CENTER FOR DRUG EVALUATION AND RESEARCH
`
`APPLICATION NUMBER: 20-965
`
`PHARMACOLOGY REVIEW(S)
`
`

`

`REVIEW AND EVALUATION OF PHARMACOLOGY/1‘OXICOLOGY DATA:
`
`KEY WORDS:
`Reviewer Name:
`Division Name:
`
`5-aminolevulinic acid, ALA, photodynamic therapy, actinic keratoses
`Amy Nostrandt
`Division of Dermatologic and Dental Drug Products
`HFD# 54o
`Review Completion Date:
`11/19/1999
`
`NOV I 9 |999
`
`Review number:
`2
`IND/NDA number: NDA 20-965
`Serial number/date/type of submission:
`
`resubmission (AZ), response to AE letter, received
`10/4/1999
`
`Information to sponsor: Yes ( ) No (X)
`Sponsor (or agent): Guidelines, Inc. for DUSA Pharmaceuticals, Inc.
`Manufacturer for drug substanced
`5
`
`Drug:
`
`‘
`Code Name:
`Generic Name:
`Trade Name:
`
`S-ALA HCl, S-ALA, ALA
`S-aminolevulinic acid HCl
`Levulan® (aminolevulinic acid HCl) Kerastickm for topical
`solution 20% 1.
`5--aminn-4-oxopentanoic acid
`,,
`7 Chemical Name.
`CAS Registry Number:
`not includedin this submission
`Molecular Formula/ Molecular Weight/Structure:
`(S-aminolevulinate)
`C5H9N03, MW = 131.13
`
`Relevant INDs/NDAs/DMFs:
`1n HFD 150
`
`if
`‘ooc—CHZ-CHZ-c—CHZ—NH;
`
`
`
`in HFD-540:
`INDt
`
`-
`
`)-
`
`’
`-
`for topical photodynamic therapy ofactinic
`keratosis
`
`—~——7r————————-————j
`
`
`
`

`

`NDA 20-965 (AZ)
`
`Review of Pharmacology and Toxicology bita
`
`‘
`
`Page 2 of 4
`
`in HEB-580:
`
`1ND.
`
`Drug Class: photodynamic therapy
`
`Indication:
`
`for the treatment of
`
`)actinic keratoses of the face and scalp
`
`constituted Levulan® (S-ALA HCl)
`
`Clinical formulation:
`Ingredient
`Levulan (S-ALA HCl)
`Alcohol, USP
`Purified water, USP
`Laureth—4
`
`Isopropyl alcohol, USP
`
`Polyethylene glycol -
`
`Ola
`
`
`
`An ampoule containing S-ALA HCl in powder form is broken to
`Route of administration:
`allow admixture with the vehicle to form a 20% solution. The solution is immediately applied
`.. topically to thelesionronly. Aftepgheursnhesite is irradiated with the applicant’s
`companion devrce, a blue light source With an emissmn peak of4.17 nm and a bandw1dth ofl_)
`nm.
`
`7
`
`The current submission is a resubmission of an approvable
`Introduction and drug history:
`NDA. The only changes that impact pharmacology and toxicology are in the label. Those
`changes are addressed below.
`
`OVERALL SUMMARY AND EVALUATION:
`Communication Review:
`- Labeling Review (NBA):
`1. Under “Carcinogenesis, Mu" enesis, Impairment ofFertility,” all references to “5-
`ALA” were changed to read
`by the applicant; References to the drug
`substance, as opposed to the drug product, were changed to ALA HCl, as proposed by the
`chemistry reviewer. For descriptions of genotoxicity studies, which were conducted
`using a solution of 5-ALA in an acetate-Puffer, the wording should be changed back to
`read ALA.
`
`2. The sentence, “PpD( formation was not demonstrated in the in vitro studies” should
`be moved to'a position immediately after the descriptions ofthose studies and “the”
`should be changed to "these.”
`'
`
`3. Other minor changes, such as more concise wording, case corrections, etc., are made
`below.
`'
`
`

