4. What bioanalytical methods are used to assess concentrations?
`
`See the response to section IVE] above.
`
`51
`
`

`

`V. LABELING RECOMMENDATIONS
`
`Please refer to Appefidix A, Annotated Label.
`
`_Hv
`u'fi-v-
`
`52
`
`

`

`3 Ci
`
`pages redacted from this section of
`
`the approval package consisted of draft labeling
`
`

`

`Appendix B. Individual Study Reviews
`
`An investigation ofthe potential for daptomycin to inhibit cytochrome P450 1A2, 2A6, 2C9, 2C19,
`2D6, 2E], and 3A4 in cryopreserved human hepatocytes (ADME Report #12)
`
`.
`OBJECTIVE:
`The purpose of the study was to assess the potential of daptomycin to inhibit hepatic cytochrome P450
`mediated metabolism via CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYPZEI, CYP 3A4.
`
`FORMULATION:
`
`Daptomycin (Lot No. X800384)
`
`STUDY DESIGN:
`
`.
`
`The in vitro evaluation of daptomycin as an inhibitor of human cytochrome P450 (CYP P450) isozymes
`was performed using daptomycin concentrations of 2.5, 10, and 40 ug/mL Human hepatocytes were
`pooled from five adult male (all Caucasian) and five adult female (4 Caucasian, 1 Hispanic) subjects.
`Hepatocytes were incubated in protein-free Krebs-Henseleit buffer (KHB) containing 0.] M CaClz.
`
`Isolated hepatocytes were diluted with suspension media (Dulbecco's modified Eagle medium) to
`determine viability using Tryptan Blue. Cells were then centrifuged and diluted with incubation medium
`(KHB) to prepare a cell suspension (2 x 106 cells/ml).
`
`The CYP form-specific activities were evaluated using the following probe substrates: 50 uM phenacetin
`(CYP1A2). 50 uM coumarin (CYP2A6), 75 [AM tolbutamide (CYP2C9), 50 pM S-mephenytoin
`(CYP2C19), 8 uM dextromethorphan (CYP2D6), 50 uM chlorzoxazone (CYPZEI), and 50 uM
`testosterone (CYP3A4). A negative control consisted of incubation media alone (Kl-1B buffer). Positive
`control inhibitors consisted of 10 pM furafylline (CYP1A2), 50 uM diethyldithiocarbamate (CYP2A6), 1
`uM sulfaphenazole (CYP2C9), 10 {AM omeprazole (CYP2C19), 1 uM quinidine (CYP2D6), 100 uM 4-
`methylpyrazole (CYPZEI), and 1 uM ketoconazole (CYP3A4).
`
`The rate of enzyme activity was assessed by the rate of formation of acetaminophen (CYP1A2), 7-
`. hydroxycoumarin (CYP2A6), 4-hydroxytolbutamide (CYP2C9), 4-hydroxymephenytoin (CYP2C19),
`dextrorphan (CYP2D6), 6-hydroxychlorzoxazone (CYPZEI), and 6B-hydroxytestosterone (CYP3A4).
`The percentage of the activity remaining was calculated as the ratio of enzyme activity in the presence of
`daptomycin relative to the enzyme activity in the presence of negative control. The impact of the positive
`control was calculated using the vehicle control (incubation medium + 0.1% DMSO) instead of the
`negative control.
`
`RESULTS:
`
`The viability of hepatocytes in suspension media using Tryptan blue was 73%.
`
`The positive controls yielded greater than 50% inhibition of enzyme activity and adequately inhibited the
`activity of the isoforms.
`
`The rate of enzyme activity (pmol/million cells/min) in the presence of negative control, positive control,
`and daptomycin is shown in Table I. The percent of the activity remaining for each P450 isozyme is
`shown in Table 2.
`
`Daptomycin, at concentrations of 275,10, and 40 ug/mL, did not appreciably inhibit of the activity of
`CYP1A2, CYP2A6, CYP2C9, CYPZD6, and CYP3A4 in human hepatocytes. The activity of CYPZEI
`
`92
`
`

