`Apotex Inc. et al. v. Novartis AG
`IPR2017-00854
`
`
`
`148
`
`.IJ. Stepan et a]. / Clinica Chimica Acta 348 (2004) 147—154
`
`the calcium kinetics [2], indicating that the biochemical
`markers ofthe bone turnover have a potential to assess
`the rate of the bone loss and to improve the prediction
`ofthe risk of osteoporosis in postmenopausal women.
`Biochemical markers reflect the whole body rates of
`bone resorption and bone formation and may provide a
`more representative index of the overall skeletal bone
`loss than it would be obtained by measuring the rates of
`change in bone mineral density at specific skeletal sites
`containing different ratios of a trabecular to cortical
`component with different metabolic rates. In postmen-
`opausal women, a sustained increase in bone turnover
`induces a faster bone loss and therefore an increased
`
`risk of osteoporosis, and biochemical markers are
`associated with bone loss measured at
`the forearm,
`calcaneus and hip, with the progressively greater risk of
`a rapid bone loss with increasing levels of markers [3].
`Bone loss due to glucocorticoids is steep during
`the first 12 months, and more gradual but continuous
`in subsequent years [4]. The mechanism of bone loss
`in patients on glucocorticoid treatment is complex [5].
`Unlike estrogen deficiency in which both bone re-
`sorption and formation increased, a major effect of
`glucocorticoids is on osteoblast function. In addition
`to a decrease in osteoblast differentiation, glucocorti-
`c01ds increase osteoblast and osteocyte apoptosts [6].
`Depression of bone formation by glucocorticoid ex-
`cess results in disruption of the fine balance among
`rate of supply of new osteoblasts and ostcoclasts and
`the timing of the dcath of'osteoclasts, osteoblasts, and
`osteocytes by apoptosis [7]. Clinically, decreased
`bone formation can be documented by decrease in
`circulating concentration of markers of osteoblast
`function such as osteocalcin and propeptides of type
`I procollagen [8,9].
`The role of bone resorption in bone loss character-
`istic of glucocorticoid—induced osteoporosis is unex-
`plained. The carly loss of bone with glucocorticoid
`excess is caused by extension of the life span of pre-
`existing osteoclasts, an effect not preventable by
`bisphosphonates [10]. However, the effect oflong-term
`low dose glucocorticoid administration remains con~
`troversial. Long-term effects of glucocorticoids on
`osteoclast function are inhibitory rather than stimula—
`tory [11713]. Clinically, an accelerated bone resorp-
`tion can be documented by increase in serum or urinary
`concentration of collagen breakdown products such as
`
`the deoxypyridinoline cross-links and the markers
`
`the cross-linked N-
`involving the cross-linking site,
`terminal telopeptide oftype I collagen (NTX), and the
`C telopeptide of type I collagen alphal (CTX) [14].
`The contribution of the underlying disease, for
`which glucocorticoids are used, confounds the assess-
`ment of glucocorticoid effects on bone. Multiple scle—
`rosis (MS) is a gait disorder characterized by acute
`episodes of neurological defect leading to progressive
`immobilization [15]. Long-term glucocorticoid use and
`progressive immobilization, along with vitamin D
`deficiency, are likely to be determinants for osteopo-
`rosis and skeletal muscle atrophy, and the increased risk
`of fracture in patients with multiple sclerosis [16718].
`The aim of this study was to evaluate the clinical
`value of markers of bone remodeling in assessment of
`rate of bone loss in patients with multiple sclerosis
`long term treated with low dose glucocorticoids, and
`to test the relative contribution of glucocorticoid dose
`and physical inactivity to rate of bone loss in patients
`with multiple sclerosis.
`
`2. Patients
`
`The study population involved 47 women and 23
`men with multiple sclerosis (Table 1). Thirty—one
`
`40.3 i 10.2
`60.4 i 8.0
`165.1 i 6.4
`10.0 : 7.4
`
`42.4 : 12.1
`76.7 t 11.6
`180.3 : 6.9
`14.0 : 6.0
`
`41.0 :109
`65.8 i 12.0
`170.1 : 9.7
`11.3 : 7.2
`
`6.0 : 5.4
`
`6.7 : 5.1
`
`6.2 i 5.3
`
`74:29
`
`71:27
`
`73 :18
`
`Table 1
`Baseline characteristics of patients with multiple sclerosis
`(mean : SD.)