`

`NDA 20-965 (AZ)
`
`Review of Pharmacoié‘gy and Toxicology Data
`
`_ Page 3 of 4
`
`RECOMMENDATIONS:
`The following sections should be revised as follows:
`
`to
`
`Fertility: No
`Impairment
`Carcinogenesis, MutageGsis,
`ALA. No evidence of
`carcinogenicity testing has been carried out using (
`mutagenic effects was seen in four studies conducted with ALA to evaluate this
`otential. Ah the Salmonella-Escherichia colil_,'mammalian{:microsome‘.:_:.reverse
`-J_inutation-Wassay (Ames mutagenicity assay), no increases in the number of revertants
`were observed with any of
`the tester strains.
`In the Salmonella-Escherichia
`coliernammaliaCynicrosomeflzreversifinutationThssay in the 5‘ resence of:isolar
`flight Lradiation (AmesClmutagenicitybgassay witfllight).v’
`ALA did not
`cause an increase in the number of revertants per
`late of any of «the tester strains in
`
`the
`resence,r
`bsence of!
`
`
`
`iiowvard
`ymphoma‘fl
`im'ula‘te'd- solar i"ht.
`in the L51787 TK"‘,__J-Eious
`
`.assay.
`V
`REA was evaluated as negative'with and without
`:mutation
`metabolic activation under the study conditions. Pplgg fonnatign wasJQLdemonstrated
`in an 'Of‘th'e‘s‘e' 'in'4WtfiiifiUBies.
`ln theflj’n vivo-
`ilifiouse;:fiicronucleuswl'ass—ay,
`ALA was considered negative under the study exposure conditions.
`.
`in contrast. at least one report in
`théTitErature has noted genotoxic effects in cultured rathepatocytes after ALA exposure
`with PplX formation. Other studies have documented oxidative DNA damage in vivo
`,i7,i,,,,,andjayitm asaresultofALA exposure.
`_
`No assessment of effects of?
`}ALA HCI on fertility has been performed in
`
`.7
`
`- _,
`
`It is unknown what effects systemic exposure to
`laboratory animals.
`HCI might have on fertility or reproductive function.
`
`i ALA
`
`j
`
`Pre nanc Category C: Animal reproduction studies have not been conducted with
`{ALA HCI.
`It is also not known Whether‘LEVULAN'KER'A‘STICK Topical
`Solution can cause fetal harm when administered to a pregnant woman or can affect
`reproductive capacity. LEVULAN KERASTICK Topical Solution should be given to a
`pregnant woman only if cleady needed.
`"‘ ‘
`‘
`
`Nursing Mothers: The leveIsOf ALA or its metabolites in the milk of sub'ects treated
`with LEVULAN KERASTICK To ical Solution h ve not been measured[:
`
`Because many drugs are excreted in
`“ uman milk, caution "sho'UId be exercised when LEVULAN KERASTICK Topical Solution
`is administered to a nursing woman.
`
`
`
`APPEARS nus WAY
`
`0N ORIGINAL
`
`L
`
`i S]
`
`Amy C ostran D.V.M., PhD.
`Ph
`acologist/Toxicolcgist
`
`/’7/2/79
`
`

`

`NDA 20-965 (AZ)
`
`‘ --Review of Pharmacology and Toxicology Data
`
`‘ Page 4 of 4
`
`CC:
`
`NDA 20-965
`HFD-340
`HFD-540
`HFD-540/PHARM/Nostrandt
`HFD-540/TLPHARM/Jacobs
`HFD-S40/MO/Okun
`HFD-540/CHEM/Hathaway
`HFD-540/PMS/Cintron
`C:\word files\nda\n20965rel .doc
`Draft date (# of drafts):
`11/19/99 ( 1)
`
`Concurrence Only:
`HFD-540/DD/WIL
`{(1.191
`ng
`HFD-S40fl‘LPHARM/JACOBflI/lf/V/
`p/{g
`
`mam ms w
`V on ORIGINAL
`
`

`

`Evaluation of Pharmacology and Toxicology Data
`Division of Dermatologic and Dental Drug Products, RFD-540
`
`MAR
`
`8 [999
`
`NDA No.:
`
`20965
`
`Date Submitted:
`
`Date CDER Received:
`
`Date Assigned:
`Date Review Completed:
`
`6/29/98
`
`7/1/98
`
`7/13/98
`3/4/99
`
`Name ofDrug:
`
`Levulan (aminolevulinic acid HCl) Kerastick for topical solution 20%
`
`Structure:
`
`(S-aminolevulinate)
`»
`
`'
`
`C,H,,NO3
`MW 131.13
`
`'OQC—CHz-CHz—C—CHz'NH3+
`
`—-—w--"~A'A?hmmaco’r6gicafeateng‘—plfitbfifiafifié therapy (PDT)
`
`Sponsor:
`
`Guidelines, Inc.
`10320 USA Today Way.
`Miramar, FL 33025
`Attn: Samuel D. Swetland
`954-433-7480
`
`.»
`
`for
`' "
`
`DUSA Pharmaceuticals, Inc.
`400 Columbus Avenue
`Valhalla, NY 10595
`914-747-4300
`
`Indication:
`
`for the treatment of}
`
`Bactinic keratoses 'of the face and scalp
`
`An ampoule containing 5-ALA in powder form is broken to allow
`Administration:
`admixture of the vehicle to form a 20% solution. The solution is immediately applied topically
`to the lesion only. Afien.
`"hours, the site is irradiated with the applicant’s companion
`device, Blu-U, a blue light source withian emission peak of417 nm and a bandwidth oh:
`
`)
`
`Formulation: constituted Levulan (S-ALA HCl)
`In
`ient
`Levulan (S-ALA HCl)
`Alcohol, USP
`
`“theoretical quantity” mg/ml
`
`"
`
`‘
`
`Purified water, USP
`Laureth-4
`
`Isopropyl alcohol, USP
`Polyeth lene 1 col
`55
`
`I
`
`Total
`
`

