Chromosomal damage and micronucleus induction by MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor: Evidence for a non-DNA-reactive mode of action

Mutation Research 782 (2015) 1–8

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Chromosomal damage and micronucleus induction by MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor: Evidence for a non-DNA-reactive mode of action Eiji Yamamura ∗ , Shigeharu Muto, Katsuya Yamada, Yuko Sato, Yumiko Iwase, Yoshifumi Uno Safety Research Laboratories, Research Division, Mitsubishi Tanabe Pharma Corporation, 1-1-1 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan

a r t i c l e

i n f o

Article history: Received 30 October 2014 Received in revised form 23 February 2015 Accepted 28 February 2015 Available online 5 March 2015 Keywords: Genotoxicity Mode of action Chromosomal damage Micronucleus induction Nicotinic acid

a b s t r a c t MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that competes with the binding of the PARP substrate nicotinamide adenine dinucleotide (NAD), is being developed as a neuroprotective agent against acute ischemic stroke. MP-124 increased structural chromosomal aberration in CHL/IU cells, but showed negative results in the bacterial reverse mutation test, and the rat bone marrow micronucleus (MN) and the rat liver unscheduled DNA synthesis tests after the intravenous bolus injection. Thus, MP124 did not appear to be direct-acting mutagen. Since, PARP-1 is a key enzyme in DNA repair, the effect of continuous PARP-1 inhibition by MP-124 was further examined in the rat MN test under 24-h intravenous infusion, and an increase in micronucleated immature erythrocytes (MNIE) was observed. The increase was clearly reduced by co-treatment with nicotinic acid, which resulted in increased intracellular NAD levels. This is consistent with the established activity of MP-124 as a competitive inhibitor of PARP and provides strong evidence that the DNA-damaging effect that leads to the increase in MNIE is a secondary effect of PARP-1 inhibition. This mechanism is expected to result in a threshold for the induction of MNIE by MP-124, and allows for the establishment of a safe margin of exposure for the therapeutic use of MP-124. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear protein in mammals (about 106 molecules/cell) with a long halflife. This enzyme is found in most eukaryotes except for yeast and is responsible for post-translational modification of proteins in response to numerous endogenous or exogenous genotoxic damages [1–5]. It is now recognized that there are 17 genes in humans encoding the protein potentially responsible for synthesizing poly(ADP-ribose), and catalytic activity has been proven in eight cases [6]. Among them, PARP-1 has been recognized for 20 years and makes the majority of poly(ADP-ribose). PARP-1 detects and binds to DNA strand breaks, and catalyzes poly(ADP-ribosyl) ation of histone, DNA repair enzymes (DNA polymerase, DNA ligase, DNA topoisomerase, etc.) and further promotes relaxation of the condensed structure of chromatin to facilitate access of DNA repair enzymes to their substrates [7].

∗ Corresponding author. Tel.: +81 438 52 3562; fax: +81 438 52 3542. E-mail address: [email protected] (E. Yamamura). http://dx.doi.org/10.1016/j.mrgentox.2015.02.006 1383-5718/© 2015 Elsevier B.V. All rights reserved.

Activation of PARP-1 results in the consumption of nicotinamide adenine dinucleotide (NAD), and excessive activation of PARP-1 depletes NAD and ATP. These subsequent changes lead to energy failure in the cell and cell death [8]. This is supported by the finding that PARP-1 knockout mice showed reduced ischemia injury in the middle cerebral artery occlusion (MCAO) model [9,10]. Inhibition of PARP-1 is considered to exert neuroprotective effects, and inhibition is expected to be a beneficial agent for the treatment of acute ischemic stroke (AIS). We recently identified a novel PARP1 inhibitor, MP-124 (an isoquinoline derivative) [11], which acts as a competitive antagonist to NAD and exhibits neuroprotective effects in rodent and monkey models [12,13]. The compound is now being developed as a neuroprotective agent against AIS at the clinical stage. Since PARP-1 is a key enzyme of DNA repair, it was speculated that inhibition of this enzyme might produce a genotoxic event in parallel with the neuroprotective effect. Therefore, we carefully investigated the genotoxicity of MP-124 and effects of PARP-1 inhibition following treatment with MP-124 in vivo, and also considered whether there was a margin of safety between the expected pharmacological effects and potential genotoxic events.