`

`was reduced in the presence of daptomycin (67.5% to 73.2%), although the inhibition was less than 50%
`of the positive control (4-methylpyrazole).
`
`Table 1. Effects of daptomy'cin on in vitro metabolism rates of major human CYP P450 specific
`activities
`
`
`
`
`
`
`
`
`Enzyme activity umol/million cells/min
`
`( ) Control
`13.5+o3
`3.64+Ol7
`IO 2 + 0 3
`
`869:043
`10010.3
`6.61 + 0.25
`20.1 :t 3.2
`
`(+) Control
`l.02i0.06
`0.49:0.04
`1.98 i 0.04
`
`2.02:0.15
`1.15:0.16
`0.49 i 0.04
`0.77 1 0.15
`
`
`
`2.5 almL
`141+02
`2.
`94+013
`
`10 4 i 0.3
`
`805+04l
`117+04
`4.84 + 015
`20.7 1 3 5
`
`_/mL
`10
`14410.3
`343:0.30
`10.4 i 0.2
`
`8.411015
`124:0.2
`4.60 i 0.23
`20.6: 2.6
`
`
`
`
`4
`141+02
`l
`3.21010
`10.9 :t 0.3
`
`853+03l
`124+03
`4.46 i 0 52
`32.6: 13 6
`
`
`
`CYP P450
`
`Probe Substrate
`
`2A6
`
`2Cl9
`
`Coumann
`Tolbulam1de
`
`S-Mephenytoin
`Dextromethorphan
`
`
`
`
`
`
`
`
`
`
`
`
`Table 2. Percentage of activity remaining of major human CYP P450 isoenzymes in the presence of
`daptomycin
`
`
`Activity remainin_ %
`Control
`
`206
`2E1
`
`Dextrometho ohan
`Chlorzoxazone
`
`Testosterone
`
`
`
`
`
`
`
`
`
`
`
`
`Following the administration of daptomycin IV 4 mg/kg q24h, anticipated peak plasma concentrations of
`daptomycin are approximately 50 ug/ml (total) and 5 ug/ml. (unbound). Thus, the maximum
`concentration of daptomycin assessed in the study exceed the anticipated peak plasma concentrations of
`daptomycin by approximately 8-fold.
`
`CONCLUSIONS:
`
`Based on the in vitro results, daptomycin IV 4 mg/kg is unlikely to inhibit the metabolism of drugs
`dependent on P450 isoforms CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYPZEl , or CYP3A4.
`
`COMMENTS:
`
`Although the sponsor states that the activities of all substrates were analyzed using proprietary 8
`methods, assay validation data were not provided with the study report. The sponsor is encouraged to
`provide the full assay validation data with the study report in the future.
`
`h.“_,
`._ “q.
`
`93
`
`

`

`An investigation of the potential for daptomycin to induce cytochrome P450 1A2, 2A6, 2C9, 2C19,
`206, 2131, and 3A4 in cultured human hepatocytes (ADME Report #13)
`
`OBJECTIVE:
`
`The purpose of the study was to assess the potential of daptomycin to induce hepatic cytochrome P450
`mediated metabolism on P450 CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYPZEl, and
`CYP3A4 isoforms.
`
`FORMULATION:
`
`Daptomycin (Lot No. X800384)
`
`STUDY DESIGN:
`
`The in virro evaluation of daptomycin as an inducer of human cytochrome P450 (CYP P450) isoforrns
`was performed using daptomycin concentrations of 25, 100, and 400 ug/mL. Fresh liver tissue was
`obtained from one male and one female donor, plus one additional male donor for CYP2A6 and CYPZEI
`(since one donor was lacking CYP2A6 and CYPZEI activity). Portions of the liver were processed into
`hepatocytes by collagenase-based digestion of connective tissue, followed by manual and mechanical
`separation and washing with media. Isolated hepatocytes were diluted with suspension medium
`(Duibecco‘s modified Eagle‘s medium) and counted to determine yield. Viability was measured with
`Tryptan blue exclusion.
`
`Hepatocytes were incubated with test or control article for 48 hrs at 37°C and then incubated with the
`following CYP P450 isoforrn-specific substrates: 2 uM ethoxyresorufin (CYP1A2), 100 uM coumarin
`(CYP2A6), 50 uM tolbutamide (CYP2C9), 100 uM S-mephenytoin (CYP2C19), 16 uM
`dextromethorphan (CYP2D6), 300 uM chlorzoxazone (CYPZEI), and 125 uM testosterone (CYP3A4).
`The negative control consisted of incubation media alone (Dulbecco‘s modified Eagle medium). Positive
`control inducers consisted of 50 uM omeprazole (CYP1A2) and 25 uM rifampin (CYP2C9 and
`CYP3A4). The sample size was n=3 for samples incubated with daptomycin and n=6 for each control.
`
`The induction potential of daptomycin was compared with that of known inducers omeprazole (CYP1A2)
`and rifampin (CYP2C9 and CYP3A4). Inducers of CYPZC 19, CYP2D6, and CYPZEl were not
`available. Daptomycin-related induction was considered biologically relevant if a dose-related increase
`was observed and the response was 250% of the isoform-specific positive control. For isoforms with no
`known inducers, response was considered relevant if the induced level was at least 150% of the
`corresponding negative control.
`
`RESULTS:
`
`The viability of hepatocytes from the three donors ranged from 88% to 89% using Tryptan Blue
`exclusion.
`
`Donor 1 lacked measurable activity in all daptomycin samples for CYP2A6 and CYP2E1 isofonns.
`
`The positive controls (omeprazole and rifampin) yielded greater than 200% of vehicle control activity for
`CYP1A2 and CYP3A4 , respectively. Rifampin yielded a modest induction of CYP2C9 activity in donor
`1 (143%) and donor 2 (195%), although the activity was 200% of vehicle control. Omeprazole and
`rifampin were associated with induction of CYP2A6 in donor 3 (662%) and donor 2 (224%). No
`induction of the other isofomis was observed.
`
`94
`
`

