APOTEX ET AL. - EXHIBIT 1058
`Apotex Inc. et al. v. Novartis AG
`IPR2017-00854
`
`

`

`130
`
`controls were selected from a cohort of healthy subjects recruited
`by the Regional Bone Center for the establishment of a reference
`range. The study was approved by the Institutional Review Board
`of Helen Hayes Hospital and all patients provided informed con-
`sent.
`Patients with MS were divided into two groups: ambulatory
`and nonambulatory, based on the Kurtzke Expanded Disability
`Status Score (EDSS) [29]. For purposes ofthe study, patients were
`characterized as ambulatory iftheir EDSS score was 0 to 6.5 and
`nonambulatory if their EDSS score was greater than 6.5. All pa-
`tients with MS received pulsed pharmacological and supraphysi-
`ological doses of glucocorticoids according to the following pro-
`tocol: one steroid month was composed of Solumedrol that was
`administered intravenously at 1.0 g for 1 week, then 0.5 g for 3
`days, and 0.25 g for another 3 days, followed by oral Prednisone
`at 80 mg for 1 week, 60 mg for 1 week, 40 mg for 4 days, 20 mg
`for 4 days, 10 mg for 4 days, and 5 mg for 4 days. Glucocorticoid
`use was therefore expressed as duration of use (months) and was
`determined from a interviewer-administered questionnaire. We
`have previously reported that the mean 25(OH)D levels in this
`group of patients with MS were in the insufficient range, and 12
`patients (23%) had frank vitamin D_ deficiency [2]. The vitamin D
`status in both the ambulatory and nonambulatory MS patients did
`not differ between the groups. Likewise,
`ionized calcium was
`within the normal range and did not differ between ambulatory and
`nonambulatory patients with MS.
`
`Measurement ofBone Mass and Body Composition
`
`total body fat-free
`Total body bone mineral content (TBBMC),
`mass (FFM), and total body fat mass (FM) was measured in the
`patients with MS using dual X—ray absorptiometry (Norland XR—
`26, Fort Atkinson, WI). For the control group,
`total body bone
`mass and body composition was measured using dual X—ray ab-
`sorptiometry (Norland XR-26, Fort Atkinson, WI,
`in 41 subjects
`and Lunar DPX-L, Madison, WI, in 30 subjects). The results for
`the 30 control subjects measured on the Lunar densitometer were
`adjusted to Norland equivalent values usmg linear regressron
`analysis based on 78 healthy female subjects. The conversion
`equations are shown below:
`
`TBBMCXR (g) = 1.103(TBBMCDPH) + 79.01527;
`r = 0.90, P < 0.01
`
`FatXR (00) = 1.032 (‘VoFatDPX_L) + 9.23;
`r 2 0.95, P < 0.01
`
`FMXR (g) = 1.156 (FMDPX_L) + 2334.6;
`r : 0.98, P < 0.01
`
`FFMXR (g) 2 0.7152 (FFMDPX_L) + 2908;
`I”
`0.83, P < 0.01
`
`Statistical Analysis
`
`Comparisons between MS patients and age—comparable controls
`were done using unpaired t tests. Linear regression analysis was
`used to determine the relationship between total body bone mineral
`content and fat-free mass with EDSS score. Comparisons of non-
`ambulatory patients with MS, ambulatory patients with MS, and
`age-comparable controls were performed using analysis of vari‘
`ance with Tukey HSD post—hoc tests. The effect of covariates on
`the difference between ambulatory and nonambulatory patients
`with MS was determined using analysis of covariance. Bone mass
`and fat-free mass were expressed in absolute terms (kg) and as
`Z-scores (SD). Z-scores were calculated as the difference between
`the observed and predicted value (based on the fitted equations
`adjusting for the covariates, age, and menopausal status in the
`control group), divided by the square root ofthe estimated variance