`
` Women Men All patients
`
`No
`47
`23
`70
`Age (years)
`Weight (kg)
`Height (cm)
`Duration of MS
`(years)
`Glucocorticoids
`(years)
`Mean dose
`(mg/day)
`Cumulative
`dose (g)
`KEDSS
`BMD lumbar
`spine (T—score)
`BMD total
`femur (T-score)
`BMD femoral
`neck (T—score)
`
`44,7Smokers (%) 47.1 52.2
`
`29.9 i 22.5
`
`42.1 i 25.8
`
`33.9 i 24.2
`
`4.4 i 1.9
`5.0 i1.9
`4.1 i 1.8
`— 1.29 t 1.27 ~1.81:1.33 — 1.46 i130
`
`4125:134 71.74:1.17 771.41:1.30
`
`v 1.67 11.33
`
`— 2.()1i1.36
`
`71.78 i134
`
`
`
`
`
`
`
`./.J. Slcpan el al. / Clim'ca Clzimir‘u Acta 348 (2004/ [4777/54
`
`149
`
`women were premenopausal (mean age, 35.1 i 8.1
`years), 16 postmenopausal women (mean age,
`50.3 t 5.0 years) were on regular hormone replace-
`ment therapy. The patients received the daily recom—
`mended dose of calcium (500 mg) and vitamin D (400
`1U). Excluded were patients with diseases, disorders or
`therapy with other drugs known to influence bone
`metabolism.
`
`3. Materials and methods
`
`3.1. Clinical assessment
`
`Motor function of the patients was evaluated using
`the Kurtzke Expanded Disability Status Scale
`(KEDSS), a scale useful in measuring the ability to
`walk that is decisive for normal remodeling of bone
`[19]. Scoring agreement for trained examining physi-
`cians has been repeatedly confirmed [20,21]. Cut—off
`KEDSS 6 represents reasonable end of motor perfor-
`mance of the patient. 6.5 means only several meters
`with bilateral support, mogtly within the flat, and 7 is
`only the ability of transfer to wheelchair from the bed.
`Glucocorticoid use was determined from an interview-
`
`tar-administered questionnaire
`
`3 .2. Bone densitometly
`
`BMD (g/cmz) was determined using Hologic
`4500A (Waltham, MA) densitometer. Normative
`values providcd by Hologic (NHANES 111 nor-
`mative values for
`the proximal
`femur) were used
`for the determination of T—scores (comparison with
`a gender-matched young normal
`reference popula-
`tion). The short—term precision in vivo error
`for
`BMD at the lumbar spine (L1«L4), total femur, and
`femoral neck was 0.7%, 0.9%, and 1.9%,
`respec-
`tively;
`the long-term precision in vitro error was
`0.31%. BMD was measured at baseline and after
`
`1.8i0.8 years.
`
`3.3. Biochemical analysis
`
`Blood specimens were collected in the morning,
`after an overnight fast. Biochemical markers of bone
`turnover were measured at
`the baseline. Normative
`
`values for this laboratory were determined in age-
`
`matched healthy subject (94 premenopausal women,
`and 46 men).
`The serum concentrations of intact aminoterminal
`
`propeptide of type I procollagen (PTNP) was assessed
`by radioimmunoassay (Procollagen Intact PINP, Orion
`Diagnostica, Finland). The assay is not sensitive to
`the small molecular weight‘ degradation products of
`the propeptide. The within run imprecision was
`below 5%, and between run imprecision was below
`7% at concentrations between 20 and 90 ug/l. Normal
`values (mean and 1 SD. range) determined in the
`control group were 33.1 ug/l
`(25.3—43.1 ug/l) and
`35.2 ug/l (27.07459 ug/l)
`in women and men,
`respectively.