`

`The rate'of enzyme activity (pmol/million cells/min) in the presence of negative control, positive control
`1 and 2, and daptomycin is shown in Table l. The enzyme activity for each P450 isoform compared to
`vehicle control (incubation media + 0.1% DMSO) is shown in Table 2.
`
`Table 1. Effects of daptomycin on in vitro metabolism rates of major human CYP P450 specific
`substrates
`
`
`
`Enzyme activitv nmol/million cells/min
`
`CYP P450
`
`(-) Control
`
`(+) Control
`l
`
`(+) Control
`2
`
`25 pg/mL
`
`100 pg/mL
`
`400 pg/mL
`
`1A2
`
`2A6
`
`2C9
`
`Donor l
`Donor 2
`
`Donor 1
`Donor 2
`Donor 3
`
`Donor 2
`
`0.16 i 0.0]
`0.30 i 0.01
`
`0.00 i 0.00
`8.35 i 0.68
`0.58 1 0.06
`
`3.41 1 0.62
`8.47 1 0.67
`
`2.30 i 0.33
`5.97 i 0.28
`
`0.00 i 0.00
`5.15 i 0.54
`2.92 1 0.20
`
`6.20 1 1.06
`13.2 1 0.8
`
`0.18 i 0.0)
`0.27 i 0.01
`
`0.00 i 0.00
`14.1 i 0.7
`0.79 1 0.09
`
`6.66 1 0.90
`19.7 11.0
`
`0.18 i 0.01
`0.34 i 0.01
`
`0.00 i 0.00
`9.00 i 0.33
`0.62 1 0.05
`
`3.73 1 0.61
`8.78 11.03
`
`0.21 i 0.01
`0.34 :t 0.02
`
`0.00 i 0.00
`9.24 i 0.32
`0.65 1 0.02
`
`4.20 1 0.61
`8.68 1 0.33
`
`0.24 i 0.02
`0.41 :1: 0.01
`
`0.59 i 0.60
`10.4 :1: 0.4
`0.66 1 0.09
`
`5.29 1 0.21
`8.88 1 0.62
`
`.
`
`Donor 2
`
`2.14 1 0.13
`
`0.88 1 0.05
`
`3.62 1 0.44
`
`1.76 1 0.43
`
`1.77 1 0.90
`
`1.54 1 0.12
`
`Donor2
`
`4.871020
`
`5.601022
`
`8.811020
`
`4.291019
`
`4.211019
`
`4.101046
`
`.
`
`2E1
`
`1.811018
`0.001000
`Donorl
`8.241041
`5.091028
`Donorz
`5.951067
`3.771074
`Donor3
` 3A4
`4.45 i 0.37
`5.59 i 0.51
`Donor 1
`
`29.8i2.0
`32.2i 1.8
`DOHOTZ
`
`Positive control 1=omeprazole; positive control 2=rifampin
`
`1.811 0.29
`7.211021
`6.451071
`25.4 i 4.40
`146i l-l.0
`
`
`
`0.001 0.00
`4.931036
`3.871083
`3.54 i 0.23
`39.01101
`
`0.001000
`4.831031
`2.761059
`3.80 i 0.40
`40.4:t 1.6
`
`
`0.001 0.00
`3.881027
`3.111070
`3.64 i 0.32
`38.0140
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Table 2. Range of enzyme activity (%) for human CYP P450 isoenzymes in the presence of
`daptomycin compared to negative control
`
`
`Ran_e of enzyme Activi
`'
`°/o
`
`
`CYP P450
`
`(+)Control]
`(+)Contr012
`100
`/mL
`400
`/mL
`
`
`1,503% to
`94% to 120%
`115% to 133%
`139% to 150%
`2.110%
`8294106624
`
`113% to 114%
`
`99% 161149.-
`
`105%1o155%
`72% 10123%
`
`
`
`
`2A6
`
`
`
`
`
`
`
`Positive control 1=omeprazole; positive control 2=Tifampin
`The shaded cells represent the anticipated induction for the respective CYP P450 isoforrn
`
`63%1o121%
`
`
`
`
`
`
`
`Daptomycin, at concentrations of 25, 100, and 400 pg/mL did not induce the activity of CYP1A2,
`CYP2A6, CYP2C9, CYP2D6, and CYP3A4 in human hepatocytes. For isoforms CYP1A2 and CYP3A4,
`the response to daptomycin was less than 50% of that for positive controls. For isoforms CYP2A6,
`CYP2C19, CYP2D6, and CYP2A1, the response to daptomycin was less than 150% for the negative
`control. For isoform CYP2C9, a possible induction response was evident in donor 1 only at the highest
`concentration (400 ug/mL). 'l-lowever, 400 pg/ml. exceeds the anticipated unbound maximum plasma
`concentration by approximatelyfiq-‘fold when administered as 4 mg/kg IV q24h and is unlikely to be
`clinically relevant.
`'
`
`95
`
`

`

`CONCLUSIONS:
`
`Based on the in vitro results, daptomycin IV 4 mg/kg is not anticipated to induce the metabolism of drugs
`cleared by isoforms CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYPZEI, or CW3A4.
`
`COMMENTS:
`
`, The sponsor has not provided assay validation data with the study report. The sponsor is encouraged to
`provide the full assay validation data with the study report in the future.
`
`APPEARS THIS WAY
`0N ORIGINAL
`
`MRS THIS WAY
`0“ ORIGINAL
`
`._ “M
`
`96
`
`