`
`C. A. Formica ct al.: Body Composition in Multiple Sclerosis
`
`for the control group. All analyses were performed using Systat for
`Windows.
`
`Results
`
`Mean descriptive characteristics and indices of body com-
`position and bone mass are shown in Table 1 . In the patients
`with MS, 10 had sustained fractures (4 ankle, 2 vertebral, 1
`hip,
`1 rib,
`1 Colle’s, and 1
`leg). There were no significant
`differences in age, height, or weight between the controls
`and the patients with MS. However, compared to age-
`comparable controls, BMI in patients with MS was statis~
`tically less than the BMI of controls (23.6 i 0.6 vs. 26.0 i
`1.0 kg/mz, P < 0.05). Compared to age-matched controls,
`patients with MS as a whole group had deficits in TBBMC
`and FFM when expressed as Z-scores (both —0.3 i 0.1 SD,
`P < 0.04), but not when expressed in absolute terms. After
`adjustment for the deficit in FFM, TBBMC was no longer
`significantly different in patients with MS as compared with
`age-comparable controls. As shown in Figure 1, EDSS
`score was negatively associated with both TBBMC (r =
`—0.33, P < 0.01) and FFM (r = ~0.41, P < 0.01). Total
`body bone mineral content was marginally associated with
`FFM (r = 0.23, P = 0.06), however, after adjustment for
`FFM, EDSS score was an independent determinant of
`TBBMC, and FFM failed to reach statistical significance (P
`= 0.4).
`As shown in Table l and Figure 2, patients with MS who
`were nonambulatory, had greater deficits in TBBMC as
`compared with age-matched controls, when expressed both
`in absolute terms (2.3 i 0.1 vs. 2.5 :t 0.1 kg, P < 0.05) and
`as a standardized score (‘06 i 0.1 SD, P < 0.01). Also,
`when compared to ambulatory MS patients, nonambulatory
`MS patients had a deficit in TBBMC whether expressed in
`absolute terms (2.3 i 0.1 vs. 2.6 i 0.1 kg, P < 0.05) or as a
`standardized score (‘06 i
`.1 vs. 0.0 i 0.2, P < 0.01).
`Fat~free mass in nonambulatory MS patients was signifi-
`cantly reduced as compared with age-matched controls
`when expressed in absolute terms (26.8 i 0.7 vs. 29.8 :t 0.6
`
`kg, P < 0.01) or as a standardized score (-0.6 :: 0.1 SD, P
`< 0.01). When compared with ambulatory MS patients, non—
`ambulatory MS patients had a deficit in FFM when ex-
`pressed in absolute terms (26.8 i 0.7 vs. 29.8 i 0.7 kg, P <
`0.02) and as a standardized score (706 i 0.1 vs. 0.0 i 0.2
`SD, P < 0.01). Ambulatory patients with MS were similar to
`age-matched controls for all measurements (P = NS for all,
`Table 1).
`Comparing ambulatory MS patients to nonambulatory
`MS patients,
`the duration of corticosteroid use (months)
`
`failed to reach statistical significance (3.2 i 0.8 vs. 5.4 :: 0.8
`months, P = 0.06), however, the duration of corticosteroid
`use was considered to be biologically significant and was
`treated as a possible covariate. In ambulatory MS patients,
`9 women were postmenopausal as compared with 11 post-
`menopausal nonambulatory MS patients. Years since meno-
`pause did not differ between the two groups. Following the
`results ofthe analysis of covariance, the difference in FFM
`between ambulatory and nonambulatory patients with MS
`was accounted for by the duration of glucocorticoid use
`(adjusted means: 29.1 i 0.8 vs. 27.0 i 0.8 kg, P = NS),
`whereas the difference in TBBMC between ambulatory and
`nonambulatory patients with MS was accounted for by the
`difference in FFM (adjusted means: 2.5 i 0.1 vs. 2.3 :t 0.1
`kg, P = NS).
`
`Discussion
`
`These data suggest that nonambulatory patients with MS
`
`

