4
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`Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159(3):394-399.
`
`The effect of lacosamide on bone tissue in orchidectomised
`male albino Wistar rats
`Julius Simko', Sona Feketeb, Jana Malakovab, Jan Kremlacekc, Jiri Horacekd, Helena Zivnae, Vladimir Palickab, Pavel Zivnyb
`
`Aims. While most antiepileptic drugs (AEDs) have been associated with various adverse effects on bone health, for the
`recently introduced lacosamide (LCM) no corresponding data have been published. The present study evaluates the
`effect of LCM on bone mineral density, bone turnover markers, and bone mechanical strength in a rat model.
`Methods. 16 orchidectomized Wistar rats were divided into control and experimental groups, 8 rats each. Dual energy
`X-ray absorptiometry was used to measure bone mineral density (BMD). As bone metabolism markers, the concen-
`trations of bone markers were assayed in bone homogenate. In addition, both femurs were measured and used for
`biomechanical testing.
`Results. Compared to the control group, we found lower BMD in the experimental group in the area of the left (8%) as
`well as the right femur (12%), all differences being statistically significant. In both femur diaphyses, but not in lumbar
`vertebrae, BMD was lower in the LCM group, suggesting a preferential effect on cortical bone. However, neither the
`thickness of the diaphyseal cortical bone nor the fragility in biomechanical testing was different between the groups.
`Of the bone metabolism markers, the significant decline was in procollagen type I N-terminal peptide (PINP) levels
`(37.4%), suggesting a decrease in osteoid synthesis.
`Conclusion: We assume then that long-lasting exposure to LCM can represent a certain risk to the health of bone in
`the setting of gonadal insufficiency. Further studies will be needed to confirm these findings and to determine how
`high the risk will be in comparison to the other AEDs.
`
`Key words: antiepileptic drugs, bone markers, bone mineral density, bone turnover, biomechanical strength
`
`Received: July 8, 2013; Accepted with revison: January 16, 2014; Available online: January 27, 2014
`http://dx.doi.org/10.5507/bp.2014.006
`
`°Department of Neurology, Faculty of Medicine in Hradec Kralove, Charles University in Prague and University Hospital Hradec Kralove,
`Czech Republic
`'Institute of Clinical Biochemistry and Diagnostics, Faculty of Medicine in Hradec Kralove, Charles University in Prague and University
`Hospital Hradec Kralove
``Department of Pathophysiology, Faculty of Medicine in Hradec Kralove, Charles University in Prague and University Hospital Hradec Kralove
`de Department of Internal Medicine - Hematology, Faculty of Medicine in Hradec Kralove, Charles University in Prague and University
`Hospital Hradec Kralove
`'Radioisotope Laboratories and Vivarium, Faculty of Medicine in Hradec Kralove, Charles University in Prague and University Hospital
`Hradec Kralove
`Corresponding author: Julius Simko e-mail: simkojulius@gmaiLcom
`
`INTRODUCTION
`
`Their use in epilepsy and the treatment of pain, and
`wide therapeutic use in psychiatry make antiepileptic
`drugs (AEDs) a pharmaco-epidemiologically important
`group of drugs both in the adult as well as the juvenile
`population1-3. Osteopathies occurring with long-term
`chronic antiepileptic treatment were first noted in the
`late sixties4.5. Since that time a number of theories have
`been proposed to explain why AEDs affect bone, but none
`explains all the reported effects. Most studies of the ef-
`fects of AEDs on bone tissue are cross-sectional. There
`are only a few longitudinal studies, and there are limited
`data regarding the newer AEDs (ref.6).
`Lacosamide (LCM) (SPM 927, formerly harkoseride),
`the R-enantiomer of 2-acetamido-N-benzy1-3-methoxypro-
`pionamide, is a chemical compound with anticonvulsant
`and anti-nociceptive properties. In November 2007, a new
`
`drug application was filed with the FDA for use of LCM
`as adjunctive therapy in the treatment of partial-onset
`seizures in adults with epilepsy. LCM was approved in
`Europe on September 3, 2008 as adjunctive therapy in
`the treatment of partial-onset seizures, with or without
`secondary generalization, for patients with epilepsy of
`16 years or older'. Sex hormone deficiency increases the
`risk of developing antiepileptic drug-induced osteopathy
`(AEDs-0) (ref.8).