`

`NDA 20-965
`
`Review of Pharmacology and Toxicology Data .‘.
`
`.
`
`Page 2 of 25
`
`Related IND’s/NDA’s:
`in HFD-ISO:
`
`_ If
`
`mumw '
`
`
`
`Jr
`
`in HFD-540:
`
`IND
`
`) DUSA
`
`MM)
`'
`
`m.-.”
`
`1ND
`
`for topical photodynamic therapy ofactinic
`
`keratosis
`
`
`____\
`_/
`
`x
`
`C
`
`‘ I
`
`ndex of Nonclinical Studies:
`Nonclinical Toxicology studies
`Acute toxicology studies of S-ALA:
`1.
`Acute intravenous toxicity study with S-aminolevulinic acid
`hydrochloride in mice ija-lm)
`- Acute intravenous toxici
`study with S-aminolevulinic acid
`hydrochloride in rats .
`6703-102)
`Acute intravenous toxicity study with S—aminolevulinic acid
`hydrochloride in dogs 1-
`5703-103)
`Acute toxicology studies ofpyrazine 2,5-dipropionic acid:
`4.
`Acute oral toxicity study ofpyrazine 2,5-dipropionic acid in mice
`70104863)
`Acute oral toxicity study ofpyrazine 2,5.dipropionic acid in rats
`)1
`‘ 170104862)
`Acute intraperitoneal toxicity study ofpyrazine 2,5-dipropionic
`acid in micei’
`i70104865)
`Acute intraperito eal toxicity study ofpyrazine 2,5-dipropionic
`acid in rats(
`i70104864)
`Genotoxicity studies:
`»
`8.
`Salmonella - Escherichia coli/mammalian-microsome revels-
`mutation assay with a confirmatory assay (‘
`18554.0-
`409R)
`
`2.,
`
`3.
`
`5.
`
`6.
`
`7.
`
`

`

`NDA 20-935
`
`ReView of Pharmacology and Toxicology Data 4
`
`..
`
`_
`
`Page 3 of 25
`
`9.
`
`10.
`
`Salmonella — Escherichia coli reverse mutation assay in the
`presence of solar light radiation with a confirmatory assay
`51 8554-1-409RSL)
`E
`L5178Y TK +/- mouse lymphoma forward mutation assay with a
`confirmatory assay $318554-0-431R)
`In vivo mouse micronucleus assayD 18554-0-4SSOECD)
`11.
`Special toxicity studies:
`12.
`Acute subcutaneous toxicity study of 5-aminolevulinic acid (ALA)
`in rats it;
`521202228)
`Acute dermal toxicity study of 5-aminolevulinic acid (ALA)
`
`formulations in rabbits (solution)u \6703-100)w
`Acute dermal toxicity study of 5-aminolevulinic acid (ALA)
`formulations in rabbits (creamy
`.|21202229)
`Intravesical stability and absorption of 5-aminolevulinic acid in
`beagle dogs'
`“\6703-105)
`Acute intravesical toxicity study with S-arninolevulinic acid
`hydrochloride in dogs!
`)6703-104)
`Assessment of an intravesical dose administration procedure in
`female dogs,t/V~fi7O3-107)
`l8. Acute intravesical toxicity and toxicokinetic study with 5-
`
`,, 7,4,,
`_ _
`.
`,aminolevulinic acid hydrochloride in dogsC:36703-106)
`Pharmacokinetic studies
`
`13.
`
`14.
`
`15.
`
`16.
`
`17.
`
`1.
`
`Pharmacology and pharmacokinetics of 5-aminolevulinic acid in beagle
`dogs after oral and intravenous administration.
`Nonclinical Pharmacology studies
`
`INTRODUCTION
`
`Arninolevulinic acid (S-ALA, S-ALA, or ALA) is an endogenous precursor in the heme
`synthetic pathway. It is enzymatically converted to protoporphyn'n D( (PplX), which is
`photoactive. The absorption peaks for PpIX are at 405 (largest), 505, 540, 575, and 630 nm.
`Administration of exogenous ALA bypasses the feedback inhibition ofALA synthesis by the
`presence of excess heme. According to the sponsor, ALA and PpD( are selectively accumulated
`in epithelial tissues. It has been reported in the literature that PpIX accumulates in some tumor
`tissues that lack the enzyme ferrochelatase, which catalyzes the formation ofheme from PpIX.
`The applicant’s product consists of S-ALA in powder form in an ampoule, which is
`broken to allow admixture of the vehicle to form a 20% solution. Packagin includes an
`applicator tip, which is used to apply material to the lesion only. Afi
`ours, the site is
`irradiated with the applicant’s companion device, Blu-U, a blue light source with an emission
`peak of417 nm and a bandwidth 0Q nm.
`A number of references fiom the scientific literature were supplied by the applicant to
`supplement their nonclinical information. These include review articles related to the clinical
`presentation and pathogenesis ofporphyrias in human patients. Additionally there are research
`articles describing studies in animals to elucidate the mechanisms by which the classical signs of
`porphyria are developed.
`
`