`The concentration of type 1 collagen cross—linked
`C-telopeptide (beta CTX) and N-MID osteocalcin
`(OC) in plasma was assessed using electrochemilumi-
`nesce-based immunoanalysis (the Elecsys 1010 Ana-
`lyzer, Roche Diagnostics, Germany). The within run
`imprecision of the CTX was below 5% for samples
`>500 pg/ml, below 7% for samples between 200 and
`500 pg/ml and below 10% for very low CTX con-
`centrations samples. The between run imprecision
`results were below 7% for samples >500 ng/l and
`below 9% for samples between 200 and 500 ng/l. The
`detection limit was <10 rig/1 Normal values (mean
`and 1 SD. range) determined in the control group
`were 267 ng/l (2044350 ng/l) and 294 ng/'1(2204393
`ng/l), in women and men, respectively. The within run
`imprecision for the OC was <5%, and between run
`imprecision was <6% at concentrations between 11
`and 40 ug/l. Normal values ‘(mean and 1 SD. range)
`deterrnincd in the control group were 20.6 ug/l (15.5
`27.4 ug/l) and 21.2 ug/l (15.6—28.9 ug/l),
`in women
`and men, respectively.
`
`3.4. Statistical analysis
`
`Data were expressed as mean valueistandard
`deviation if not otherwise stated. Logarithmic trans-
`formation was used to normalize non-normal distri-
`
`butions. The original skewed data were tested to
`confirm the validity of the transformation. The differ-
`ences in clinical and biochemical characteristics be-
`tween groups were compared using t-test. The
`differences in biochemical markers between groups
`of patients with MS and controls were compared
`using one-way ANOVA. Subsequent Bonferroni t—test
`
`
`
`150
`
`Table 2
`
`JJ. Stepzm e! a]. / Clinica Chimica Acm 348 (2004) 1474154
`
`SigmaStat statistical software version 3.0 (Jandel,
`San Rafael, USA).
`
`4. Results
`
`the lumbar spine and
`BMD T—scoreZI S.D. at
`proximal femur was found in 20% patients (11 women
`and 3 men, KEDSS, 3.3 i 2.0; bone loss at the total
`femur, ~1.10 i 2.06% per year). BMD T—score be-
`tween -—1 and —2.5 SD. was found in 45.7% patients
`(23 women and 9 men, KEDSS, 4.0 i 1.8; bone loss,
`—1.52i 1.97% per year). BMD T—score below ~25
`SD. was found in 34.3% patients (13 women and 11
`men, KEDSS, 5.6i 1.3; bone loss, 43.47i 3.63%
`per year).
`the variables are shown in
`Mean values for
`Tables 1 and 2. The biochemical markers are com-
`
`pared with the corresponding values in the control
`group.
`The correlation matrix for the variables is shown
`
`Kurtzke Expanded Disability Status Scale (KEDSS), biochemical
`markers of bone remodeling and rate of bone loss (RBL, % per
`year) in patients with multiple sclerosis
`
`Women
`Men
`All patients
`47
`23
`70
`29.8
`56.5
`38.6
`
`No
`KEDSS 2 6
`("/0)
`RBL lumbar
`spine
`RBL total
`femur
`RBL femoral
`neck
`Osteocalcin
`(ug/l)
`PlNP(ug/1)
`
`beta CTX
`
`v 0.69 t 2.53 . — 1.29 i 3.50
`
`v 0.89 i 2.87
`
`—1.69i2.37
`
`42,96:3.46 —2.10i2,82
`
`— 2.31 i 279
`
`19.2
`(10.0~36,9)
`33.3
`(1917584)
`318
`
`
`
`~ 2.84 : 3.19
`
`20.6
`(13.47318)
`38.5
`(24.6~-60,3)
`387
`
`— 2.49 i 2.90
`
`19,7
`(10.9 «35.4)
`350
`(20.67592)
`349
`
`(ng/l)
`
`(1887538)*
`
`(234~~640)*
`
`(2137570)
`
`*p < 0.05 as compared with the normal values
`laboratory, in healthy subjects (t-test).