`We report here our findings on the impact of LCM
`on bone mineral density (BMD), bone mineral content
`(BMC), bone metabolism markers, and bone biomechani-
`cal properties in orchidectomised (ORX) rats fed on LCM-
`enriched diet for 12 weeks. Apart from this study, to our
`knowledge there are no other full-text studies evaluating the
`effect of LCM on bone tissue. We have found one study re-
`ferring to the absence of changes in BMD in juvenile dogs
`
`that has been published only in the form of an a race. 0
`
`394 ARGENTUM Exhibit 1071
` Argentum Pharmaceuticals LLC v. Research Corporation Technologies, Inc.
`IPR2016-00204
`
`EXHIBIT
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`Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159(3):394-399.
`
`METHODS
`
`Animals
`All animals received humane care in accordance with
`the guidelines set by the institutional Animal Use and
`Care Committee of Charles University in Prague, Faculty
`of Medicine in Hradec Kralove, Czech Republic. The pro-
`tocol of the experiment was approved by the same com-
`mittee. The experiments used eight-week-old male albino
`Wistar rats (Biotest s.r.o., Konarovice, Czech Republic).
`The animals were housed in groups of 4 in plastic cages.
`During the experimental period the animals were main-
`tained under controlled conventional conditions (12 h
`light and 12 h dark, temperature 22±2 °C, air humidity 30-
`70%). Tap water, standard laboratory diet (SLD, VELAS,
`a.s., Lysa nad Labem, Czech Republic) and SLD enriched
`with LCM were given ad libitum. The weight of the rats
`was monitored once a week.
`
`Experiment
`Rats weighing (270±7 g) at the beginning of the experi-
`ment were divided into two groups of 8 animals:
`1. CON-ORX: orchidectomised control fed with SLD
`2. LCM-ORX: orchidectomised rat fed with SLD en-
`riched with LCM (18 mg/25 g of the diet; Lacosamid,
`UCB Pharma)
`At the beginning of the experiment the rats (CON-
`ORX and LCM-ORX) underwent bilateral orchidectomy
`under ether anaesthesia. On the second day after opera-
`tion the LCM-ORX began to receive SLD enriched with
`LCM and the CON-ORX only SLD, both diets ad libitum.
`After 12 weeks, the animals were sacrificed by exsangui-
`nation from the abdominal aorta under ether anesthesia,
`and the obtained serum was aliquoted and stored at -80 °C
`for ensuing biochemical analyses. Both tibiae and femurs
`of the sacrificed rats were dissected free of soft tissue,
`wrapped in gauze moistened with saline and frozen to
`-80°C till the time of analysis.
`
`Analysis of serum and bone homogenates
`In the blood serum, the levels of osteoprotegerin
`(OPG) and insulin-like growth factor 1 (IGF-1) were de-
`termined by the ELISA (Enzyme-Linked Immunosorbent
`Assay) method. The blood serum levels of LCM were
`determined in the middle and at the end of the experi-
`ment. LCM was assayed by modified high-performance
`liquid chromatography with diode array detection'''.
`Sample preparation included precipitation of plasma
`proteins: 200 AL of acetonitrile and 20 AL of zinc sul-
`phate solution (10%) were added to 100 µ1_, of plasma
`samples in 1.5-mL polypropylene centrifugation tubes.
`The tubes were vortexed for 120 seconds and centrifuged
`at 15,000 rpm for 10 minutes. The supernatant (30 µL)
`was injected into the HPLC system. Analysis was per-
`formed on a 2695 Waters Separations Module equipped
`with 996 photodiode array detector and Peltier column-
`thermostat Jet-Stream (Thermotechnic Products). Data
`acquisition and processing were provided with Empower
`Software (Waters). The analytical column was Zorbax
`SB-C8 (Agilent Technologies) - 150 x 4.6 mm, 3.5 µm.