`

`NDA 20-965
`
`Review of Pharmacology and Toxicology Data ,., .
`
`Page 4 of 25
`
`A number of the articles dealt with experimental models of neurotoxicity associated with
`porphyria. In a number of in vitro preparations, ALA was shown to impair neuromuscular signal
`transmission, to inhibit neurotransmitter release at the motor endplate, and to affect membrane
`ion conductance. ALA inhibition ofNa-K-ATPase (with no effect on Mg-ATPase until high
`concentrations are reached) has been demonstrated and shown to be reversible. Effects on
`neuronal membrane electrical resistance appear to be similar to those produced by GABA. In
`fact, competition by ALA has been demonstrated for GABA receptors in CNS at ALA
`concentrations that are consistent with those thought to exist in the CNS of porphyric patients.
`Several researchers speculate that ALA may be a GABA receptor agonist- Low concentrations
`of ALA displaced GABA at specific GABA binding sites; it was concluded that ALA may be
`specific for presynaptic GABA receptors. High ALA concentrations have been shown to
`stimulate GABA release and inhibit GABA uptake. Some studies have shown similar effects on
`glutamic acid. The effects demonstrated in vitro appear to be predominantly prejunctional and
`occur at concentrations that might be reasonably expected in patients with porphyria.
`In vitro studies in liver tissue demonstrated inhibition of liver tryptophan pyrrolase
`activity by ALA, which was thought to possibly lead to alterations in tryptophan and serotonin
`levels. Other studies of effects on transmembrane and mitochondrial membrane potentials in
`liver showed calcium-dependent oxidative damage to the mitochondria at 50-100 uM ALA.
`Those concentrations were estimated to occur in the livers of porphyric patients. Lipid
`peroxidation demonstrated in vitro resulted in increasedpermeability in liposomes and may
`7-»represent the potential for alteredrnembrane permeability in vivo.
`In vivo, ALA has been found in the cerebrospinal fluid of porphyria patients and can
`cross the blood-brain barrier. In a study of motor activity in mice injected ip with 0.76 mong
`ALA, marked depression of spontaneous activity relative to controls was seen in the first five
`minutes afier injection. By 30 minutes after injection, activity was significantly increased
`relative to controls and persisted to at least 90 minutes afier injection. In rats treated with 40
`mg/kg ALA every other day for 2 weeks, there were indicators of oxidative stress in brain.
`ALA in alkaline solution condenses tolform pyrazine 2,5- dipropionic acid (PDPA). One
`article provided by the applicant suggests that alternative forms and autocondensation products
`may serve as the active toxin in porphyrias. Another study determined optimal conditions to
`minimize condensation of ALA forpreparation of a product for clinical use. Those optimal
`conditions were: 0.18 M (3%) ALA, pH 5, 4°C. The authors speculated that it also might be
`important to control urine pH in patients treated with ALA.
`ALA clearance is by glomerular filtration with limited tubular reabsorption. One
`reference stated that,in overwhelmingneuropathy. cases, the maximum plasma concentrations of
`ALA are 9-12 umol/L.
`According to the applicant, the clinical trials submitted to this NDA demonstrate 80-90%
`complete response after a single use and 90-1100% complete response rate after the second use.
`The human dosers estimated to be 7-15 mg Levulan per patient, or approximately 2-4% of the
`estimated 358 mg ALA synthesized daily by the human body.
`
`APPEARS THIS WAY
`0” ORIGINAL
`
`