`

`“C-LY146032: Distribution and metabolism (Protocol BSB-LC-AVAC)
`
`Uate: Nor stated
`
`Clinical Site: Lilly Laboratory for Clinical Research, Wishard Memorial Hospital, Indianapolis, IN
`46202
`
`Analytical Site: Not stated
`
`OBJECTIVES:
`
`The objectives of the current study were 1) to determine the distribution, excretion, and metabolism of
`NC-I.Y146032 using a single IV dose of] mg/kg, and 2) to identify and measure, ifpossible, any
`metabolites of LY146032.
`
`-
`FORMULATION:
`The batch No. of the LY146032 43 formulation was not stated
`
`STUDY DESIGN:
`
`This study was a single center, open-label, non-randomized,'single dose study in five healthy adult males
`between the ages of 25 and 36. Following an overnight fast, three subjects received LY146032 and NC-
`tryptophan labeled LY146032 (1 mg/kg containing approximately 50 uCi labeled l“C-LY146032,
`equivalent to approximately 13.88 mg of material with a specific activity of 3.6 pCi/mg) infused in 50 rnL
`5% dextrose over a 30 min period. An additional two subjects were admitted to the protocol and received
`the same dose of drug in 50 ml. of 0.9% sodium chloride solution. The structure of MC-tryptophan
`labeled LY 146032 is shown in Figure 1.
`
`Figure 1. LY146032 labeled with “C tryptophan
`
`/
`
`/
`. /
`
`Blood samples were collected every 10 min during the infusion and at 30 min, 45 min, and l, 2, 3, 4, 6, 8,
`12, 18, 20, 24, 30, 36, and 48 hrs after the end of the infusion, and every 24 hrs thereafter until
`radioactivity had approached background levels.
`
`Urine samples were collected during the following intervals: -2 to 0, 0 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10,
`10 to 12, 12 to 18, and 18 to 24 hrs after the start of the infusion. Specimens were collected in 6 hr
`increments thereafter until radioactivity had returned to near baseline background counts.
`_-~_..,
`
`97
`
`

`

`A control fecal sample was obtained within 24 hrs prior to the study for background radioactive
`determination. All bowel movements were collected until the radioactivity approached background
`levels.
`
`Breath and saliva samples were obtained at one-half hr intervals commencing with the end of the infusion
`period and continued for 8 hrs for the three subjects receiving ”C-LY146032 in 5% dextrose. Breath
`samples were collectedin a hyamine hydroxide solution for liquid scintillation counting. For the two
`subjects who receivedC-LY146032m 0.9% sodium chloride, the collection period for breath and saliva
`samples was extended through 120 hrs.
`
`a
`Protein binding:
`Plasma samples were ultracent'n'fuged to separate plasma water from protein. The radioactivity in the
`plasma water was measured and the data used to estimate protein binding of the radioactive materials
`present.
`
`Liquid Scintillation Counting:
`Thel C-label1n plasma, urine, saliva, and expired air was measured using liquid scintillation counting
`Whole blood and fecal homogenate samples were dried, combustedm an oxygen atmosphere, and the
`resulting carbon dioxide trapped in the scintillant counted to determine the radiocarbon content.
`
`Microbiologic Assay:
`. Plasma and urine samples were assayed for LY146032 using a microbiologic assay with Sarcina [urea as
`the indicator organism for measuring active drug.
`s“
`
`-——
`The presence of rad1ocarbon labeled drug and metabolites1n urine was examined using
`Eluant from the column was collectedin timed fractions which were combined with liquid
`scintillation fluid and examined1n a liquid scintillation counter for radiocarbon content
`~
`
`The metabolic patterns in urine samples were examined with silica gel plates using a solvent containing
`acetonitrile, water, and acetic acid. Radioautography was used to locate areas of radioactivity and
`unlabeled drug and potential metabolites were added for reference purposes.
`
`DATA ANALYSIS:
`
`Daptomycin concentration data were analyzed by non-compartmental pharmacokinetic analysis. The
`following parameters were determined for plasma daptomycin concentration data: the maximum plasma
`concentration (Cm), time to Cm, (Tm), the area under the plasma concentration-time curve from zero to
`the last quantifiable concentration (AUCM), AUC fiom zero to infinity (AUCo..), plasma clearance (CLT),
`renal clearance (CLR), non--renal clearance (CLW), volume of distribution (V5), amount of drug excreted
`in urine (Ae), and terminal elimination half-life (B)
`
`STATlSTICAL ANALYSIS:
`
`Phannacokinetic parameters were summarized as mean, SD, median, and range.
`
`A. “_r
`._-«___,.
`
`98
`
`

