`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926
`
`2.6.6 TOXICOLOGY
`
`2.6.6.1 Overall toxicology summary
`
`2.6.6.2 Single-dose toxicity
`No single-dose toxicity studies were included in this submission.
`
`2.6.6.3 Repeat-dose toxicity
`
`No repeat-dose toxicity studies were included in this submission.
`
`2.6.6.4 Genetic toxicology
`
`The following genetic toxicology studies were submitted and are reviewed in this section:
`0
`1:1 combination of Naproxen sodium and Sumatriptan succinate: In Vitro
`Chromosome Aberration Assay with Cultured Chinese Hamster Ovary (CHO) Cells
`1:1 combination of Naproxen sodium and Sumatriptan succinate: In Vitro Mutation
`Assay with L5178Y Mouse Lymphoma Cells at the TK Locus
`
`0
`
`IS
`
`on Org-norm
`
`10
`
`

`

`
`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926
`
`1:1 combination of Naproxen sodium and Sumatriptan succinate: In vitro
`Chromosome Aberration Assay with Cultured Chinese Hamster Ovary (CHO) Cells
`
`Study no.2 .3 Study 2990/7; GSK Document WD2006/03218/00
`
`Volume # , and page #: eNDA 21-926 #016, Module 4, Section 4.2.3.311, pages 1-76
`Conducting laboratory and location: “ North Yorkshire, UK
`Date of study initiation: 04 AUG 2006
`GLP compliance: Yes, UK 1999/2004, signed 31 OCT 2006 by Study Director
`QA reports: yes, statement signed 31 OCT 2006 by the QA Representative
`Drug, lot #, and % purity: Naproxen Sodium (NAP) Lot #NPXNAM-631 and
`#NPXNAM-635, Purity 100%; NAP doses were calculated as the free acid, using a
`correction factor of 1.1. Sumatriptan Succinate (SS) Lot #K058945 and #K026882, Purity
`98.7% and 99.2%, respectively. SS doses were calculated as the free base, using a
`I conversion factor of 1.4.
`
`Methods
`
`Strains/species/cell line:
`CHO cells, originally supplied by w were maintained at
`— in tissue culture flasks containing - ,5A medium with 10%
`(v/v) heat inactivated fetal calf serum, and 100 ug/mL gentamycin. The doubling time of
`this cell line is ~13 hrs, and its modal chromosome number is 21.
`
`Doses used in definitive studies:
`
`-S9: NAP alone at 1920 and 2500 ug/mL
`SS alone at 1920 ug/mL
`SS/NAP together at 1710/1710, 1815/1815, 1850/1850, and 1920/1920 ug/mL
`+S9: NAP alone at 1780 and 2500 ug/mL
`SS alone at 1780 ug/mL
`SS/NAP together at 1640/1640, 1710/ 1710, 1745/1745, and 1780/1780 ug/mL
`
`Basis of dose selection:
`The primary measure of cytotoxicity in this study was mitotic index (M1), the percentage
`of cells in mitosis, based on scoring of at least 1000 cells per culture. Slides showing >
`61% reduction in M1 were not scored for chromosomal aberrations. The concentrations
`
`selected for analysis of chromosomal aberrations was agreed with the sponsor before
`scoring. Cytotoxicity was also measured based on cell counts (compared to mean vehicle
`control) and population doublings (PD = [log (N/Xo)]/log 2, where N = mean final cell
`count/culture at harvest, and X0 = starting count at beginning of treatment).
`
`Negative controls:
`Purified water was used as the solvent control.
`
`11
`
`