`

`NDA 20-965
`
`Review of Pharmacology and Toxicology Data V
`
`,. .
`
`Page 5 of 25
`
`NONCLINICAL TOXICOLOGY STUDIES
`Acute toxicology studies of S-ALA:
`1. Study title:
`Acute intravenous toxicity study with S-aminolevulinic acid hydrochloride
`in mice
`56703-101
`
`1
`Study number:
`Performing organization:
`Drug lot and batch:
`lot # L2260-401-007
`Date of study:
`9/4/96 — 9/24/96
`GLP compliance:
`yes
`Study design:
`single intravenous bolus into lateral tail vein on day 1
`Dosing:
`Dose mugs: 10 ml/kg ofvehicle, 30, 100, or 300 mg/kg ALA (90, 300, 900 mg/m’, or human
`equivalent doses of 2.5, 8, or 25 mg/kg)
`Formulation: S-ALA in 0.2 M sodium acetate buffer; final pH was approximately 5.
`Test animals: Crl:CD-l°(ICR)BR VAF/Plus' mice, 10 /sex/group, 6 weeks old at the initiation
`of treatment, 20-29 g. Animals were observed for clinical signs at 1, 2, and 4 hours post-dose on
`the day oftreatment and at least once daily thereafter. The mice were kept under low lighting for
`the first two days after treatment. Body weight and feed consumption were recorded weekly.
`Blood samples were collected on day 16 prior to sacrifice; samples from 5/sex /group were used
`for hematological evaluation, and samples from 5/sex/group were used for abbreviated clinical
`mufflghemistry evaluation (glucose, BUN, total protein, albumin, globulin, ALT).
`Findings:
`Deaths:
`No signs were observed that were related to treatment. No significant
`Clinical signs:
`effect was noted in body weight, body weight gains, or food consumption.
`Clinical chemisfl and hematology: No effects were seen on hematological or examined serum
`chemistry parameters.
`All animals were examined macroscopicallly, and macroscopic
`Pathological examination:
`lesions identified at necropsy were examined microscopically. No effect ofthe test material was
`noted.
`
`none
`
`The NOEL was determined to be 300 mg/kg (900 mg/m’, RED = 25 mg/kg).
`
`2. Study title:
`
`Acute intravenous toxicity study with S-aminolevulinic acid hydrochloride
`in rats
`
`6703-102
`
`Study number:
`Performing organization:
`Drug lot and batch:
`lot # L2260-401-007
`Date of study:
`9/9/96 — 9/25/96
`GLP compliance: ‘
`yes
`Study design:
`single intravenous bolus of 10 ml/kg into lateral tail vein on day 1
`Dosing:
`Dose ggoups: vehicle, 30, 100, or 300 m;’kg (0, 180, 600, 1800 mg/m’, or human equivalent
`doses of 0, 5, 16, or 50 mg/kg)
`Formulation: S-ALA in 0.2 M sodium acetate buffer; final pH was approximately 5.
`
`

`

`NDA 20-965
`
`Review of Pharmacology and Toxicology Data .
`
`,_
`
`-
`
`Page 6 of 25
`
`Test animals: Crl:CD°(SD)BR VAF/Plus' rats, 6 weeks of age (150-209 g) at the beginning of
`treatment, 5 /sex/group. The rats were observed for clinical signs at 1, 2, and 4 hours post-dose
`- on the day of treatment and at least once daily thereafter. Animals were kept under low lighting
`for the first 2 days after treatment. Body weight and feed consumption were recorded weekly.
`Blood and urine samples were collected on days 3 and 17. Animals were sacrificed on day 17,
`followed by gross necropsy and histopathological examination ofmacroscopic lesions.
`Findings:
`One male at 100 mg/kg died after blood sample collection on day 3. There was
`Deaths:
`no evidence that this was related to treatment.
`Clinical signs:
`No clinical signs were noted; no test material-related differences were
`noted in body weight, body weight gains, or food consumption.
`Clinical chemisfl and hematology: No test material-related differences were noted in results of
`hematology, serum chemistry, or urinalysis.
`Pathological examination:
`No test material related differences were noted.
`The NOEL was determined to be 300 mg/kg (1800 mg/m’, HED=50 mg/kg).
`
`3. Study title:
`
`Acute intravenous toxicity study with 5-aminolevulinic acid hydrochloride
`in do 5
`
`703-103
`
`‘)
`
`Study number:
`Performing organization: (
`Drug lot and hamlet it L2260401-007
`Date of study:
`8/28/96 — 9/16/96
`GLP compliance:
`yes
`Study design:
`single intravenous bolus of 5 mllkg into a cephalic vein on day 1
`Dosing:
`Dose goups: vehicle, 10, 30, or 100 mg/kg (200, 600, 2000 mg/m’, or human equivalent doses
`ofS, 15, or 50 mg/kg)
`Formulation: 5-ALA in 0.2 M sodium acetate buffer; final pH was approximately 5.
`Test animals: beagles, 3/sex/group, observed for clinical signs at 1 hour post-dose on the day of
`treatment and at least once daily thereafier. The animals were kept under low lighting for the
`first 2 days after treatment. Body weights and feed consumption were recorded weekly. Blood
`and urine samples collected twice before dosing and on days 3 and 19. During week 3, the
`animals were sacrificed and necropsied. Livers and tissues with macroscopic lesions were
`examined histologically.
`Findings:
`.
`’
`Deaths:
`Excessive salivation was noted during dosing of 16/18 ALA-treated
`Clinical signs:
`animals and 1/6 vehicle-treated animals. One male at 10 mg/kg and one female at 30 mg/kg
`vomited within 1 hour of dosing. 14/18 ALA-treated animals were noted to have vomited within
`first 24 hours, including all high dose animals. No treatment-related effects were seen on body
`weight or food consumption.
`Clinical chemistry and hematology: Significant increases were seen in mean AST and mean
`ALT on day 3 in females at 100 nag/kg (Individual values for the animals in this group were
`within normal limits with the exception of 1/3 animals for AST and with the exception of 2/3
`
`none
`
`

