111 j11i111 p;1in or
`
`-....... .
`
`
`4 of 8
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1005
`
`
`
`•.
`
`ORAL IRON CHELATION IN THAl.ASSEMIA INIERMEDIA
`
`2745
`
`Fig 3. MRI of cardiac iron before l 1 therapy (A).
`lnitlal T 1-welghted spln echo MAI in the cardiac short
`axis view demonstrated a depressed myoc•rdial sig(cid:173)
`nal. (B) After 9-month L 1 therapy, T 1·weighted spin
`echo MRI in the cardiac short axis view demonstrated
`improvement, indicating that the iron content was
`reduced as compared whh that in the previous study.
`
`'• '
`
`,1jff111:~s.:• "' ~ll ch:lll!!l' in hi.:01:1111l111!il· or hi11chc111icd
`paranic1•·rs was nb~c;,.·c:d <lurill)! •/ 1~1t1111hs ,,f 1Jt,.:,1py.
`;\CTI I 1c,1in)! rcpt·:1tnl 4 nllllllh' :1flc:r ha,t·li1w ~lmwctl n11
`d1;J11j.ll' in th.: \'al11c~ nhscr\'cd hl'l'or•· I.I ;1d111i11is1ratiou.
`A111inurk;ir an1ih11dy ;1ml rhc11111a1nid fac:t11r ha\'.: ri.:·
`111aincc.J nc::gati,·c.
`
`DISCUSSION
`Thou s and~ o f Thiru World pa tie nts w it h 111aj<>1
`.,tJ;alassc:mia S)'nclmmcs. induding those who develop irn11-
`., rl'lall'd !c>xil'ity in asS<><:iation wilh gastrnintcMinal hypcrab(cid:173)
`~<•rption uf irnn. 11 .:xhihit scriou:; inn1-rc::latc:d nwrhidity
`and ruortality. F<•r lh~·sc palicnl'-, ;111 orally ~t·tivi.: iron ·
`
`
`5 of 8
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1005
`
`
`
`2746
`
`OUV!ERI ET AL
`
`Fig 4. Hepatic Iron stain and
`histology at low and high power
`IA) before l 1 therapy. Histology
`of the liver showed more then
`90'1. olhepatocvtes strongly pos·
`ltive for cytoplasmic Iron by Prus(cid:173)
`sian blue sulnlng. Iron was also
`abundant In groups of portal
`macrophages; liver architecture
`was altered with moderate por·
`tal fibrosis. (8) After 9 months of
`L 1 therapy. light and electron
`microscopic examination of the
`liver showed approximately 30%
`of hepatocytes staining posl·
`tively for Iron, with • marked
`reduction in the amount of Iron
`in each hepotocyte.
`
`chclnling agent would provide life-sa~ing. chdati<•n thi:rapy.
`Fnr palicnts of the <lcvd0pcd world. freedom from nightly.
`11f1,·n p<1inful infusions w.rnld represent a wck(lme normal(cid:173)
`iz<iiion of lifestyle. An nwlly availahlc iron-d1clalin~ :1gc11t
`has thus been souchl at·li,·ci\· for more lhan.a dcc;idc.
`The l>idcntalc h)•drm:ypyrid1•ncs show t:onsidcrahk poten(cid:173)
`tial as orally availahlt: iron chl"l~h•rs . t\dmini~tratinn Pl
`l.2-tlimc1hyl-:\-hydrnxypyriJ·-l-1iril0 or 1.1 1' · : 11 n:sull..:<l in
`urinary iron cxcrctinn comparable 1111hal 11h~l'incJ with SC
`dcfcroxaminc.: in thalassc.:rnic p:iticms. Although conllicting
`ohsc:rva1ions of acutc toxicity have hecn rc:pnr1l·d wilh usc
`
`of LI in animals.·" ·'~ administration t1f tlll." drui: ha' been
`<t!\SO<:iatcd with infrcqu..:nt bul s..:ri<HI' t:11111plica1io11!-. in
`hum;i111ri<1b. including agr;111ulnc.:ytosis and 1hro111ho<:ywpl."·
`nia which rc.:\'l"rsc:d :1f1-c·r wirhdraw;tl of I.I." ;111<l in lnng·
`te rm <:linit·:il lriab l11:111atolo~k par:11111."lcrs will h;i\l" t11 he
`m1•ni1orc:d l·:1rcfully to tktt· rminl" thL· risk ,,r aµran11!11,)'h•(cid:173)
`si, and wh..:1111.:r 1hc <lrtrl! is ;Kn·ptahl\' :oak lc1r dinit'al u,c.
`\Ve.: repc>rt ll<>rmalizaiinn uf S l'rtl01° krritin n111ccntra1ion
`and rcdl•l'ti••n in tissue.:
`iron ston:s in .1 p:llil."nl with
`lhalasscniia in1cr111l·dia 111;iint:ti1wd on I . I f1>r
`•J months.
`Like lhis p:i1ic111. indi<idu:ol< with th:il:i~"l"1lli:1 i111c·rm~·Jia
`
`....... , -.
`
`
`6 of 8
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1005
`
`
`
`ORAL IRON CHFLA TION IN THALASSE MIA INTEAM EOIA
`
`2747
`
`Fig S. Hep•tlc; Iron stain and histology (Al before
`l 1 therapy and (8) after 9 months of l 1 therapy. w ith
`a marked reduction In nalnable Iron.