`
`The analytical precolumn was Symmetry C18 Guard
`Column - 20 x 3.9 mm, 5 µm (Waters). The mobile phase
`was pumped at flow rate 0.8 mL/min and consisted of
`acetonitrile:formic acid 0.1 % (30:70, v/v). Temperature
`on the column was set at 30 °C, and injection volume was
`30 µL. LCM concentration was determined at a wave-
`length of 215 nm (ref.m).
`In bone homogenate, the levels of the markers car-
`boxy-terminal cross-linking telopeptide of type I colla-
`gen (CTX-I), aminoterminal propeptide of procollagen
`type I (PINP), bone alkaline phosphatase (BALP) and
`bone morphogenetic protein 2 (BMP-2) were determined
`also using the ELISA method. The homogenate was pre-
`pared from the tibia. After animal sacrifice, both tibiae
`were carefully excised; after removal of all the surround-
`ing skin, muscle and other soft tissue, they were stored
`at -80 °C until required. The proximal part of the tibia
`(0.1 g) was disrupted and homogenized in 1.5 mL of phos-
`phate buffer (PBS, PAA Laboratories GmbH, Pasching,
`Austria) with MagNA Lyser instrument (Roche Applied
`Science, Germany) at 6500 rpm for 20 s and cooled on
`the MagNA Lyser Cooling Block. This procedure was
`repeated three times.
`The tissue homogenate was centrifuged at 10,000 g
`at 4 °C for 10 min. The supernatant was obtained and
`stored at -80 °C. Determination of the levels of bone mark-
`ers was carried out using kits from Uscn Life Science
`Inc., Wuhan, China (PINP, Procollagen I N-Terminal
`Propeptide, pg/mL; OPG, Osteoprotegerin, pg/mL; IGF-
`1 Insulin Like Growth Factor 1, pg/mL; CTX-I, Cross
`Linked C-Telopeptide Of Type I Collagen; pg/mL; BALP,
`Alkaline Phosphatase, Liver/Bone/Kidney, ng/mL; BMP-
`2, Bone Morphogenetic Protein 2, pg/mL).
`
`Dual energy X-ray absorptiometry analysis
`The rat bone mineral density (BMD, g/cm2) was
`measured by means of dual energy X-ray absorptiometry
`(DEXA) on a Hologic Delphi A device (Hologic, MA,
`USA) at the Osteocentre of the Faculty Hospital Hradec
`Kralove, Czech Republic. Before measurements, a tissue
`calibration scan was performed with the Hologic phantom
`for the small animal. Bone mineral density of the whole
`body, in the area of the lumbar vertebrae and in the area
`of the femur was evaluated by computer using the ap-
`propriate software programme for small animals (DEXA;
`QDR-4500A Elite; Hologic, Waltham, MA, USA). All
`animals were scanned by the same operator.
`
`Biomechanical testing procedure
`Mechanical testing of the rat femoral shaft and fem-
`oral neck was done with a special electromechanical
`custom-made testing machine (Martin Kosek & Pavel
`Trnecka, Hradec Kralove, Czech Republic) as described
`in a previous report". For the three-point bending test,
`the femur was supported in the anteroposterior direc-
`tion on a holding device, with the two support points
`18 mm apart. A small stabilizing preload to 10 N was
`used to fix the bone between the contacts. A constant
`deformation rate of 6 nun/min as applied until maximal
`load failure and breaking strength (maximum load, N)
`
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`
`were recorded. Once the bone was broken, the thickness
`of the cortical part of the bone was measured by means
`of a sliding micrometer (OXFORD 0-25MM 30DEG
`POINTED MICROMETER, Victoria Works, Leicester,
`Great Britain). The proximal part of the femur was used
`for compression test of the femoral neck. The diaphysis
`of the bone was embedded into a container using a meth-
`acrylate resin and a vertical load was applied to the top
`of the femoral head. A small stabilizing preload to 10 N
`was applied and then advanced at a constant speed of
`6 mm/min until failure of the femoral neck. The break-
`ing strength (maximum load, N) was recorded by the
`measuring unit (Digitalanzeiger 9180, Burster praezisios-
`messtechnik Gmbh & co kg, Gernsbach, Germany). All
`bones were analyzed by the same operator.