`

`NDA 20-965
`
`Review of Pharmacology and Toxicology Data ...
`
`r
`
`Page 7 of 25
`
`animals for ALT). Additionally, one male at 30 mg/kg and one male at 100 mg/kg had increases
`in serum ALT. By day 19, AST values in treated animals were essentially recovered; mean
`values for mid and high dose femaleswere statistically significantly different from controls, but
`the differences were not biologically significant. Mean ALT values were not significantly
`different fiom controls at day 19, but the individual value for one high dose female was slightly
`greater than normal.
`No treatment-related changes were seen either on gross
`Pathological examination:
`examination or on microscopic examination of the liver and gross lesions.
`The NOEL was not determined, but findings were limited to salivation and emesis at 10
`mg/kg (200 mg/m’, HED 5 rug/kg).
`
`Included in the submission were a number ofjournal articles, including one authored by
`Kennedy et al. (Food Cosmet. Toxicol. 14:45-47, 1976) in which the subacute toxicity ofup to
`100 mg/kg (300 mg/m’, HED=8 mg/kg) S-ALA injected ip three times per week for 13 weeks
`was assessed. No toxic effects were noted. Fertility was reported to be unaffected, and there was
`no evidence of embryotoxicity or reduced numbers of fetuses. Urinary excretion of ALA was
`evaluated, and a dose-related increase in that parameter was noted.
`
`Acute toxicology studies of pyrazine 2,5-dipropionic acid:
`Reviewer ‘5 comment: None ofthefour studies conducted included an untreated or vehicle
`—#“~~Ieontrolgroupr
`"*Amfi”
`" " “ ’
`,__7,_, '
`4. Study title:
`Acute oral toxicity study of pyrazine 2,5-dipropionic acid in mice
`i
`Study number:
`370104863
`
`Performing organization:
`l
`Drug lot and batch:
`lot # RD 85/229
`Date of study:
`4/18/97 - 6/3/97
`GLP compliance:
`yes
`Study design:
`single dose by gavage of 10 or 15 ml/kg '
`Dosing:
`range—finding phase — 500, 1000, 3000, 5000 mg/kg; definitive study - 5000
`Dose groups:
`mg/kg (15,000 mg/mz, HED= 417 mg/kg)
`_
`Formulation: pyrazine 2,5-dipropionic acid in 0.5% carboxymethylcellulose (w/v) in distilled
`water
`
`_.>
`
`Test animals: Crl:CD°-I(ICR)BR mice, 4-6 weeks of age, 22-29 g; llsex/group for the range-
`finding phase and 5/seX' for the definitive study. Animals were observed at 1, 2.5 and 4 hours
`after test article administration and once daily thereafier. Body weights were determined weekly.
`Animals in the definitive study were sacrificed and subjected to an abbreviated gross necropsy on
`day 14.
`‘
`Findings:
`Deaths:
`
`‘
`none
`
`In the definitive study, soft stools were noted in 4/5 males and 2/5 females
`Clinical signs:
`on the day of treatment. Another female exhibited hypoactivity and staggered gait on the day of
`treatment. Body weight gain was seen throughout the study.
`.
`Pathological examination:
`No lesions were noted on gross examination.
`
`

`

`NDA 20-965
`
`Review of Pharmacology and Toxicology Data ,.. .
`
`Page 8 or'és
`
`The LD,o was determined to be >5000 mg/kg.
`
`Acute oral toxicity study ofpyrazine 2,5;dipropionic acid in rats
`l7010486
`
`5. Study title:
`(
`Study number:
`Performing organization:
`Drug lot and batch:
`lot # RD 85/229
`Date of study:
`4/18/97 — 6/3/97
`GLP compliance:
`yes
`Study design:
`single dose by gavage of 10 or 15 ml/kg
`Dosing:
`range-finding phase — 500, 1000, 3000, 5000 mg/kg; definitive study — 5000
`Dose groups:
`rug/kg (30,000 mg/mz, HED= 833 mg/kg)
`Formulation: pyrazine 2,5-dipropionic acid in 0.5% carboxymethylcellulose (w/v) in distilled
`water
`
`Test animals: Crl:CD°(SD)BR rats, 9-13weeks ofage, 220-300 g; 1/sex/group for the range-
`finding phase and S/sex for the definitive study. Animals were observed at l, 2.5 and 4 hours
`after test article administration and once daily thereafier. Body weights were determined weekly.
`Animals in the definitive study were sacrificed and subjected to an abbreviated gross necropsy on
`day 14.
`Findings:
`am new
`Red-stained face, sofi stool, and dark- or yellow-stained urogenital areas
`Clinical signs:
`were noted, but all animals were normal in appearance by day 3. Body weight gain was seen in
`all treated animals throughout the study.
`Pathological examination:
`No lesions were noted on gross examination.
`The LD,o was determined to'be >5000 mg/kg.
`
`
`
`6. Study title:
`
`Acute intraperitoneal toxicity study ofpyrazine 2,5-diprOpionic acid in
`mice
`'
`)70104865
`
`[
`Study number:
`Performing organization:
`Drug lot and batch:
`lot # RD 85/229
`Date of study:
`4/18/97 — 6/3/97 _
`GLP compliance:
`yes
`Study design:
`single intraperitoneal injection of 10 ml/kg
`Dosing:
`range-finding phase - 100, 250, 500, 1000 mg/kg; definitive study — 1000 mg/kg
`Dose groups:
`Formulation: pyrazine 2,5-dipropionic acid-in 0.5% carboxymethylcellulose (w/v) in distilled
`water
`~
`.
`Test animals: Crl:CD°-1(ICR)BR mice, 4~6~weeks of age, 21-29 g; I/sex/group for the range-
`finding phase and 5/sex for the definitive study. Animals were observed at 0.5, l and 4 hours
`after test article administration and once daily thereafier. Body weights were determined weekly.
`Animals in the definitive study were sacrificed and subjected to an abbreviated gross necropsy on
`day 14. Gross lesions were collected for possible microscopic examination.
`
`