`
`for
`
`this
`
`was used to determine differences between the groups.
`The correlation was analyzed using least square linear
`regression analysis. A p<0.05 was considered statis—
`tically significant. Analyses were made using the
`
`In patients with multiple sclerosis, a
`in Table 3.
`significant positive correlation was found between
`biochemical markers of bone remodeling (Fig.
`1).
`However, only the marker of bone resorption (plas-
`
`Table 3
`l‘he correlation matrix and the statistics for the “best" subset in the multiple linear regression analysis for rate of bone loss (RBL) at the
`proximal femur as dependent variable in patients with multiple sclerosis
`
`Variable
`1
`2
`3
`4
`5
`6
`7
`8
`l RBL femur
`2 RBL neck
`
`,.
`0.44*
`
`3 RBL spine
`4 KEDSS
`5 CTX
`6 PlNP
`7 Osteocalcin
`8 CC total dose
`9 BMI
`Multiple correlation
`Adjusted squared niult. correlation
`F—statistics (11/267)
`Significance
`
`0.24
`7056*
`—0.45*
`7021
`70.12
`7 0.25
`0.13
`
`0.34*
`—0.39*
`7046*
`~ 0.22
`“0.11
`., 0.23
`W 0.02
`
`70.11
`—0.12
`7 0.08
`70.03
`W 0.01
`(),()l
`
`0.46“
`0.26
`0.24
`042*
`.,. 0.11
`0,60
`0.34
`19.12
`<0.001
`
`055*
`046*
`0.02
`.. 0.04
`
`086*
`., 0.05
`0.01
`
`A 0.07
`0.08
`
`7* 0.08
`
`
`Variable
`Coefficient
`Standard error
`2
`Significance
`
`<0.001
`—4.14
`0.165
`70.682
`KEDSS
`0.03
`— 2.19
`0,001
`r 0,003
`Beta-CTX
`
`Constant 0.005 2.172 0.744 2.92
`
`
`
`
`*Correlation coefficients at the 0005 level of significance,
`
`
`
`151
`J.J. Slepan 61 ul. / Clinica Cliimitiu Acta 348 (2004) 1477154
`
`
`r—
`1500 ~
`
`A
`
`
`
`BaselineserumCTX(ng/l
`
`1000
`
`700
`
`200 l—r—
`
`
`
`
`
`500 300
`
`a
`
`O
`
`I
`
`I
`
`.-
`
`\
`
`4—...
`1
`
`I
`2
`
`L
`l
`L
`5
`4
`3
`Kurtzke EDSS
`
`I
`6
`
`L
`7
`
`Fig. 3. Relationship between the Kurtzke EDSS and plasma CTX.
`Dotted line: prediction intervals.
`In y=—0.093x+0.027x2+5.64,
`r=0.51, 11:70, p<0.001. The solid bars indicate normal
`range
`(x i 2 SD.) for this laboratory (light: women, dark: men).
`
`bone remodeling which correlated with the rate of
`bone loss and with the motor function of the patients
`(KEDSS) (Table 3). None of the remaining variables
`(age, weight, height, duration of multiple sclerosis,
`duration of GC treatment and the average and total
`dose of GC) entered the regression model. After
`adjustment for BMI,
`the plasma CTX concentration
`remamed significant predictor of the rate of bone loss
`(2141-05).
`.
`Mean values for the biochemical markers of bone
`
`remodeling and rate of bone loss by KEDSS are
`shown in Table 4.
`
`Table 4
`Biochemical markers of bone remodeling and rate of bone loss
`(RBL, "/0 per year) by Kurtzke Expanded Disability Status Scale
`(KEDSS) in patients with multiple sclerosis
`KEDSS 175.5
`KEDSS>55
`
`27
`43
`No
`7383 i289"
`7— 1.02t2.18
`RBL femur
`, 3.70 i 258*
`-1.74 i 2.85.