`

`
`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926
`
`Positive controls:
`
`4-Nitroquinolone 1-oxide (NQO, 0.25 and 0.30 ug/mL final concentration, fi'om stock in
`DMSO, - , was used as the positive controls in the absence of metabolic activation.
`
`Cyclophosphamide (CPA, 6.25 and 12.5 ug/mL final concentration, from stock in
`DMSO, - i was used as the positive control in the presence of metabolic activation.
`
`Incubation and sampling times:
`Duplicate cultures were prepared and treated with test article or positive control i S9
`metabolic activation (rat liver, — , 2% final concentration) for 3 hrs as described
`above (vehicle controls were tested in quadruplicate cultures). Cells were harvested 17
`hrs after the beginning of treatment, and 1.5 hrs after the addition of colchicine (1 ug/mL
`final concentration) to arrest dividing cells in metaphase. A cell count was determined
`from an aliquot of each cell suspension prior to centrifugation and resuspension of the
`cells '
`g
`
`, followed by centrifugation and resuspension
`-
`several times to clean the cells. Slides were prepared after several drops of 45% (v/v)
`aqueous acetic acid were added to enhance chromosome spreading. Dried slides were
`stained for 5 minutes in filtered 4% (v/v) Giemsia stain in Gurr’s buffer (pH 6.8), rinsed,
`dried, and mounted in DPX under coverslips.
`
`The top four or five concentrations without excessive toxicity were scored for
`chromosome aberrations (100 metaphases from each of the duplicate flasks, providing
`200 per concentration level, and 400 from the four vehicle-treated cultures). Only cells
`with 19-23 chromosomes Were considered acceptable for analysis of chromosomal
`aberrations. The frequency of hyperdiploid, polyploid and endoreduplicated cells was
`also scored for each culture.
`
`A 20—hr treatment in the absence of S9 was also performed, but was not analyzed for
`chromosomal aberrations because the 3-hr incubations without S9 were clearly positive.
`
`Results
`
`Study validig
`Criteria for a valid assay were met for the 1:1 combination:
`0 The highest concentrations analyzed (1920/1920 ug/mL —S9, 61% MI; 1780/1780
`ug/mL +59, 57% MI) both showed greater than the minimum 50% requirement for
`mitotic inhibition.
`
`0 At least 80% of the intended total cells per treatment (200 for test article and positive
`controls; 400 for vehicle controls) were scored, except at the highest dose —S9
`(1920/1920), where only 151 of the intended 200 cells were analyzed. This was not
`important since that concentration was clearly positive.
`0 The percentage of cells with aberrations in the solvent controls were within or close
`to laboratory historical control ranges.
`Positive control cultures showed clear, unequivocal positive responses as expected.
`
`0
`
`12
`
`

`

`
`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926
`
`Study outcome:
`Criteria for a positive response (chromosomal aberration (CA) frequency (excluding
`gaps) falling outside the historical vehicle control range, and statistically increased over
`vehicle controls) were met for the combination at 2 1815/1815 ug/mL without S9, and at
`2 1745/1745 ug/mL with S9. No increase in the frequency of chromosomal aberrations
`was observed with NAP alone at up to 2500 ug/mL i S9 or with SS alone at 1920 ug/mL
`(-S9) or 1780 (+89). Both cytotoxicity (as measured by % Reduction in Cell Count) and
`frequency of chromosomal aberrations increased with increasing concentrations of
`NAP/SS. The frequency of numerical aberrations was also significantly increased at 2
`1815/1815 ug/mL (-S9), primarily due to increased endoreduplication, but not in a clearly
`dose-related manner. Similarly, numerical aberrations were significantly increased at
`1745/ 1745 ug/mL (+S9), but not at 1780/1780 ug/mL.
`
`% Mitotic % Reduction % Population % of Cells w/
`Inhibition
`In Cell
`Doubling
`Structural
`Count
`Inhibition
`Aberrations
`(excluding
`
`(II
`
`U]
`
`DJ
`b)b)
`
`‘
`
`'
`
`‘
`' '
`
`Reduction
`In Cell
`Count
`
`Population
`Doubling
`Inhibition
`
`% of Cells w/
`Aberrations
`(excluding
`gaps)
`
`Treatment
`(3 hr Incubation
`Without S9)
`
`Purified Water
`NAP/SS
`NAP/SS
`NAP/SS
`NAP/SS
`NAP/SS
`NAP
`
`——_—
`
`Treatment
`(3 hr
`Incubation
`With S9)
`
`1640/1640 n_——
`
`Purifiedwmer “—“-__
`NAP/SS
`
`* Statistically significant: p<0.001 #Statistically significant: p< 0.05, but Within historical control range.
`
`13
`
`