`

`C. A, Formica ct al.: Body Composition in Multiple Sclerosis
`
`131
`
`Table 1. Age, height, weight, glucocorticoid use, bone mass, fat mass, and fat-free mass in
`_________________________——————————-——-
`age-comparable controls and women with multiple sclerosis
`MS Patients
`
`Contro s
`Ambulatory (39) Nonambulatory (32)
`Total (71)
`(71)
`______________________________.__.—————-——-
`
`
`
`47.7 it 1.2
`l6l.0 221.2
`66.3 :: 1.6
`26.0 :: 1.0
`
`l
`45.6 :: l
`163.5 :: 0,8
`63.0 :: 1.7
`23.6 :: 0.6fll
`
`
`
`
`
`46.5 221.8
`l62.0 221.3
`60.3 :: 2.4
`23.0 :: 0.8
`7.8 :: 02b
`5.4 :: 0.8C
`
`
`
`44.8 :: 1.6
`164.7 :: l.l
`65.2 :: 2.2
`241 :: 0.9
`5.8 :: 0.2
`3.2 :: 0.8
`
`
`
`Age (years)
`Height (cm)
`Weight (kg)
`BMI (kg/m)
`EDSS score
`Steroid use (months)
`Disease duration
`11.6 :: 1.3
`8.5 ::1.2
`(years)
`2.3::0.1“'d
`2.6 :: 0.1
`24:01
`TB BMC (kg)
`—06
`0.1"~r
`0.0 i 0.2
`—0.3
`01°
`Z-score
`48.6 i 1.9
`47.6 :: 1.7
`48.0 :t 1.2
`Fat mass (%)
`0.0 i 0.2
`70.15: 0 2
`0.1 i 0.2
`Z-score
`29.4 :: 2.1
`31.2 :: 2 0
`30.4 ::1.5
`Fat mass (kg)
`-0.2 i 0.2
`701 i 0.2
`~01 :: 0.1
`Z-score
`26.8 i 0.7f'g
`29.8 :: 0 7
`28.5 :: 0.5
`Fat-free mass (kg)
`—0.6 i 0.1“,f
`0.0 i 0.2
`—0.3 i 0.15
`Z-score
`7.9 i 0.4
`8 0 :: 0.3
`8.0 i 0.2
`8.2 i 0.2
`TB BMCzFFM (%)
`-0.2 i 0.2
`‘02 i 0.2
`-0.2 i 0.1
`Z-score
`________—____—__—__———————————-———-—
`
`2.5:: 0.1
`
`47.8 :: 1.2
`
`
`
`.
`
`31.8 221.6
`
`29.8 i 0.6
`
`a P < 0.05 compared to controls
`b P < 0.01 compared to ambulatory patients with MS
`C P = 0.06 compared to ambulatory patients with MS
`d P < 0.05 compared to ambulatory patients with MS
`c P < 0.04 compared to controls
`fP < 0.01 compared to controls
`3 P < 0.02 compared to ambulatory patients with MS
`
`Total Body Bone Mineral Content
`
`4.0
`
`60
`
`Total Body Fat-Free Mass
`
`kg 35
`
`3.0
`
`2.5
`
`2,0
`
`1.5
`
`1.0
`
`0.5
`
`50
`
`4O
`
`10
`
`30
`
`20
`
`0'0
`
`2
`
`4
`
`6
`
`e
`
`10
`
`O
`EDSS Score
`
`2
`
`4
`
`6
`
`8
`
`1O
`
`Fig. 1. Total body bone mineral content and fat-free mass as a function of disability status in patients with multiple sclerosis.
`
`may be at increased risk of fracture caused by a reduction in
`bone mass and lean body mass. The severity ofthe deficit in
`bone mass was related to the degree of physical disuse. By
`contrast, ambulatory patients with MS had no difference in
`bone mass or body composition as compared with age-
`comparable controls, suggesting that either the time since
`diagnosis or the disease process may be different. In addi-
`tion, glucocorticoid use had minimal effects on bone mass
`and fat—free mass in mobile patients. In nonambulatory pa
`tients with MS, immobility and corticosteroid use, possibly
`
`reflecting a more severe disease condition, accentuated the
`deficit in bone mass.
`Prolonged immobility in clinical cases such as spinal
`injury and stroke has been shown to lead to osteoporosis
`[3A8]. Generalized immobilization, such as with quadriple-
`gia, leads to generalized osteoporosis, whereas hemiplegia
`causes osteoporosis in the affected limb. Nonambulatory
`patients with MS have generalized immobility and, in this
`study, total body bone mineral content was reduced by 8%
`as compared with ambulatory patients with MS, and by
`
`