`

`RESULTS:
`
`Three subjects received a single dose of MC-LY146032 (approximatelyl mg/kg) infused in 5% dextrose
`solution, whereas two subjects received a single dose of HC-LY146032 (approximatelyl mg/kg) infused
`in 0.9% sodium chloride solutibn. The mean (SD) age, weight, and height for the five subjects were 31.2
`(4.7) yrs, 65.8 (1.8) kg, and 68.0 (2.9) inches. The concentration-time profiles of LYl46032 in plasma
`and whole blood determined using a microbiological assay (shown as bioassay) and total MC (shown as
`C14) are shown in Figure 2 (n=5).
`
`Figure 2. Mean LY146032 concentration-time profiles in plasma (microbiological assay and "C-
`equivalents) and whole blood ("C—equivalents) for the five subjects
`
`+ Plasma - C14 -o- Plasma - bioassay +Whole blood - C14
`
`14
`
`
`
`
`
` Concorequivalents(mcglmL)
`
`Time (hrs)
`
`The reviewer combined the concentration-time data from the five subjects since no appreciable
`differences were noted between subjects receiving 5% dextrose solution and 0.9% saline solution.
`Overall, the mean LY146032 concentration-time profiles determined by microbiologic assay and total l‘C
`were similar in plasma. However, it is unknown if active metabolites of LY146032 are present in plasma
`since no attempt was made to identify metabolites in plasma. The concentrations of LY146032 in whole
`blood were substantially lower than plasma. The mean whole blood/plasma concentration determined by
`total I‘C was 0.53 and ranged from
`—-—-
`Thus, it is likely that LY146032 remains in the
`extracellular compartment due to poor penetration across cell membranes.
`
`The mean cumulative excretion profiles of LY146032 in urine (determined by microbiologic assay and
`total MC) and feces (total I4C) are shown in Figure 3. Approximately 78% of the administered dose was
`recovered based on total HC, whereas approximately 52% of the administered dose was recovered using
`the microbiologic assay. The mean percentage of the administered dose excreted in feces collected for up
`to nine days was 6.1% and ranged from.
`h——-
`
`Although results were not reponedfornrine samples analyzed by r" to identify metabolites,
`the urine concentration of LYI4§O§3 determined with the microbiologic assay was less than the
`concentration based total l‘C. Although this supporLs the presence of inactive metabolites in urine, the
`
`99
`
`

`

`sponsor states that as much as 10% of the administered dose NC activity may have been composed of
`impurities. Thus, the percentage of the administered dose excreted as inactive metabolites in urine may
`be substantially lower than the difference between the cumulative un'ne excretion profiles.
`
`Figure 3. Mean cumulative excretion (°/o) of LY146032 in urine (microbiologic assay and total I‘C)
`and feces (total ”C) in five subjects
`'
`
`
`+Urine - C14
`-0- Urine - bioassay +Feoes - C14
`
`80
`
`
`
`I
`
`'
`
`i
`;
`'i
`
`Cumulative%ofDose
`
`so
`50 I
`40 [r
`.0 It
`'I
`.ll
`
`20
`
`'
`
`10
`
`.
`‘
`
`0
`
`O
`
`24
`
`48
`
`72
`
`96
`
`120
`
`144
`
`168
`
`192
`
`'
`
`Time (hrs)
`
`The phannacokinetic parameter estimates for the three subjects administered drug in 5% dextrose solution
`are shown in Table l. The concentration-time data from the two subjects administered study drug in
`0.9% saline solution were not analyzed by the sponsor.
`'
`
`Table I. Mean (SD) pharmaeokinetic parameter estimates for LY146032 in plasma (total "C and
`micro assay) and whole blood (total HC)
`
`
`
`
`Parameter
`Plasma ~ Micro assay
`=3
`
`
`a/mL
`Cm“
`rm. hrs
`
`Plasma - Total I‘C
`n=3
`
`Whole blood - Total I‘C
`n=3
`
`
`
`
`
`51.7 43
`
`78.] 0.5
`
`78.1 05
`
`
`
`
`
`
`
`
`
`
`
`
`’”\
`
`Based on plasma concentrations determined by microbiologic assay and total 14C, LY146032 has a mean
`apparent volume of distribution that-is less than 9.0 liters and is barely greater than the volume of the
`
`100
`
`

`

`intravascular compartment. The mean total plasma clearance is approximately 0.01 l L/hr/kg (11.7
`mL/min) and is substantially lower than either the renal or hepatic blood flow. The mean renal clearance
`comprises approximately 48% of the total clearance (6.0 mL/min) when determined using the
`microbiologic assay and 75% of‘the total clearance (9.2 mL/min) based on total I"C.
`
`The total I‘C activity of expired air was determined from samples over 8 hrs. The recovery of the I"C
`label from expired air was approximately 3%. The recovery of the 1"'C label from feces was
`approximately 6%. Thus, less than 10% of the administered dose of LY 146032 is eliminated through
`these pathways. The mean non-renal clearance of LY146032 ranged from 5.6 mL/min (determined using
`the microbiological assay) to 6.1 mL/min (based on total l“C). However, the non-renal clearance
`comprises 51% and 24% of the mean total clearance based on the plasma concentrations determined with
`the microbiological assay and total MC, respectively. Thus, the elimination of greater than 10% of the
`administered dose was unaccounted for.
`
`_
`Protein binding
`The plasma protein binding of LY146032 was assessed in one subject over an eight hr period by
`comparing the total disintegrations per min (DPM) from the total sample to the DPMs obtained from only
`the plasma water. The protein binding ranged from 69% to 81% with a mean value of 76%. Although it
`appears that the protein binding decreases with time (and thus concentration), the decline in protein
`binding may be due to the presence of radiolabeled metabolic products and not a decrease in protein
`binding.
`
`Protein bindin_ %
`
`
`
`CONCLUSIONS:
`Following the administration of l“C~1.Y146032, approximately 78%, 6%, and 3% of total ”C was
`eliminated in urine, feces, and expired air, respectively.
`
`Concentrations of LY1446032 were similar in plasma based on microbiological assay and total "C. No
`attempt was made to identify the presence of active metabolites of Ly146032.
`
`Concentrations of LY1446032 were lower in urine based on microbiological assay than total l"C and may
`be due to the presence of inactive metabolites. However, no attempt was made to identify the presence of
`inactive metabolites of LY146032.
`
`._ “u—
`__ ____
`
`101
`
`