`

`
`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926
`
`Sponsor’s Conclusions:
`The sponsor concluded that the 1:1 combination of Naproxen Sodium and Sumatriptan
`Succinate was clastogenic when incubated with CHO cells for 3 hours in the presence
`and absence of metabolic activation. However, the sponsor also noted that no induction
`of chromosomal aberrations was observed in cultures with cytotoxicity S 54% as
`measured by inhibition of population doublings (PD, “a more reliable and robust measure
`of cytotoxicity,”page 22, and that this assay would be considered negative if > 50%
`inhibition of PD were the cytotoxicity target used in dose selection instead of mitotic
`inhibition. The sponsor believes that this indicates that the chromosomal aberrations
`observed at higher concentrations of NAP/SS were caused by a non-genotoxic
`mechanism dependent on cytotoxicity, as described in Greenwood et al (lay/foflmmtzz/
`a/zd/I/o/e‘cz/[ar/l/u/age/zeflk 43 :3 6-44, 2004).
`
`Reviewer’s Conclusions:
`
`FDA has not yet adopted the cytotoxicity target of > 50% PD inhibition for the CHO
`chromosomal aberrations assay. Our current guidance states:
`
`“The desired level of toxicity for in vitro cytogenetic tests using cell lines should be greater than
`50% reduction in cell number or culture confluency. For lymphocyte cultures, an inhibition of
`mitotic index by greater than 50% is considered sufficient.”
`{Gul'a’efl'lzefarl/Idz/Jt/y, SpeaficAspect! o/A’egz/la/o/y Geno/aavl’l'g/ flJ/JflrPédr/aflcey/Ikak
`[07524, Jprll lflflofpage 3)
`
`Therefore, in the present study, the most appropriate measure of cytotoxicity is reduction
`in cell number. The combination of NaprOxen Sodium and Sumatriptan Succinate should
`be considered positive for clastogenicity in CHO cells, since statistically significant dose-
`related increases in the frequency of cells with structural aberrations were observed
`without S9 at NAP/SS 1815/1815 ug/mL and 1850/1850 ug/mL associated with
`reductions in cell number of 50% and 56%, respectively; and with S9 at NAP/SS
`1745/1745 ug/mL and 1780/1780 ug/mL associated with reductions in cell number of
`42% and 52%, respectively.
`
`The data also confirm suggestions fi'om previous studies that the combination of NAP
`and SS produces a synergistic effect on both toxicity (a cytotoxic and/or cytostatic effect
`as measured by the reduction in cell number) and clastogenicity. Without S9, 1920
`ug/mL NAP + 1920 ug/mL SS reduced cell number by 68%, more than the sum of NAP
`and SS alone at the same dose (30% and 24%, respectively; sum = 54%). Similarly, with
`S9, the combination at 1780/ 1780 ug/mL reduced cell number by 52%, more than the
`sum of NAP and SS alone (31% + 0% = 31%). The frequency of cells with chromosomal
`aberrations was increased 20-40-fold with the highest dose of the combination, but not at
`all with the same doses of NAP and SS alone.
`
`14
`
`

`

`Reviewer: David B. Hawver, Ph.D.
`
`NDA No. 21-926
`
`1:1 combination of Naproxen sodium and Sumatriptan succinate: In Vitro Mutation
`Assay with L5178Y Mouse Lymphoma Cells at the TK Locus
`
`Study no.: — = Study 2990/25; GSK Document WD2006/03038/00
`
`Volume #, and page #: eNDA 21-926 #016, Module 4, Section 4.2.3.312, pages 1-75
`Conducting laboratory and location: —
`Date of study initiation: 04 AUG 2006
`GLP compliance: Yes, statement signed 30 OCT 2006
`QA reports: yes, statement signed 30 OCT 2006
`Drug, lot #, and % purity: Naproxen Sodium (NAP) Lot #NPXNAM-631, Purity
`100%; NAP doses were calculated as the free acid, using a correction factor of 1.1.
`Sumatriptan Succinate (SS) Lot #K026882, Purity 99.2%. SS doses were calculated as
`the fi'ee base, using a conversion factor of 1.4.
`
`Methods
`
`Strains/species/cell line:
`The original cultures of mouse lymphoma L5178Y (TK+") cells were obtained from
`—
`and stocks were stored frozen in
`liquid nitrogen. Each batch of frozen cells was purged of L5 1 78Y (TK"') mutants,
`checked for spontaneous mutant frequency and that they were mycoplasma free.
`
`Doses used in definitive studies (combinations were tested in a separate study ):
`:I:S9, 3 hr: SS/NAP together at 400/400, 800/800, 1200/1200, 1350/1350, 1400/1400,
`1500/1500, 1550/1550, and 1700/1700 ug/mL; NAP alone at 400, 800, 1200, 1350,
`1400, 1500, 1550, and 1700 ug/mL; SS alone at 400, 800, 1200, 1350, 1400, 1500,
`1550, and 1700 ug/mL
`-S9,’ 24 hr: SS/NAP together at 75/75, 150/150, 200/200, 250/250, 300/300, 350/350, and
`400/400 ug/mL; NAP alone at 75, 150, 200, 250, and 300 ug/mL; SS alone at 75,
`150, 200, 250, 300, 350, 400, 450, 500, 600 ug/mL.
`
`Basis of dose selection:
`
`A preliminary cytotoxicity test assayed concentrations of NAP and SS alone and together
`from 78.13 to 2500 ug/mL (~10 mM NAP; ~ 6 mM SS). Cytotoxicity (reduction in
`Relative Total Growth, RTG) was observed at 2 1250/1250 ug/mL in the 3 hr treatment
`iS9, and at 2 312.5 ug/mL in the 24 hr treatment —S9. No precipitates were observed.
`Based on these results, maximum doses of 1800/1800 ug/mL for the 3 hr assays and
`600/600 ug/mL for the 24 hr assay were chosen.
`
`Negative controls:
`Purified water was used as the vehicle control.
`
`15
`
`