`

`NDA 20-965
`
`I_-
`Review of Pharmacology and Toxicology Data ,. -
`
`Page 9 of 25
`
`Findings:
`Deaths:
`
`none
`
`Hypoactivity was noted in 4/5 males and 5/5 females on the day of
`Clinical signs:
`treatment. Body weight gains were seen throughout the study.
`the right median and
`Pathological examination:
`One gross lesion was seen in one female:
`lateral lobes of the liver were adhered with multiple gray fibrous adhesions that were thought to
`be due to inflammatory reaction to the test material in the peritoneal cavity.
`The LD,o was determined to be >1000 mg/kg.
`
`7. Study title:
`
`Acute intraperitoneal toxicity study of pyrazine 2,5-dipropionic acid in
`rats
`
`770104864
`
`2
`
`‘i
`Study number:
`Performing organization: t
`Drug lot and batch:
`lot # RD 85/229
`Date of study:
`4/18/97 - 6/3/97
`GLP compliance:
`yes
`Study design:
`Dosing:
`single intraperitoneal injection of 10 ml/kg
`Dose groups:
`range-finding phase— 100, 250, 500, 1000 mg/kg; definitive study -— 1000 mg/kg
`Formulation: pyrazine 2,5-dipropionicacidin 0.5% carboxymethylcellulose (w/v)1n distilled
`water
`est animals. Crl:CD”(SD)BR rats, 9-13 weeks ofage 230-300 g; l/sex/group for the range-
`finding phase and 5/sex for the definitive study. Animals were observed at 0.5,] and 4 hours
`after test article administration and once daily thereafier. Body weights were determined weekly.
`Animals in the definitive study were sacrificed and subjected to an abbreviated gross necropsy on
`day 14. Gross lesions werecollected for possible microscopic examination.
`Findings:
`‘
`-
`,
`-
`.
`_
`_
`Deaths:
`none
`
`- T
`
`On the day of treatment, signs were observed in 2/5 males and 4/5 females
`Qlinical signs:
`consisting of hypoactivity, staggered gait, red-stained face, sofi stool, and wet urogenital area.
`All animals exhibited body weight gain during the study.
`
`Patholocdcal examination:
`0n gross examination, lesions were seen in 2/5 males and 5/5
`females, consisting of multiple grayrfibrous adhesions between the spleen, stomach serosa,
`diaphragm, or liver, and between some lobes of the liver. The splenic capsules were described as
`opaque and some liver lobeswere found to be irregularly shaped, possibly because of adhesions
`to the capsular surface. All findings werethou’ght to be due to inflammatory reaction to the
`material injected into the peritoneal cavity.
`The LD,0 was determined to be >1000 mg/kg.
`
`Genotoxicity studies:
`8. Study title:
`
`Study number:
`(
`Performing organization:
`
`Salmonella - Escherichia coli/mammalian-microsome reverse mutation
`assay with a confirmatory assay
`'
`18554-0-409R
`
`