`RBL neck
`~ 1.00 t 2.28
`0.82 t 3.21
`RBL spine
`455 (287 720)M
`295 (190 77458)
`beta»CTX
`40.7 (23.2~71.4)***
`31.8 (196' 51.71
`PlNP
`
`
`17.9 (10.3 80.9)Osteocalcin 23.0 (12.3 42.8)
`
`*p<0.005 as compared with KEDSS 175.5 (Hest).
`**p<0.05 as compared with KEDSS 1m5.5 and with control
`group (ANOVA).
`‘
`***p<0,05 as compared with KEDSS 1755, not with control
`group (ANOVA).
`
`150
`
`
`
`PINP(ug/l)
`
`100E80
`60 '
`50
`40 [30
`20 -
`
`‘10P
`
`
`
`5CL__L| “flux—1.41144
`3
`5
`10
`20
`30
`50
`70 100
`150
`Osteocalcin (ug/l)
`
`1. Correlation between circulating osteocalcin and PINP
`Fig.
`(y: — 46.0 — 3.86x, r2046.
`)1: 120, p<0.001).
`ln y=0.7681n
`.r+ 1.269, r:0.86, n : 70, p<0.001. The solid bars indicate normal
`range (.x i 2 SD.) for this laboratory (light: women, dark: men).
`
`ma CTX) correlated significantly with the rate of
`bone loss at
`the proximal femur (Fig. 2) and with
`KEDSS (Fig. 3). The rate of bone loss at
`the
`proximal
`femur was not significantly different
`be—
`tween tertiles of plasma OC concentrations
`(ANOVA).
`The multiple regression analysis of the results in
`multiple sclerosis indicated that
`the plasma CTX
`concentration was the most significant parameter of
`
`_
`
`3000
`
`92000 —
`
`serumCTX(ng/ 100 50._:iiiL__L_IiiiiJ__.liIil
`Baseline
`
`r...
`
`10005
`
`500
`
`300
`200
`
`-12
`
`-10
`
`-8
`
`.6
`
`-4
`
`-2
`
`0
`
`2
`
`4
`
`6
`
`Change from baseline in BMD (% / year)
`
`Fig. 2. Relationship between the rate of BMD change at the total
`proximal femur and plasma CTX. Dotted line: prediction intervals.
`In y: ‘ 0.079x+5.867, r=0.59, n:70, p<0.001. The solid bars
`indicate normal range (x i 2 SD.) for this laboratory (light: women,
`dark: men).
`
`
`
`152
`
`J.J. Slepan er a/ / Clinica Chimica Arm 348 (2004) [477/54
`
`5. Discussion
`
`Results in this study are in agreement with the
`hypothesis that long-term effects of low—dose gluco-
`corticoids on osteoclast function and biochemical
`
`marker of bone resorption are not stimulatory, al-
`though such effects may'possibly occur at an early
`and/or transient phase of glucocorticoid-induced os-
`teoporosis [10].
`In patients long-term treated with
`glucocorticoids, osteoclast surface is not
`increased
`[13], and serum concentrations of markers of bone
`resorption have been shown to be inhibited rather than
`elevated [11,12]. Similar results have been reported in
`patients with Cushing’s syndrome [8,9].
`In our
`patients with a good physical activity (KEDSS l~
`5.5), all markers were within normal limits, Only in
`patients with poor physical activity (KEDSS>5.5)
`plasma CTX was significantly increased as compared
`with the control group. .Thus,
`this study confirms
`detrimental effects of physical
`inactivity on bone
`mass.