`

`COMMENTS:
`
`1. The results from this study should be interpreted with caution since the sponsor did not provide a
`description of the analytical-methods or assay validation data for any of the methods used in this study.
`Thus. the data from this study should be considered supportive of other data reported elsewhere.
`
`2. The Sponsor stated the presence of radiocarbon labeled drug and metabolites in urine was examined
`
`using '
`_
`Using ~ eluant from the column was to be collected in timed
`fractions, which were combined with liquid scintillation fluid and examined in a liquid scintillation
`counter for radiocarbon content. However, the results from these analyses are not reported in the study
`report and it appears that no attempt was made to identify potential metabolites of LY146032 in urine.
`Based on the difference in urine concentrations of LY146032 determined with the microbiological assay
`and tom] l“C, inactive metabolites may be present in urine.
`
`3. The sponsor states that up to "" of the MCI-LYl46032 dose may have been composed of impurities.
`Since the sponsor used a microbiologic assay to determine LY146032 concentrations in plasma and urine,
`the discrepancy between mean urine LY146032 concentrations determined by microbiologic assay and
`total MC may be due to inactive metabolites. However, the actual contribution due to inactive metabolites
`is unknown due to the known presence of MC-containing impurities.
`'
`
`APPEARS THIS WAY
`0N ORIGINAL
`
`_ H,
`._-h.,
`
`102
`
`

`

`A randomized, double~blind, multiple-dose, pharmacokinetic and safety study of ascending dosesd‘
`daptomycin in healthy volunteers (Protocol DAP-OO-OZ)
`
`Dates: June 29, 2000 to August 30, 2000
`Clinical site: .
`" '
`
`
`Analytical site:
`Protein binding:
`
`OBJECTIVES:
`
`f.‘
`
`The objectives of this study were 1) to assess the safety and tolerability of intravenous (IV) dosing of
`daptomycin at 4, 6, 8, and 10 mg/kg q24h in healthy volunteers for periods of either 7 or 14 consecutive
`days, and 2) to assess the pharmacokinetic profile of multiple doses of daptomycin in healthy volunteers
`at doses OH, 6, 8, and 10 mg/kg q24h.
`
`FORMULATION:
`
`Daptomycin 250 mg/l 0 mL vial (lot #8013l0)
`0.9% Sodium Chloride Injection USP, 50 m1. bag (lot #J00929)
`
`STUDY DESIGN:
`
`This study was a single center, double-blind, randomized, multiple-dose study in 32 healthy male and
`female subjects with four dosing cohorts and eight subjects per cohort. Subjects in cohorts l, 2, 3, and 4
`were to receive 4 mg/kg q24h, 6 mg/kg q24h, 8 mg/kg q24h, and 10 mg/ltg q24h, respectively
`administered by IV infusion over 30 min. Within each cohort of eight subjects, six subjects were
`randomized to receive daptornycin and two subjects were to receive control vehicle (0.9% normal saline).
`Subjects in Cohorts 1 and 2 were dosed for seven days, whereas subjects in cohorts 3 and 4 were to be
`closed for 14 days. Subjects were sequentially assigned in groups of eight to receive a given dose, with
`the first eight eligible subjects assigned to receive the lowest dose. However, the study was terminated
`after all eight subjects in cohort 3 completed the study and no subjects were enrolled into Cohort 4 to
`receive 10 mg/kg q24h.
`
`Subjects in cohorts 1 and 2 had plasma and urine sampling performed on Day 1 (24 hrs)_and Days 7-10
`(72 hrs). In addition, plasma trough concentrations were obtained on Days 2-10.
`
`Subjects in cohort 3 had plasma and urine sampling performed on Day I (24 hrs) and Days 7-10 (72 hr
`period). In addition, plasma trough concentrations were obtained on Days 2-10 and 14-17.
`
`Blood samples for 24 hr and 72 hr pharmacokinetic analysis were collected at -30 min (baseline), ~15 min
`(mid-infusion), 0 (end of infusion), and 0.5, 1, 2, 4, 6, 8, 10, I2, and 24 hrs (48 and 72 hrs, if applicable)
`after the end of the infusion.
`
`Blood collection for protein binding determinations was performed on days I, 7, and 14 (if applicable) at
`time 0 (end of infusion), 2, and 8 hrs for all Cohorts. Plasma protein binding was determined using
`equilibrium dialysis against 0.2 M phosphate buffer at 37°C for 3 i 0.5 hrs. All samples were adjusted to
`pH 7.4 using either 1.0 N BC] or 0.1 N NaOH prior to dialysis. The percent unbound (% free) was
`calculated from the ratio of free drug concentration (buffer side) to total drug concentration (serum side)
`multiplied by 100.
`
`Urine samples were collected on Days 1 and 7 during the following intervals: baseline (-30 min), 0 to 2, 2
`to 4, 4 to 6, 6 to 8, 8 to 10, 10 to_12._an5_l 12 to 24 hrs (24 to 48 and 48 to 72 hrs, ifapplicable) based on
`_.._~_,_
`the end of the infusion.
`
`103
`
`