`

`
`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926
`
`Positive controls:
`
`Methyl methanesulphonate (MMS, — in DMSO) was used as the positive control in
`the absence of metabolic activation, diluted with water to final concentrations of 5 or 15
`ug/mL.
`
`Benzo[a]pyrene (BaP, - in DMSO) was used as the positive control in the presence
`of metabolic activation, diluted with water to a final concentration of 3 ug/mL.
`
`Incubation and sampling times:
`Cultures were treated with test articles or controls for 3 hrs in S9 (rat liver, 2% final
`concentration) or 24 hrs —S9, then replated and cultured for 48 hrs, then replated into 96-
`well plates to determine viability (for 3 days) and mutation frequency (for 1-2 weeks).
`
`Results
`
`Study validity
`Criteria for a valid assay were met for the combination doses:
`0 The highest combination doses tested in each of the three assays resulted in toxicity
`within or near the desired range of 10-20% RTG (NAP/SS RTG = 23%, 15%, and
`16% in the 3-hr ~89, 24-hr —S9, and 3-hr +S9 assays, respectively).
`Results with vehicle and positive controls were within the expected ranges.
`0 No confounding factors or excessive heterogeneity were observed.
`
`However, the highest doses of NAP alone evaluated did not induce toxicity close to the
`desired range of toxicity in two of the three assays (NAP RTG = 68% at 1500 ug/mL
`afier 3 hrs —S9; 17% at 300 ug/mL after 24 hrs —S9; and 59% at 1700 ug/mL after 3 hrs
`+S9). Similarly, the highest doses of SS alone evaluated did not induce toxicity close to
`the desired range of 10-20% (SS RTG = 82% afier 3 hrs —S9; 73% afier 24 hrs -S9; and
`64% after 3 hrs +S9). For definitive results, NAP and SS alone should be tested in each
`assay up to concentrations associated with a RTG of S 20% or 5000 ug/mL Or 10 mM,
`whichever is lowest (10 mM = ~2300 ug/mL NAP and ~2940 ug/mL SS).
`
`Study outcome:
`In the 3-hr study without S9, the mutation frequency was not significantly increased with
`NAP/SS up to the limit of toxicity, 1500/1500 ug/mL, at which RTG was 23% of control.
`RTG at 1600/1600 ug/mL was 4%, below the acceptable limit of 10%. In the 24-h'r study
`without S9, the mutation frequency was not significantly increased up to the limit of
`toxicity, 400/400 ug/mL, at which RTG was 15% of control. In the 3-hr study with S9,
`the mutation frequency was not significantly increased up to the limit of toxicity,
`1700/1700 ug/mL, at which RTG was 16% of control.
`
`NAP alone did not significantly increase the mutation frequency after 3 hrs —S9 (RTG
`68% at 1500 ug/mL), after 24 hrs -S9 (RTG 17% at 300 ug/mL), or after 3 hrs +S9 (RTG
`59% at 1700 ug/mL).
`
`16
`
`

`

`
`
`% Relative
`Total
`Growth
`
`Mutation
`Frequency
`(x 10“)
`
`% Relative
`Total
`Growth
`
`Mutation
`Frequency
`(x 10“)
`
`
`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926
`
`SS alone did not significantly increase the mutation fi'equency afier 3 hrs -—S9 (RTG 82%
`at 1500 ug/mL), after 24 hrs -S9 (RTG 73% at 600 ug/mL), or after 3 hrs +S9 (RTG 64%
`at 1700 ug/mL).
`
`Treatment
`
`3 hr Incubation
`Without S9
`
`3 hr Incubation
`With S9
`
`1
`
`
`
`
`Purifiedwmer n— 58.65 .m- 5451 ~
`NAP/SS
`400/400
`54.31 m. 51.10
`NAP/ss
`_uu
`NAP/ss
`
`was _———I_
`
`__——_._
`
`NT=Not Treated; NP=Not Plated; NE=Not Evaluated
`
`Treatment
`
`24 hr Incubation
`Without S9
`
`% Relative
`Total
`Growth
`
`Mutation
`Frequency
`(x 10“)
`
`_-—I-
`
`—_—-1-
`
`'
`
`NT=Not Treated; NP=Not Plated; NE=Not Evaluated
`
`17
`
`