`
`Analysis
`Data were analyzed using Statistica v.10 (USA) soft-
`ware. Because the Shapiro-Wilk W-test for normality of
`data indicated that normal distribution was unlikely, non-
`parametric tests were used in subsequent analyses. Results
`were expressed in the form of medians, as well as lower
`and upper quartiles. Data of CON-ORX and LCM-ORX
`groups were compared using the Mann-Whitney U test to
`assess differences. Statistical significance was indicated
`by P-values <0.05 in all calculations.
`
`RESULTS
`
`Comparison of body composition showed a signifi-
`cantly lower fat mass in total (16.5 g) as well as in rela-
`tive (4.1%) expression in those rats supplied with LCM.
`The contrast in fat expressed as a percentage was 18.3%
`(100*(1-fatLcmR/fatcoNR) between the groups. Also, the
`weight of rats in the CON-ORX groups was found to be
`higher (37 g) compared to the LMC-ORX group, but this
`difference was not statistically significant - for details see
`Table 1.
`Among the tested bone markers only aminoterminal
`propeptide of procollagen type I was significantly lower
`in the LCM-ORX group, by 29.6 pg/L, representing an
`intergroup contrast of 37.4%. Particular values and a de-
`tailed list of the other followed parameters are displayed
`in Table 2.
`The mineral density of bone evaluated in the whole
`body and in the area of the lumbar vertebrae did not
`show any significant differences between the groups;
`however in the area of the left as well as the right femur
`we found significantly lower density (left 0.18 g/cm2 and
`right 0.17 g/cm2) in the LCM-ORX group compared to
`the control group (left BMD 0.20 g/cm2 and right BMD
`0.20 g/cm2). The contrast between groups was 8.8% in
`the left BMD and 12.0% in the right BMD. The mineral
`content did not differ statistically between the groups. The
`listed values are medians; for interquartile intervals and
`other significant parameters see Table 3.
`
`Parameter
`
`Weight (g)
`
`Fat (g)
`
`Fat (%)
`
`Table 1. Body weight and fat mass.
`
`CON-ORX (n=8)
`[25% - 75%]
`
`523 [489 - 543]
`
`90.3 [83.1 - 127.2]
`
`22.1 [20.1 - 26.7]
`
`LCM-ORX (n=8)
`[25% - 75%1
`
`486 [471 - 499]
`
`73.8 [65.1 - 85.2]
`
`18.0 [16.1 - 20.0]
`
`Lean body mass (g)
`
`326.2 (310.9 - 378.8)
`
`340.9 (326.9 - 350.9)
`
`Data are expressed as median (25th - 75' percentiles)
`
`Parameter
`
`OPG (pg/mL)
`
`IGF-1 (pg/mL)
`
`CTX-1 (pg/mL)
`
`PINP (pg/mL)
`
`BALP (ng/mL)
`
`BMP-2 (pg/mL)
`
`Table 2. Levels of bone markers.
`
`CON-ORX (n=8)
`[25% - 75%]
`
`54.7 [51.9 - 58.5]
`
`1.11 [1.06 - 1.21]
`
`0.97 [0.76 - 1.57]
`
`79.0 [71.9 - 81.5]
`
`1.16 [0.76 - 1.46]
`
`876 [610 - 1010]
`
`LCM-ORX (n=8)
`[25% - 75%]
`
`57.9 [51.1 - 54.5]
`
`1.17 [1.05 - 1.29]
`
`0.90 [0.64 - 1.63]
`
`49.4 [34.5 - 67.6]
`
`0.88 [0.70 - 1.14]
`
`824 [763 - 1055]
`
`Man-Whitney U Test
`1/1
`0.189
`
`0.024
`
`0.041
`
`0.495
`
`Man-Whitney U Test
`[P]
`
`0.372
`
`0.564
`
`0.772
`
`0.005
`
`0.495
`
`0.958
`
`OPG, osteoprotegerin; IGF-1, insulin-like growth factor 1; CTX-I, carboxy-terminal cross-linking telopeptide of type I collagen; PINP, aminoter-
`minal propeptide of procollagen type I; BALP, bone alkaline phosphatase; BMP-2, bone morphogenetic protein 2.