`

`NDA 20-965
`
`Review of Pharmacology and Toxicology Data .
`
`.,
`
`. Page 10 of 25
`
`Drug lot and batch: Batch # 335
`Date of study:
`5/21/97 — 6/19/97
`GLP compliance:
`yes
`Study design:
`Dose groups: A dose range-finding study was performed using tester strains TA100 and
`WP2uvrA and 10 doses ranging from
`pg/plate, one plate/dose in the presence and
`absence of 89. For the initial mutageniCity assay, doses of 33.3, 100, 333, 1000, 3330, and 5000
`ug/plate, 3 plates/dose in the presence and absence of S9 were used. For the confirmatory assay,
`doses of 33.5, 100, 335, 1000, 3350, and 5020 rig/plate in the presence and absence of S9 were
`used. Each assay included appropriate vehicle and positive controls. The positive control in
`presence of 89 was 2-aminoanthracene, and the positive control in absence of S9 was 2-
`nitrofluorene (TA98), sodium azide (TA100, TA1535), ICR-l9l (TA1537), or 4-nitroquinoline-
`N-oxide (WPZuvrA).
`Formulation: 5-ALA in 0.2 M acetate buffer
`Test strains:
`S. typhimurium TA98, TA100, TA1535, TA1537; E. coli WP2uvrA
`Findings:
`Under the conditions ofthe assay, S-ALA did not cause a positive increase in the number
`of revertants per plate in any strain in the presence or absence of S9.
`
`Salmonella - Escherichia coli reverse mutation assay in the presence of
`, ~ reisolariightradiation with a confirmatory assay
`8554~l~409RSL
`
`9. Study title:
`signifier
`Study number:
`Performing organization: i
`Drug lot and batch: Batch # 335
`Date of study:
`7/17/99 — 8/5/99
`GLP compliance:
`yes
`Study design:
`Dose groups: Doses were 3.33, 10.0, 33.3, 100, 333, 1000, 3330, and 5000 pg/plate for the
`initial mutagenicity assay (Reviewer ’5' comment: Analysis ofsolutions usedfor dosing in the
`initial assay resulted in no detectable 5-ALA in samples ofthe three lowest concentrations.) and
`100, 333, 1000, 3330, and 5000 ug/plate for— the confirmatory assay. Doses of test article and
`vehicle controls were administered to cultures in the absence of light and in the presence of 2
`solar light doses (the first was that which was demonstrated to result in a 2-fold or greater
`increase in the number of revertants/plate, the second dose was half of the first; 8 and 4 seconds
`for TA1537, 16 and 8 seconds for TA102 and WP2(pKM101)) and absence of directed solar
`light radiation. The vehicle for S-ALA was 0.2M acetate buffer, pH 5.0, and the positive control
`was 8-methoxypsoralen in DMSO. Additional positive controls included in non-light exposed
`groups only were mitomycin C (TA102), ICR-l9l (TA1537), and 4anitroquinoline-N-oxide
`(WP2(pKM101)). -'
`Formulation: S-ALA in 0.2 M acetate buffer
`.7
`.
`Test strains:
`S. typhimurium ‘TA102, TA1'537; E. coli WP2(pKM101)
`Reviewer 's comment: The study report .Jescribes in detail the mutations present in these
`bacterial strains and their sensitivity to various mutagens, but does not describe why these
`particular strains were more appropriatefor this assay than thefive strains nonnally
`
`

`

`NDA 20-965
`
`§
`
`Review of Pharmacology and Toxicology Data _ ..
`
`Page 11 of 25
`
`recommendedfor the standardAmes assay. Infact, lack ofpositive response ofTAI537 when
`exposed to 8-methox3psoralen and solar radiation when compared to vehicle control-treated
`TA1537 exposed to solar radiation (This strain is unusually sensitive to UVlight) may indicate
`that this strain is not appropriatefor a photogenotoxicity assay. Also, it is not stated whether or
`not any ofthese bacteria possess metabolic capabilityfor PpIXproduction.
`Findings:
`Under the conditions ofthe assay, there was no demonstration ofmutagenic
`photoproducts of S-ALA.
`,
`Reviewer '3 comment: The assay was done only in the absence ofS9. Some sort ofmetabolic
`activation would have been required to assess thephoto-genotoxicity ofthephotoactive moiety,
`PpIX. However, at sufi'iciently high doses, photoactivation ofPpIXshould result in cytotoxicity.
`Perhaps a lower dose of5-ALA and a lower resultant dose ofthe metabolite, PpIX, in the
`presence ofan appropriate metabolic activating system or low doses ofPpIXonly would have
`been non-cytotoxic and more informative.
`
`10. Study title:
`
`L5178Y TK +/- mouse lymphoma forward mutation assay with a
`confirmatory assay
`’
`18554-0-431R
`.
`Study number:
`Performing organization:—
`Drug lot and batch: batch # 335
`Datepf study;
`-. _-_,5120192;719197
`GLP compliance:
`yes
`Study design:
`Dose groups: The preliminary dose range-finding experiment included 10 treatments ranging
`from
`ug/ml, and vehicle and untreated controls. In the definitive study, two trials were
`performed at doses up to 5000 ug/ml. In the both trials, doses were 78.5, 157, 313, 625, 1250,
`2500, and 5000 ug/ml. Only the six highest concentrations were evaluated. Cells were
`incubated for 4 hours in the presence and absence ofS9 followed by 2-day expression period.
`Vehicle and positive controls (methyl methanesulfonate without S9, methylcholanthrene with
`S9) were included.
`Formulation: S-ALA in 0.2 M sodium acetate buffer, pH 5.0
`Test culture: L5178