`

`Reviewer: David B. Hawver, Ph.D.
`
`NDA No. 21-926
`
`Test Artiele
`
`3 hr Treatment +SS-mix
`3 hr Treatment -SB—mlx
`Mean
`Mean
`Dose
`Relative
`Mean Mutant
`Relative
`Level‘
`Total
`Frequency
`Total
`L
`Growth
`mo‘)
`Growth
`"1"“
`1%]
`1%!
`100
`70.57
`100
`0
`Punfied water
`82
`55.13
`111
`400
`Naproxen
`62
`62.66
`116
`600
`Napmxen
`NE
`70.98
`133
`1200
`Naproxen
`67
`NT
`NT
`1350
`Naproxen
`NE
`59.34
`73
`1400
`Napmxen
`NE
`75.58
`66
`1500
`Napmxen
`TI
`NT
`NT
`1550
`Napmxen
`59
`NE
`NE
`1700
`Naproxen
`NT
`446.96
`44
`15
`Methyl methane sulphonate
`NT
`NT
`NT
`NT
`Meaty! methane sulphonate
`36
`NT
`NT
`3
`Home a
`e
`1. All concentrafions of Naproxen sodium are expressed in terms 01 parent compound
`NT Not treated
`NE Not evaluated
`
`Mean Mutant
`Frequency
`(x108
`56.64
`48.85
`65.53
`NE
`7226
`NE
`NE
`51.58
`52.62
`NT
`NT
`646.93
`
`24 hr Treatment -SS—rnlx
`Mean
`Relative
`Total
`Growth
`1%!
`100
`43
`61
`58
`NE
`17
`NT
`NT
`NT
`NT
`21
`NT
`
`Mean Mutant
`Frequency
`(x10‘)
`60.66
`51.18
`53.40
`52.26
`TE
`70.95
`NT
`NT
`NT
`NT
`579.11
`NT
`
`Dose
`Level‘
`I L
`”g "‘
`0
`75
`150
`200
`250
`300
`NT
`NT
`NT
`NT
`5
`NT
`
`{Page [2 off/2m}A’qpanfl
`
`I
`
`2! hr Treatment oss-mix
`3 hr Treatment ~39-mix
`Mean
`Mean
`Relative
`Mean Mutant
`Relative
`Mean Mutant
`.
`Total
`Frequency
`Total
`Frequency
`Levet
`Te" Ame“
`Growth
`(no‘)
`Growth
`(1110‘)
`“9"“
`'5
`54
`100
`41.23
`»
`1m
`6026
`0
`Punfied water
`74
`52.30
`95
`56.39
`400
`Sumatrlptan
`75
`42.62
`107
`57.13
`600
`Sumatrlptan
`NE
`NE
`120
`5066
`1200
`Sumatriptan
`68
`66.09
`NT
`NT
`1350
`Sumatriptan
`NE
`NE
`86
`44.34
`1400
`Sumetriptan
`NE
`NE
`62
`70.08
`1500
`Sumatrlptan
`89
`56.74
`NT
`NT
`1550
`Sumatrlptan
`64
`66.34
`NE
`NE
`1700
`Sumatriptan
`NT
`MT
`NT
`NT
`NT
`Sumatt'iptan
`NT
`NT
`NT
`NT
`NT
`Sumatriptan
`NT
`NT
`28
`410.73
`15
`Methyl methane sulphonate
`NT
`NT
`NT
`NT
`NT
`Methyl methme sulphmato
`24
`877.66
`NT
`NT
`3
`Ben: I
`one
`1. All concentrations a! Sumatriptan succinct: are expressed in terms of parent compound
`NT Not treated
`NE Not evaluated
`
`Dose
`
`I
`
`”WI
`"9“”-
`0
`75
`150
`200
`250
`300
`350
`400
`450
`500
`600
`NT
`5
`NT
`
`24 hr Treatment -S$mix
`Mean
`Relative
`Mean Mutant
`Total
`Frequency
`Growth
`(“0‘1
`'A
`100
`66.36
`94
`43.63
`108
`44.67
`94
`65.11
`77
`57.46
`103
`57.69
`100
`53.74
`112
`67.5,
`94
`70.32
`71
`66.13
`73
`60.40
`NT
`NT
`27
`1087.49
`NT
`NT
`
`{Page I} off/my Report]
`
`Sponsor’s Conclusions:
`The 1:] combination of NAP and SS was not genotoxic in the mouse lymphoma L5178Y
`TK +/- assay in the presence or absence of S9 metabolic activation, when tested up to the
`limits of cytotoxicity.
`
`Reviewer’s Conclusions:
`
`This study presented valid negative results for the 1:1 combination of NAP and SS up to
`appropriate levels of toxicity. However, the study did not evaluate sufficiently high doses
`of NAP and SS alone to conclude that they are not genotoxic in this assay, except in the
`20-hr assay without S9 for NAP. It is not clear why NAP was not genotoxic in this assay
`at concentrations much higher than those that were shown to be genotoxic in a previous
`mouse lymphoma assay — #AA33KS, JL, KC, .7040002.BTL, Pozen
`#MTl 00—T26) submitted to support _ (dose-dependent increases in mutation
`frequency were observed at 50, 150, and 300 ug/mL NAP alone in a 4-hr assay with $9).
`
`18
`
`