`Data are expressed as median (25th - 75th percentiles)
`
`396
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`Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159(3):394-399.
`
`Table 3. Comparison of values from DXA analysis and values of biomechanical testing.
`
`CON-ORX (n=8)
`[25% - 75%]
`
`LCM-ORX (n=8)
`[25% - 75%]
`
`Man-Whitney
`U Test [P]
`
`DEXA
`Whole body BMD, g/cm2
`W BMC (g)
`LF BMD, g/cm2
`LF BMC (g)
`RF BMD, g/cm2
`RF BMC (g)
`Lumbar columna (L3-L5) BMD (g/cm2)
`Lumbar columna (L3-L5) BMC (g)
`
`Biomechanical testing measurement
`LF length, mm
`RF length, mm
`LF diameter, mm
`RF diameter, mm
`Cortical LF thickness, mm
`Cortical RF thickness, mm
`Maximal load of the left femoral shaft, N
`Maximal load of the right femoral shaft, N
`Maximal load of left femoral neck, N
`Maximal load of right femoral neck, N
`
`0.175 [0.171 - 0.176]
`14.0 [13.3 - 14.,4]
`0.197 [0.192 - 0.199]
`0.276 [0.258 - 0.290]
`0.198 [0.188 - 0.211]
`0.259 [0.236 - 0.280]
`0.214 [0.210 - 0.223]
`0.571 [0.546 - 0.598]
`
`37.5 [36.6 - 38.0]
`37.4 [36.6 - 37.8]
`3.63 [3.51 - 3.95]
`3.63 [3.53 - 3.72]
`0.720 [0.665 - 0.740]
`0.705 [0.665 - 0.735]
`214 [197 - 236]
`216 [203 - 230]
`144 [135 - 152]
`160 [138 - 178]
`
`0.169 [0.165 - 0.171]
`13.5 [13.1 - 13.9]
`0.179 [0.169 - 0.185]
`0.266 [0.259 - 0.279]
`0.175 [0.160 - 0.179]
`0.259 [0.248 - 0.284]
`0.212 [0.207 - 0.223]
`0.575 [0.542 - 0.614]
`
`37.3 [36.9 - 38.6]
`37.4 [37.2 - 37.8]
`3.64 [3.62 - 3.,67]
`3.65 [3.47 - 3.95]
`0.705 [0.695 - 0.750]
`0.695 [0.680 - 0.740]
`218 [207 - 229]
`224 [204 - 235]
`146 [128 - 163]
`149 [141 - 161]
`
`0.066
`0.318
`0.004
`0.793
`0.001
`0.564
`0.793
`0.495
`
`0.495
`0.958
`0.958
`0.753
`0.263
`0.958
`1.000
`0.564
`0.897
`0.355
`
`LF- left femur, RF - right femur, BMD - bone mineral density, BMC - bone mineral content. Data are expressed as median (25th - 75th percentiles)
`CON-ORX, orchidectomised control fed with standard laboratory diet; LCM-ORX, orchidectomised rat fed with standard laboratory diet enriched
`with lacosamide.
`
`The Mann-Whitney U test also showed that medians
`of biomechanical and geometric parameters of right and
`left femurs did not differ (see Table 3).
`The level of lacosamide in LCM-ORX at the end of
`the experiment: 13.49 umol/L (12.96 - 14.59).