`

`DAPTOMYCIN ASSAY METHODOLOGY:
`
`
`
`LLOQ
`Linearity
`
`Precision
`
`
`__‘E_
`
`3.00 to 500 _./m1.
`3.00 to 500
`-/m1.
`
`
`
`/
`
`
`
`
`Satisfactory
`
`Satisfacto
`
`
`
`-
`Satisfacto
`
`Satisfactory
`
`
`Satisfactory
`
`
`
`Satisfactory
`
`
`
`DATA ANALYSIS:
`
`'
`
`‘
`
`Plasma (total and unbound) and urinary daptomycin concentration data were analyzed by non-
`compartmental PK analysis. The following parameters were determined for plasma total daptomycin
`concentration data:
`the area under the plasma concentration-time curve from zero to the last quantifiable
`concentration (AUC0_,), plasma concentration-time curve from zero to infinity (AUCP), maximum
`plasma concentration (Cm), time of Cm, (Tm), clearance of daptomycin from plasma (CLT), renal
`clearance of daptomycin (CLR), mean residence time (MRT), terminal elimination half-life (ti/2),
`terminal exponential volume of distribution (V2), and volume of distribution at steady-state (Vss). The
`Cm, CLR, CLNR, terminal exponential volume of distribution (V2), and volume ofdistribution at steady-
`state (Vss) were determined for unbound plasma daptomycin concentration data.
`In addition, the fi'action
`of the dose excreted unchanged in the urine (Ae) was determined.
`'
`
`STATISTICAL ANALYSIS:
`
`Pharmacokinetic parameters were summarized as mean, SD, coefficient ofvariation (CV), median,
`minimum, maximum, SD oflog transformed data, geometric mean and 95% confidence interval (CI) for
`each study day in each dosing cohort. ANOVA or non-parametric analyses were performed as
`appropriate.
`
`RESULTS:
`
`The study was terminated after all eight subjects completed Cohort 3. Thus, subjects were not enrolled
`into Cohon 4 because the anticipated clinical development plan includes a dosing range up to 6 mg/kg
`q24h and the sponsor felt that adequate data had been obtained to evaluate the PK profile of once daily
`dosing at or above the anticipated clinical dosing range.
`
`The mean (SD) age, weight. and height for the subjects in Cohorts 1—3 are show: in the table below. The
`age, weight, and height were similar among Cohorts.
`
`
`“mm-
`1
`(4 mE/RE‘,
`2F/4M
`33.4 3.6
`69.2 12.2
`164.5 114
`2 6mg’k-)
`3F/3M
`364 81
`696 11.7
`1651 12.0
`70.4 (4.7
`3 (8 ma/k_)
`3F/3M
`37.5 49
`171.0 7.5
`
`
`
`
`
`
`
`The plasma concentration-time profiles following once-daily administration of daptomycin IV 4 mg/kg
`q24h, 6 mg/kg q24h, and 8 mg/kg q24h for 7 to 14 days are shown in_Figure I.
`.. “7
`
`_~‘.-
`
`104
`
`