`

`
`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926
`
`2.6.6.5 Carcinogenicity
`
`No carcinogenicity studies were included in this submission.
`
`2.6.6.6 Reproductive and developmental toxicology
`
`No reproductive and developmental toxicology studies were included in this submission.
`
`2.6.6.7 Local tolerance
`
`No local tolerance studies were included in this submission.
`
`2.6.6.8 Special toxicology studies
`
`No special toxicology studies were included in this submission.
`
`2.6.6.9 Discussion and Conclusions
`
`See Overall Conclusions and Recommendations.
`
`2.6.6.10
`
`Tables and Figures
`
`Tables and Figures were included within the text.
`
`2.6.7 TOXICOLOGY TABULATED SUMNIARY
`
`19
`
`

`

`
`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926 .
`
`
`
`OVERALL CONCLUSIONS AND RECOMlVIENDATIONS:
`
`In the original NDA submission, an in vitro chromosomal aberration assay in CHO cells
`(Study MT400/T07, #0735/0736-3110) demonstrated greater clastogenic effects with the
`combination of naproxen and sumatriptan compared with naproxen alone, raising the
`concern that the two compounds together may have carcinogenic effects not observed
`with either drug alone. The clastogenic effects were observed only at concentrations
`producing substantial cytotoxicity, making the biological significance of the effects
`unclear.
`
`In the approvable letter dated 08 JUN 2006, the sponsor was asked to attempt to clarify
`this issue by repeating the chromosomal aberrations assay testing concentrations of the
`1:1 NAP/SS combination between those exhibiting minimal toxicity (1250/1250 ug/mL)
`and those inducing substantial toxicity (2500/2500 ug/mL without S9 activation, and
`2000/2000 ug/mL with S9 activation), and by conducting an in vitro mouse lymphoma tk
`assay testing naproxen and sumatriptan alone and in combination.
`
`The current submission contains final reports from the requested genotoxicity studies
`with NAP and SS alone and in combination at 1:1.
`
`In the new assay for chromosomal aberrations in CHO cells, the frequency of cells with
`structural aberrations was increased dose-dependently by NAP/SS at concentrations that
`reduced the total cell count by 50-68% in the absence of S9 metabolic activation
`(1815/1815 to 1920/1920 ug/mL), and by 42-52% in the presence of S9 (1745/1745 to
`1780/1780 ug/mL). Neither NAP nor SS alone at the same concentrations resulted in
`significant clastogenicity. NAP alone was also negative at the highest concentration
`tested, 2500 ug/mL, which is equivalent to ~10.9 mM, exceeding the recommended
`maximum concentration of 10 mM (Note: these calculations are based on MW = 230.266
`for naproxen free acid, and 295.406 for sumatriptan free base). These data demonstrate a
`synergistic clastogenic effect of the combination of the two drugs at concentrations
`greater than or equal to ~76 mM NAP and ~5.9 mM SS, associated with reductions in
`cell number of 2 42%. NAP and SS also had synergistic effects on cytotoxicity at these
`concentrations. The sponsor argues that using reduction in cell count underestimates the
`cytotoxicity, and that this assay would be considered negative if inhibition of population
`doublings (PD) were used instead. However, the current ICH guidance recommends
`testing test article concentrations that cause “greater than 50% reduction in cell number
`or culture confluency.” /G}:/1k’efi/zefor//zdzxyt/y Spec??? Aspects of/Pegzz/m‘o/y
`Geno/01187? [kyflflrPflamacez/lzba/J; [677524, Aan 1990:page!)
`
`20
`
`

`

`
`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926
`
`In the new in vitro mouse lymphoma assay, the 1:1 NAP/SS combination was not
`genotoxic at concentrations up to those inducing cytotoxicity within (or close to) the
`desired range of 10-20% Relative Total Growth (1450/1450 ug/mL afier 3 hrs —S9;
`1700/1700 ug/mL alter 3 hrs +89; and 400/400 ug/mL after 24 hrs —S9). It is not clear
`why NAP was not genotoxic in this assay at up to 1700 ug/mL +S9 when a previous
`mouse lymphoma assay showed dose-dependent increases in mutation frequency at 50,
`150, and 300 ug/mL NAP alone after 4-hr treatments with S9 .
`
`The original NDA submission for Trexima included negative results for a valid bacterial
`reverse mutation assay (up to 2500 ug/plate NAP/SS) and for a valid in vivo mouse
`micronucleus assay (up to an MTD of 500/1500 mg/kg NAP/SS (M) or 375/1625 mg/kg
`(F)). Negative carcinogenicity studies are described in the current labeling for SS
`(Imitrex; rat and mouse) and NAP (Anaprox; rat). In addition, the current sponsor
`conducted a two-year rat study to support _ that
`demonstrated no increases in neoplasms in rats receiving NAP alone at the MTD of 8
`mg/kg/day. Finally, a 26-week carcinogenicity study in p53+/' mice was negative in mice
`given 50 mg/kg/day NAP in combination with 50 or 1.6 mg/kg/day metoclopramide (see
`-
`
`The approval letter of 08 JUN 2007 also included the following statement:
`
`You need to include the results of the in vitro mouse lymphoma tk assays (Studies MT 100 T25,
`MTlOO T26) and the carcinogenicity study in p53+" heterozygous mice (Study MT 100 T35) for
`naproxen in product labeling.
`
`The sponsor has agreed to include the following statement in labeling to address the first
`point:
`
`21
`
`