`
`DISCUSSION
`
`The aim of this study was to evaluate the effect of
`LCM on BMD, bone markers, and biomechanical qual-
`ity using the mature ORX rat model. The effect of LCM
`on bone tissue has not yet been investigated, except for
`studies Cornet et al 2010 in juvenile dogs (published only
`in the form of an abstract) (ref.9). In this study, ORX
`rats fed with SLD enriched with LCM (LCM-ORX) had
`significant loss of BMD at the left and right femur after
`12 weeks when compared to the control (CON-ORX).
`However, no significant differences in biomechanical and
`geometric parameters of rat right and left femurs were
`observed. Evaluation of bone turnover using biochemical
`markers specific for both bone formation (BALP, BMP-2,
`PINP, OPG, IGF-1) and bone resorption (CTX-I) was
`without significant difference with the exception of PINP.
`With respect to AEDs, phenytoin (PHT), phenobarbi-
`tal (PB), and primidone (PRM) are most consistently as-
`sociated with a negative impact on bone. Carbamazepine
`(CBZ) and valproate (VPA) may also result in bone ab-
`normalities, but the data are mixed. Current studies sug-
`gest that lamotrigine (LTG) has limited (if any) effect,
`
`397
`
`Fig. 1. Evaluation of BMD in three areas of the rat skeleton.
`RI - lumbar columna (L3-L5);
`R2 - left femur,
`R3 - right femur
`
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`
`but again, the data are inconsistent. Other AEDs have
`received limited study' but there is increasing evidence
`supporting the notion that topiramate (TPM) may have
`negative impact on bone health's-15.
`The significance of bone turnover markers for diagno-
`sis of antiepileptic drug-induced osteopathy (AEDs-0) is
`controversial: although PHT is well-known to cause sig-
`nificant loss of BMD and BMC, only modest changes
`in markers of bone turnover have been observed in ani-
`malsl". Similarly, in a longitudinal study of premeno-
`pausal women treated with PHT, bone turnover markers
`remained unchanged after 1 year, except for a significant
`decline in urine N-telopeptide. This result is unclear and
`difficult to explain, particularly in view of the significant
`observed femoral neck bone loss". Conflicting data exist
`regarding the effects of CBZ on BMD and bone turn-
`over8s9-21. WA in animals reduced BMD and BMC and in-
`creased bone turnover's. There are mixed data in humans.
`Some have observed that WA monotherapy decreased
`BMD and increased both markers of bone formation
`and resorption significantly22.23, but in the longitudinal
`study of young women mentioned above, the BMD was
`stable and bone turnover markers remained unchanged
`after 1 year of WA treatment's. There are scarce data
`regarding LTG. So far LTG has not been shown to cause
`significant effects on BMD and bone turnover18'21, except
`significantly increased osteocalcin, a marker of bone for-
`mation referred to in one study".
`In our study we noticed a significant change in the
`PINP. PINP is a marker of bone formation, which was
`significantly lower in LCM-ORX. To our knowledge, there
`have appeared no studies with AEDs (or with CYP2C 19
`inhibitors) in which changes in the level of PINP were
`monitored. Some further research will be necessary to ver-
`ify the role and the importance of PINP in the diagnosis,
`and more precisely in the pathophysiology of AEDs-0.
`We discovered only one prospective study testing the in-
`fluence of LCM on BMD in gonadally intact subjects, in
`which the authors claim an absence of influence of LCM
`on BMD (refs). However, we monitored the significant
`decline of BMD at the left and right femur. We assume
`then that long-lasting exposure to LCM can represent a
`certain risk to the health of bone in the setting of the
`gonadal insufficiency. It is complicated to determine how
`high the risk will be in comparison to the other AEDs. In
`the case of levetiracetam (LEV) we monitored a signifi-
`cant decline of BMD on the same model in only 1 out of
`4 locations of measurement (left femur), and moreover,
`we did not record significant changes in the mechanical
`durability of the bone". In the case of LTG and TPM, we
`recorded on the same model both significant decline of
`BMD (at 3 measurement locations out of 4) and signifi-
`cant reduction in the mechanical durability of the bone
`(supplied data). The old AEDs have not been tested on
`the ORX rat model so far. However, in models of gonad-
`ally intact rats, significant reduction of BMD after using
`the old AEDs has been observed (see text above).