`

`C. A. Formica ct 211.: Body Composition in Multiple Sclerosis
`
`<0.0l
`
`132
`
`kg
`
`SD
`
`0.3
`
`0.0
`
`-0.3
`
`—0.6
`
`-0. 9
`
`.
`
`'
`
`1
`
`-
`
`'p<0.01 compared to
`age-comparable controls
`
`
`
`
`
`0.3
`
`0.0
`
`—O.3
`-0.6
`
`'0‘ 9
`~1 .2
`
`///
`,, /, ,,/,,, “HQ/25%
`' p<0.0l compared to
`age-comparable conuols
`
`-1 .2
`
`p <0.0l
`
`Z
`
`p <0.01
`
`;
`
`l:l Age—comparable controls - Ambulatory patients with MS
`Fig. 2. Total body bone mineral content and fat-free mass in age—comparable controls, ambulatory patients with multiple sclerosis, and
`nonambulatory patients with multiple sclerosis, expressed in absolute terms (kg) and as a Z-score (SD).
`
`Non-Ambulatory patients with MS
`
`l 15% as compared with age—comparable controls. it is un—
`certain whether this reduced bone mass is reversible. The
`
`bone loss associated with physical disuse is possibly caused
`by an increase in bone turnover or as a result of altered bone
`cell function, which may make the bone loss irreversible.
`Increased urinary calcium excretion has been demonstrated
`in metabolic studies, suggesting an increased bone turnover
`state [3—4, 9, 16]. Whereas the mechanisms may be unclear,
`these observations highlight the importance of mechanical
`usage in patients with MS and highlight the need to imple~
`ment appropriate loading in the management of these pa-
`tients.
`
`Skeletal muscle depletion is a consequence of physical
`disuse and glucocorticoid usage [11, 17—24]. Nonambula—
`tory patients with MS had reduced fat—free mass (:100/0) as
`compared to ambulatory patients with MS and age-
`comparable controls. Whereas both physical disuse and glu-
`cocorticoid use in this group of patients would largely ac—
`count for the deficit in fat-free mass, it would appear, based
`on the analysis of covariance, that the duration of glucocor-
`ticoid use is the main determinant for this deficit. Prolonged
`use of glucocorticoids causes catabolism of skeletal muscle
`[11, 19—24]. Decreased amino acid transport into muscle
`and increased glutamine synthesis activity with resultant
`muscle atrophy are some of the concomitant effects of glu—
`cocorticoid use on skeletal muscle.
`
`Endogenous glucocorticoid excess also produces gener-
`alized osteoporosis, most prevalent in trabecular-rich skel—
`etal regions [13, 15, 25—28]. The osteoporosis is most likely
`multifactorial, because of increased renal calcium losses,
`decreased gastrointestinal calcium absorption, secondary
`hyperparathyroidism, and increased bone turnover with de—
`pression of bone formation. Resorption cavities of greater
`depth may occur and may result in more rapid bone loss and
`possibly trabecular perforation.
`In both ambulatory and
`nonambulatory patients with MS, glucocorticoid use was
`not associated with total body bone mass. However, our
`group has previously reported that bone mass at the lumbar
`spine, proximal femur, and total body was higher in patients
`with previous steroid use [2]. This was due, at least in part,
`to the fact that glucocorticoid treatment is generally admin-
`istered to younger patients. Furthermore, the beneficial ef-
`fects of pulsed steroids on mobility in patients with MS may
`offset the deleterious pharmacological effects on bone and
`skeletal muscle. Obtaining an accurate history of glucocor—
`ticoid use from questionnaire data is inherently difficult,
`therefore we need to confirm these hypotheses with longi-
`tudinal data.
`Deficits in bone mass and fat—free mass were associated
`with the severity of multiple sclerosis. Using mobility as
`one means of defining disease severity, we showed that
`ambulatory patients with MS were no different than age—
`
`

`

`C. A. Formica ct al: Body Composition in Multiple Sclcrosis
`
`comparable controls. By contrast, nonambulatory patients
`with MS had a deficit in bone mass that would increase
`fracture risk approximately two-fold. This risk of fracture
`may be fiiither increased because of the increased risk of
`falls associated with deteriorating visual and motor perfor-
`mance in patients with MS
`In summary, nonambulatory patients with MS have gen»
`eralized deficits1n bone mass and fat- free mass, increasing
`the risk of falls and fractures. Glucocortieoid catabolism of
`skeletal muscle largely accounted for the deficit in fat-free
`mass, and the deficit in fat-free mass largely accounted for
`the deficit in bone mass. In conclusion, in patients with MS,
`immobilization and glucocorticoid use are the main deter-
`minants for the decrease in fat-free mass and the increased
`risk of fracture and morbidity.
`
`Acknowledgments, This work was supported by NIH Grants AR
`39191 and DK 46381.
`
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