`
`who maintain a hi!lh pc:riphcral hcmoglohin conccntrMion
`without regular RDC transfusions may show variable de(cid:173)
`grees of exc:es.~iv.: food iron ahsorpli<>n which may lead to
`serious iron loading f:iy middle life. 11 TI1c~c· patients repre(cid:173)
`scnl a major puhlic health pn1hlcm in populations in which
`lhal:t ~i;em ia is w mmou.1! Although the <k grc e of iron
`owrlo:ul is less in suc:h patk nt~ than in patknts with
`tr:111sf11si11n-dep<:ndl·nt thaht~scmia .11 nur patient hnd cvi·
`<lcn.:c of inm-rclaktl nrgan wxicity with carF:i- hepatic:
`cirrhosis :111d mi(.! eardi;ic: diastolic (lysfunl·tion with la.:k nr
`systolic incrca'c: with c~l·rt"i'l' . I.I thl·rapy ha~ rcduc:c:d hi~
`ti~~11e irnn fo;1tf suhManti:1lly. a~ dcmon~t ra t e<.l hy reduction
`in tran~fcrrin ~;1t11ra t i1111 and ~c:rum krritin conc:cntration.
`i111pruvc111c111 in lin·r l111wt ion. and inc:rca<c in I. vr:r with
`•·xcrci~.: aftl·r ''·11111111h I.I thc1ap) . impro,cml·nt in /v!RI of J.
`1
`.....
`REFERENCES
`Coh,·n 1\. l ':o.:.1t.11rir hcm:11itl••c\ . I lr111a111I On.·111 Clin N11r1h
`l ~fh:t·1 un livl·r 1r,1n c:c111l"l .. Ulration.
`llu.·rary Ill th:ila"cnua nMJtlf
`,\111 (::\:::I. l'IX'
`• •
`Ii\ er hi''"'"!!) .rn.t dtni.-:11 prllftC>'. llr Med) =::tl'I. 1'17-1
`,;. ll.1rr~ ,\I. Fl~ 1111 I>. Lt·t'~~ I:. l(i-.l11n k1\: I ""!: r.:11n d1clJ ti11n
`.1. \Volf,· LC Oli< 1cri NF. !>:111,111 Dl.. Pr11rr•·r RD. Frcc,lm;111
`
`cardiac and hepatic iron, and reduction in hepatic iron
`conccnlnition. Omtinucd monitoring of urinary iron cxcrc(cid:173)
`tfon and 1rc11ds in scrum fcrritin conccnrrntion in response
`lo a reduced daily dllSc of LI will be necessary in t11fa
`patient to prevent further iron-related organ damage.
`Our reporl is the first to present cvitlcncc in hum:ms for
`LI -induced rcJuction of iron in the liver unJ heart, the
`organs involvc1I in th..: most ~crinu~ iron-rclntcJ morbidity
`:mu m!lrtality. Although the :>afcty and dlic:acy of long-term
`use of LI has hl'l'll the suhjcct ,,f much ddiatc.~'-.':
`is "'"'
`governmental appnwal frn c:li11i.:al 11 ials t\f LI
`forthcoming. in !'cvt•r:1I crn1111rk~. indudin~ the U11i1c:d
`Stales. ;tnd ~hould he wpportcd hy the c:nt·ouragin!! result
`ohSC:f'\cd in'"" pal icnt.
`
`.•
`'•·
`
`" . 1 . ...
`l.'.
`: '~
`
`
`7 of 8
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1005
`
`
`
`...
`
`2748
`
`MH, Nathan DG: Tbe prevention of cardiac disease by subcutane(cid:173)
`ous deferoxamine in patients with thalassemia major. N Engl J
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`7. Ehlers KH, Giardina PJ, Lesser ML, Engle MA, Hilgartner
`MW: Prolonged survival in patients with beta-thalassemia major
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`12 Weatherall DJ, Clegg JB: The Thalassemia Syndromes.
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`14. Kontoghiorghes OJ: Dose response studies using desfeniox(cid:173)
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`15. Kontoghiorghcs GJ, AJdouri MA, Hoflbrand AV, Barr J,
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`16. Kontoghiorghes 01, Bartle$$ AN, Hoflbrand AV, Goddard
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`OLIVIERI ET Al
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`(1,2-dimethyl-3·hydroicypyrid-4-one) for orai" iron chelation in
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`18. Agarwal MB, Viswanathan C, Ramanathan J, Massi! DE,
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`oral iron cbelator LI in iron loaded patients. Lancet 336:1275, 1990
`21. Tanner JM, Whitehouse RH: Oinical longitudinal standards
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`22. Miller BA. Olivieri NF, Salameh M, Ahmed M, Aatogoetti
`G, Nathan DO, Orkin SH: Molecular analysis of the high F
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`potent iron chelators· l-alk:yl-3-bydroxy-2-methylpyrid-4-ones. ln(cid:173)
`org Chim Acta 136:L11, 1987
`24. Olivieri NF, Koren G, Herman C. St Louis P, Bentur Y,
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`28. Mehta J, Singhal S, Revankar R, Walvalkar A, Olablaoi A,
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`30. Olivieri NF, Koren G, Freedman MH, Roitman C: Rarity of
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`
`
`8 of 8
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1005
`
`