`
`total body bone mineral content and
`Previously,
`fat—free mass were shown negatively associated with
`the disability status (KEDSS) [18]. In this study, the
`contribution of physicalvinactivity confounded the
`assessment of glucocorticoid effects on bone markers,
`and site‘spccific effects of disability were docu-
`mented. A significant negative correlation was dem-
`onstrated between the disability status and rate of
`bone loss namely at the proximal femur but lumbar
`spine. In wheelchair bound patients, an atrophy of hip
`muscles affects proximal femur while lumbar spine is
`stimulated by the trunk and back muscles. The accel‘
`erated bone loss in the disabled patients was associ-
`ated with an increased bone resorption as indicated by
`plasma CTX. The results are in good agreement with
`increased bone loss in prolonged immobilization in
`bed rest,
`in paraplegics, or after long duration space-
`flight
`that
`is associated with the loss of skeletal
`muscle [22,23]. Biophysical
`input generated during
`normal physiological
`loading is a major determinant
`of bone mass and morphology. An inhibitory effect of
`strain on receptor activator of NF-HB ligand
`(RANKL) expression has been demonstrated [24]
`indicating that mechanical stressors utilize classical
`intracellular signaling pathways. The RANKL binds
`to its receptor on the surface of hemopoietic osteoclast
`progenitor cells and, with macrophage colony stimu—
`
`lating factor is sufficient to induce osteoclastogenesis
`[25].
`The bone loss in patients treated with glucocorti-
`coids can be accentuated by an impaired bone forma-
`tion. At pharmacological
`levels,
`the major effect of
`glucocorticoids on bone cells involves compromising
`osteoblast survival through enhancement of apoptosis
`and adipocyte differentiation at the expense of osteo-
`blast differentiation and function [26]. Despite low
`dose glucocorticoids treatment, plasma concentrations
`of osteocalcin indicating an impaired bone formation
`were decreased in 8 patients with good physical
`activity and in 3 patients with KEDSS>5.5. More-
`over, in physically disabled patients, plasma OC was
`not significantly increased despite evidence of in-
`creased bone resorption, indicating an impaired cou—
`pling between bone resorption and bone formation in
`GC treated patients.
`This study is
`limited by the precision error of
`repeated measurements of BMD in a single individ-
`ual, which is of the same order of magnitude as rate
`of bone loss over 2,4 years,
`i.e., 3—4%, by the
`precision error of repeated measurements of the
`markers, by differential rates of bone loss between
`various skeletal sites, and because it
`is not clear
`whether the bone loss at the var10us sites 1s cons1s—
`
`tent over time. Another important limitation of this
`study is that 25—hydroxyvitamin D, PTH, cytokincs,
`sex hormones, and other parameters of calcium and
`bone metabolism were not determined.
`It
`is not
`
`known whether vitamin D supplementation was
`sufficient
`in patients in this study. During the 2
`years of prospective follow-up in 54 patients with
`multiple sclerosis, bone loss at
`femoral neck was
`somewhat faster in the group with low levels of 25-
`hydroxyvitamin D (5.6% per year) compared with
`the group with high levels of 25-hydroxyvitamin D
`(4.3% per year)
`[17]. Also. decreases in various
`circulating sex steroids may potentially contribute
`to the bone loss in glucocorticoid treated patients
`[27,28]. However, changes in circulating hormones
`would cause systemic rather than the site-specific
`increase of bone loss in our patients. Postmenopausal
`women not substituted with estrogens were not
`included into this study.
`This study indicates that in patients on long-term
`GC treatment for MS, plasma CTX is associated with
`bone loss measured at the hip, with the progressively
`
`
`
`JJ. Sreprm et al. / C/i/zica C/iimica Acta 348 (2004) [4777/54
`
`153
`
`greater risk ofa rapid bone loss with increasing levels
`of the marker of bone resorption. The results support
`the view that patients having increased biochemical
`marker of bone resorption are confirmed 2 years later
`as bone losers [14]. However, the available data do
`not
`indicate that measuring the individual serum
`marker of the bone turnover can accurately predict
`rates of bone loss at the hip and spine over a 2-year
`period in the individual with sufficient accuracy to be
`used in clinical practice.
`We conclude that in adult patients with MS treated
`with low dose GC, the plasma CTX concentration was
`the most significant parameter of bone remodeling
`which correlated with the rate of bone loss and with
`
`the motor function of the patients.
`
`Acknowledgements
`
`We thank Eva Ticha, Oldriska Lukaskova, Anna
`Masatova, and Jana Krenkova for excellent technical
`assistance. The work was supported by project
`NF6780~3 by the IGA, Ministry of Health of the
`Czech Republic.
`
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