`The mechanism of the effect of LCM on bone is un-
`clear. LCM has been shown to produce a significant effect
`in rodents consistent with anxiolysis: LCM increased the
`
`suppression ratio in a conditioned emotional response
`test, and reduced the number of marbles buried in the
`marble burying assay". In rodents, physical activity pre-
`vents decrease in BMD as it does in humans, which sug-
`gests that increased physical activity could be useful in
`the prevention of bone mineral loss, regardless of gonadal
`hormone deficiency". Therefore reduced locomotor ac-
`tivity could be the factor contributing to significant de-
`crease LF-BMD and RF-BMD in LCM-ORX compared
`to CON-ORX.
`In our study, LCM-ORX had significantly lower fat
`mass compared with CON-ORX. LCM showed no po-
`tential to induce or to inhibit cytochrome P450 isoforms
`except for CYP2C19 (60% inhibition) (ref."). CYP2C19
`is one of the (most) important isoforms involved in the
`metabolism of sex hormones. CYP2C 19 catalyzes the
`1713-hydroxy dehydrogenation and 16a hydroxylation of
`estradiol, and 1713-hydroxy dehydrogenation is the main
`metabolism pathway at low estradiol concentrations293°.
`The main pathway of testosterone oxidative metabolism
`by human liver microsomes is the formation of 1 2a-
`/13-, 60-, 15f3-, and 1613-hydroxytestosterones, mainly cata-
`lyzed by CYP2C9, CYP2C19, and CYP3A4 (ref.31). In
`a study in the ORX rat model with different doses of
`testosterone replacement there were no significant dif-
`ferences in fat mass's, and in another study, 1713-estradiol
`prevented the ORX-related increase of fat mass, whereas
`5a-dihydrotestosterone did not33. Thus we propose that
`inhibition of estrogen metabolism may be the cause of
`lower fat mass in LCM-ORX rather than inhibition of
`androgen metabolism.
`There are several limitations that should be considered
`in evaluating the present study. Firstly, the three-month
`follow-up period may be too short for monitoring changes
`in biomechanical properties of the bone tissue. Secondly,
`the sample size was small: although statistical significance
`is evident, the capacity to identify possible variables of
`confusion is limited. Finally, behavioral activity was nei-
`ther controlled nor assessed.
`
`CONCLUSION
`
`In summary, in this study, LCM-ORX had significant
`loss of BMD at the left and right femur and significant
`decline in PINP compared to the control. It will be neces-
`sary to carry out further studies to validate the findings
`of this study and to elucidate the exact mechanism of
`the significant loss of femur BMD. Further studies are
`warranted to establish whether LCM has a clinically sig-
`nificant effect on BMD exclusively in the model of gonad-
`ectomized rats, or whether the effect applies also in the
`model of gonadally intact animals, and in the respective
`human models.
`Despite the above-mentioned limitations, this study
`contributes significantly to our knowledge about the effect
`of LCM on bone tissue.
`
`398
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`Page 00005
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`

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`Biomed Pap Med Fac Un iv Pa lacky Olomouc Czech Repub. 2015 Sep; 159(3):394-399.
`
`ACKNOWLEDGEMENT
`
`This study was supported by a Research Project of
`PRVOUK 37/11 Charles University in Prague project,
`SW-2011-262902, SW -2012 - 264902 and MH CZ -
`DRO (UHHK, 00179906). The authors wish to thank
`Dagmar Jezkova and Katerina Sildbergerova for their
`skilful technical assistance throughout the experiment.
`The authors are grateful to Ian McColl MD, PhD for as-
`sistance with the manuscript.
`Author contributions: JS, SF, JM: literature search,
`manuscript writing; JS, PZ, VP: study design; SF: figures;
`SF, HZ, JM: data collection; JS, JK, JH, VP, PZ: data
`interpretation; JS, JK, JH: statistical analysis.
`Conflict of interest statement: None declared.
`
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