`

`
`
`Reviewer: David B. Hawver Ph.D. NDA No. 21-926
`
`Reviewer’s Conclusion:
`
`The data demonstrate that the combination of NAP and SS induce genotoxic effects in
`CHO cells in the presence and absence of S9 that are not observed at similar
`concentrations of either drug alone. These effects occur at moderate levels of cytotoxicity
`(Z 42% reduction in cell number) and at relatively high concentrations (2 7.6 mM NAP;
`2 5.9 mM SS). This reviewer believes it might be reasonable to argue that these
`concentrations exceed the recommended maximum concentration of 10 mM because 7.6
`
`+ 5.9 = 14.5 mM. Considering the negative findings in the mouse lymphoma assay, the
`bacterial reverse mutation assay, and the in vivo mouse micronucleus assay with
`NAP/SS, and the negative carcinogenicity findings for each of the components of the
`combination, this reviewer believes that including the positive genotoxicity findings in
`the labeling for Trexima is sufficient to address this issue. No additional nonclinical
`studies are needed.
`'
`
`Unresolved toxicology issues:
`The discrepancy between the dose-dependent positive findings in a previous mouse
`lymphoma tk assay with NAP alone in the presence of S9 metabolic activation and the
`negative findings in the present mouse lymphoma tk assay with NAP alone (+S9) at
`much higher concentrations remains unexplained.
`
`Recommendations:
`
`The nonclinical package is adequate to support an approval action for NDA 21-926
`TREXIMA (sumatriptan succinate/naproxen sodium) Tablets for the acute treatment of
`migraine.
`
`22
`
`

`

`07 Page(s) Withheld
`
`Trade Secret / Confidential
`
`I/ Draft Labeling
`
`Deliberative Process
`
`Withheld Truck Number: Pharm/Tox— \6—
`
`'
`
`

`

`This'Is a representation of an electronic record that was signed electronically and
`this page is the manifestation of the electronic signature.
`
`/S/
`
`David Hawver
`
`8/1/2007 06:18:33 PM
`PHARMACOLOGIST
`
`Lois Freed
`
`8/1/2007 06:21:29 PM
`PHARMACOLOGIST
`
`Please see separate memo for comments.
`
`

`

`MEMORANDUM
`
`DEPARTMENT OF HEALTH & HUMAN
`SERVICES
`Public Health
`Service
`
`Food and Drug Administration
`
`Division of Neurology Products (HFD-120)
`Center for Drug Evaluation and Research
`
`Date:
`
`June 9, 2006
`
`From: Lois M. Freed, Ph.D.
`Supervisory Pharmacologist
`
`Subject: NDA 21-926 (TREXIMA; sumatriptan/naproxen)
`
`The nonclinical data submitted in support of the NDA for TREXIMA were reviewed by
`David B. Hawver, Ph.D. (Pharmacology/Toxicology Review and Evaluation NDA 21-
`926, June 9, 2006). Based on his review, Dr. Hawver has concluded that the nonclinical
`data support an approvable action; however, he identifies several unresolved toxicology
`issues:
`
`(a) whether or not naproxen exacerbates the cardiovascular effects (i.e., vasoconstriction,
`coronary artery vasospasm) of sumatriptan.
`
`Dr. Hawver notes that the special toxicology study to assess the effects of naproxen on
`sumatriptan—induced vasoconstriction/vasospasm was methodologically flawed and, thus,
`provided no reliable data to address the issue. However, he concludes that issues of
`technical feasibility and interpretability argue against requiring a new cardiovascular
`safety study as a condition for approval.
`'
`
`Comment: although it is not clear that the technical difficulties encountered during the
`conduct of the study necessarily means that an interpretable combination cardiovascular
`study could not be conducted, it seems unlikely that a new or repeat study would provide
`data that would cause greater concern of cardiovascular risk than that already
`acknowledged based on data in humans. Therefore, I agree that no additional nonclinical
`study is needed.
`
`(b) the potential for additive/synergistic or novel toxicities with the combination of
`naproxen and sumatriptan compared to each drug alone.
`
`