`

`Figure 1. Median daptomycin plasma concentrations on Day 1, Day 7, and Day 14 following a 30
`min infusion 0” mg/kg for 7 days, 6 mg/kg for 7 days, or 8 mg/kg for 14 days
`
`-o- Day124m9’k9
`.
`~Cr~ DB! 15'“ng
`—-fi—~ Day 113097“
`
`3'
`
`-e
`9%)-
`if: “Q
`‘ib.
`
`'.
`
`'
`
`120
`m
`so
`
`a,
`40
`
`20
`
`0
`
`o
`59.
`‘-
`D9
`5‘? “K
`.
`-_
`ii]
`
`an
`
`..
`
`E 140
`g 120
`v
`g 100
`0
`so
`E
`.5
`E so
`c
`'9.
`5
`§ 20
`o
`
`40
`
`5
`
`D
`
`—o— Day7:4mg/kg
`--0-— Day 7:6mg/‘kg
`_
`-~¢‘:- DEW-amen
`
`8I.
`
`120
`
`“D
`
`80
`
`60
`
`4o
`
`20
`
`O
`
`—o— Day14:8 rug/kg
`
`
`D D
`4.0
`a 0
`12.0
`16.0
`20.0
`24 0
`
`Time (hours)
`
`3 E8
`
`“
`
`E.uco
`
`Da
`
`:EaL
`
`n
`:1
`
`c EgaI
`
`IDc
`
`.5
`35
`
`The pharmacokinetic parameters following administration of daptomycin to healthy male and female
`subjects are shown in Table 1 (total) and Table 2 (unbound). The pharmacokinetic linearity between Day
`1 and Day 7 as well as accumulation with multiple dosing are shown in Table 3.
`
`The mean tog Cm, AUCM4, and AUCo... increased greater than proportional with increasing dose after
`the lst and 7th dose. In general, the mean V55 and V2 decreased with increasing dose. The mean total
`CLT and CLR decreased with increasing dose after both the lst and 7th dose. The greatest decline
`occurred after the lst dose. Similarly, the half-life was greatest with 8 rug/kg, followed by 6 mg/kg and 4
`mg/kg.
`
`The Day 7/Day l (and Day l4/Day l for 8 mg/kg) total Cm ratios for 4 mg/kg, 6 mg/kg, and 8 mg/kg
`were similar to the predicted accumulation ratio of -—_ whereas the mean AUG”. ratios were greater
`than predicted and ranged from 1.17 to 1.39. The mean total CLT and CL; was similar between Day I
`and Day 7 (or Day 14 for 8 nag/kg) for the 6 mg/kg and 8 mg/kg doses, whereas the mean total CLT and
`CLR decreased between Day 1 andgay 7 since the ratio was less than 1.00.
`—-H—’
`
`105
`
`
`
`
`
`
`
`MedianDaptomycinPlasmaConc(meg/mt)
`
`

`

`Similar to total concentrations, the mean unbound Cmax increased greater than proportional with
`increasing dose after the lst and 7th dose. In general, the mean unbound V55 and V; decreased with
`increasing dose. Although sporadic, the mean unbound CLT, CLR, CLNR decreased with increasing dose
`after both the lst and 7th dose.‘ When a decrease occurred, the degree ofdecline was greater with
`unbound concentrations than total concentrations.
`
`The Day 7/Day l (and Day l4/Day l for 8 mg/kg) unbound CM ratios for 4 mg/kg, 6 mg/kg, and 8
`mg/kg were similar to the predicted accumulation ratio of .
`Although the mean unbound CLT, CLR,
`CLNR ratios ranged from 0.86 to 1.22, no definitive trend was observed and the values were similar to
`1.00.
`
`
`Table 1. Mean (CV%) pharmacokinetic parameters for total daptomycin 4 mg/kg q24h, 6 mg/kg
`q24h, and 8 mg/kg q24h on Days 1 7, and 14
`
`
`
`
`—W
`Dav 7
`Dan
`
`
`
`5%
`
`494
`15%
`
`9%
`
`16%
`
`27%
`
`3%
`
`622
`7%
`9%)
`
`8%
`
`10%
`
`9%
`
`23%
`
`Vz (ng)
`
`
`
`
`
`-
`
`
`
`
`
`
`
`
`
`
`
`
`
`(12%
`
`747
`(12%
`
`i3%
`
`9%
`
`932
`(13%
`14%
`
`(14%
`
`14%
`
`10%
`
`06
`
`10%
`
`111%
`
`1,090
`10%
`
`12%
`
`13%
`
`12%15%
`
`11%10%
`
`6%
`
`6%
`
`15%
`
`20%
`
`12%12% (12%
`
`25%
`
`20%
`
`23%
`
`(15%
`
`19%
`
`11%
`
`16%
`
`13%
`
`(10%
`
`9%
`
`10%
`
`20%
`10%
`24%
`13%
`(10%
`18%
`16%
`—-IIIII”——-
`
`23%
`
`12%
`
`23%
`
`ND- only performed on Day 1
`
`
`
`
`
`
`
`
`106
`
`

`

`Table 2. Mean (CV%) pharmacokinetic parameters for unbound daptomycin 4 mg/kg q24h, 6
`mg/kg q24h, and 8 mg/kg q24h on Days], 7, and 14 (for 8 mg/kg only)
`
`
`
`
`Parameterm Dav l4
`
`Cmu(pg/ml.)
`4.78 58910331163
`16% -
`(11%
`(22%
`8%)
`12%
`11%
`
`(30%
`
`
`
`
`
`(25%
`
`24%
`131.5
`
`(11%
`101.8
`
`40%)
`
`(41%
`
`(27%
`108.8
`
`12%
`
`12%
`81.0
`
`(14%
`78.2
`
`15%
`
`(29%
`
`21%)
`
`(9%
`
`21%
`
`11%
`
`11%)
`
`(13%
`
`53%
`ND - only performed on Day 1
`
`29%
`
`26%
`
`26%
`
`24%
`
`
`wwwmmmmmm
`— (40%
`30%
`28%
`23%
`12%
`15%
`22%
`
`wmmmwmmmm
`(15%
`16%
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Table 3. Ratios of mean pharmacokinetic parameters for total and unbound daptomycin