`

`Two 90-day combination toxicity studies were conducted in mice (Study #907-009 and
`Study 04-293/MT400-T19). Study #907-009 tested sumatriptan (S) and naproxen (N) in
`combination (S/N) and each alone at oral (gavage) doses of 0/0, 25/12, 105/50, 210/ 100,
`320/150, 320/0, and 0/150 mg/kg/day in males and 0/0, 50/12, 110/25, 210/50, 320/75,
`210/0, and 0/50'mg/kg/day in females. In males, the dose of naproxen (alone and in
`combination with sumatriptan) was lowered from 150 to 100 mg/kg/day on Day 62 (after
`a drug holiday from Days 57-61) due to increased mortality. In females, the 320/75
`mg/kg/day group was terminated on Day 65 due to increased mortality; mortality was
`also increased in the 0/50 mg/kg/day group. Dr. Hawver noted that no sumatriptan-related
`toxicity was observed in either males or females. Dr. Hawver also noted that “concerns
`regarding the accuracy and homogeneity of the dosing solutions” made interpretation of
`the study difficult. It was also the sponsor’s conclusion that Study #907-009 was not
`definitive due to methodological problems.
`
`Study 04-293/MT400-T19 tested sumatriptan (S) and naproxen (N) in combination (S/N)
`and each alone at oral doses of 0/0, 30/10, 100/30, 320/ 100, 320/0, and 0/ 100 mg/kg/day
`in males and 0/0, 30/10, 100/30, 320(210)/50, 320(210)/0, and 0/50 mg/kg/day in
`females.
`In females, the high dose of sumatriptan was decreased from 320 to 210
`mg/kg/day at the start of dosing Week 4 due to death in 3/18 and 2/18 females in the
`320/50 and 320/0 mg/kg/day groups, respectively. Other than these deaths, no
`sumatriptan—related toxicity was observed. Toxicity induced by naproxen was
`characterized by Dr. Hawver as a “low level of toxicity” and by the sponsor as “subtle”.
`The sponsor also concluded that “There were no deaths or clinical signs that were
`considered to be test article-related”. There were, however, a number of unscheduled
`deaths which were attributed to gavage error or lower urinary tract obstruction.
`
`Based on the lack of clear dose—related toxicities for either sumatriptan or naproxen (or
`the combination) in the definitive 90-day study, Dr. Hawver recommends that another 90-
`day study be conducted in mice testing up to a maximum tolerated or maximum feasible
`dose of each drug alone and in combination.
`
`Comment: based on Dr. Hawver’s review of the data from the two 90-day mouse studies,
`it would appear that Sumatriptan could have been tested at higher doses. However, the
`high doses of naproxen tested in Study 04-293/MT400-T19 would appear to have been
`maximum tolerated doses (MTDs). Although 100 and 50 mg/kg were not associated with
`notable toxicity in males and females, respectively, in that study, the high doses of
`naproxen used in Study #907-009 (150 and 75 mg/kg in males and females, respectively)
`exceeded the MTD based on increased mortality. Comparing the data from the two
`studies, it would appear that the plasma exposures (AUC) for naproxen were fairly
`similar between studies.
`
`The high dose of sumatriptan used in both studies (320 mg/kg in males and females) does
`not appear to have been associated with any toxicity. Although the sponsor lowered the
`dose from 320 to 210 mg/kg/day in females in Study 04-293/MT400-Tl9 due to two
`deaths, the sponsor concluded that no drug-related deaths occurred in that study. In the
`28-day dose-range finding study in mice, a dose of 500 mg/kg was well-tolerated (i.e., no
`
`

`

`sumatriptan-related toxicity was detected). Therefore, there are no data that establish an
`MTD for sumatriptan in mice. However, there is a large safety margin between the
`plasma exposure (AUC) at the high dose in males (leO fold at 320 mg/kg/day) and
`females (z80 fold at 210 mg/kg/day) and the plasma AUC associated with the
`recommended daily dose of 85 mg/day in humans.
`
`Although I agree with Dr. Hawver that higher doses of sumatriptan should have been
`used in the combination study, I don’t believe that a repeat study is necessary since (a)
`naproxen was tested at an MTD, (b) there is a large safety margin between the plasma
`exposures at the high doses of sumatriptan used in the 90-day mouse study and those
`anticipated in humans at the recommended daily dose of sumatriptan, (o) no novel
`toxicities were detected with naproxen and sumatriptan in combination, and (d) according
`to the clinical team there is a robust safety database for the combination in humans.
`
`_(c) the effects of sumatriptan on the clastogenicity of naproxen.
`
`Dr. Hawver concludes that the possibility that sumatriptan may potentiate the clastogenic
`effects of naproxen “remains unresolved”. However, it is Dr. Hawver’s opinion that
`experiments to further investigate this possibility are not necessary since naproxen was
`not tumorigenic in carcinogenicity studies in rats at maximum tolerated doses.
`
`Comment: naproxen and sumatriptan were negative in the Ames assay and the in vivo
`micronucleus assay in mice when tested alone and in combination. However, naproxen,
`alone and in combination with sumatriptan, was clastogenic in an