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E. Yamamura et al. / Mutation Research 782 (2015) 1–8

In this paper, we review the results of preclinical genotoxicity assays with MP-124 and additional studies to clarify the mechanism of action, and show evidence for the existence of a threshold for the genotoxic events. 2. Materials and methods 2.1. Chemicals MP-124 was synthesized by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). The test chemical was dissolved in physiological saline (PS, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan). The positive control chemicals and reagents were purchased from the manufacturers: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2, CAS No. 3688-53-7), sodium azide (NaN3 , CAS No. 26628-22-8), 2-aminoanthracene (2AA, CAS No. 613-13-8), mitomycin C (MMC, CAS No. 50-07-7), benzo[a]pyrene (B[a]P, CAS No. 50-32-8) and nimustine (ACNU, CAS No. 55661-38-6) from Wako Pure Chemical Ind.-Ltd. (Osaka, Japan); 9-aminoacridine hydrochloride hydrate (9AA, CAS No. 52417-22-8) and nicotinic acid (NA, CAS No. 59-67-6) from Sigma–Aldrich Co. (St. Louis, MO, USA); cyclophosphamide monohydrate (CP, CAS No. 605519-2) from Wako Pure Chemical Ind.-Ltd. and Sigma–Aldrich Co.; 2-acetylaminofluorene (2-AAF, CAS No. 53-96-3) and dimethylnitrosamine (DMN, CAS No. 62-75-9) from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). 2.2. Cells The Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2 uvrA− were obtained from National Institute of Health Sciences, Japan. CHL/IU cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). 2.3. Animals Male Crj:CD(SD) rats were purchased from Charles River Japan Inc. (Tokyo, Japan) and used at 7–9 weeks of age after acclimatization for 1–3 week. The rats were randomly assigned to each group. Diet and water were given ad libitum, and all animals were housed in air-conditioned circumstances. Animal experiments were performed in accordance with the rules of animal welfare in the testing facility and approved by the ethical committee. 2.4. Standard battery for genotoxicity testing of MP-124 MP-124 was examined with a standard battery of genotoxicity tests in accordance with ICH-S2 guidelines [14,15] as follows: the bacterial reverse mutation test was carried out using strains of S. typhimurium and E. coli in the presence and absence of a metabolic activation system, a cofactor-supplemented post-mitochondrial fraction prepared from the livers of rats treated with a combination of phenobarbital and ␤-naphthoflavone (S9, Oriental Yeast Co. Ltd., Tokyo, Japan) using the pre-incubation method. A test compound was judged to be mutagenic if it produced, in at least one concentration and one strain, an increase equal to twice or more the number of revertant colonies compared with the negative (vehicle) control; the chromosomal aberration test was performed using CHL/IU cells in the presence and absence of a metabolic activation system, and the numbers of cells with chromosomal aberrations in the negative control and test article treatment groups were analyzed by the chi-square test and the Cochran–Armitage trend test for a doserelated increase; and the in vivo micronucleus test was carried out using rats exposed by a single intravenous bolus injection, bone marrow cells were collected at 24 h and 48 h after the dosing, and the numbers of micronucleated immature erythrocytes (MNIME)

per 2000 immature erythrocytes (IME) and the proportions of IME among 500 or 1000 erythrocytes were statistically analyzed by the methods of Kastenbaum and Bowman [16] and Student’s t-test, respectively. Additionally, the in vivo micronucleus test after continuous intravenous infusion with MP-124 was conducted using rats to investigate effects on MNIME after continuous inhibition of PARP-1. MP-124 was administered to rats by 24 h continuous intravenous infusion. On 7 days before the dosing, animals received surgical implantation of catheters into the abdominal vena cava via the femoral vein under anesthesia, and the catheters were kept patent with continuous circulation of PS until the initiation of dosing. Bone marrow cells were collected at 24 h after the termination of dosing, and the numbers of MNIME and the proportion of IME were analyzed as described above. 2.5. Follow-up genotoxicity tests with MP-124 Since a positive finding was obtained in the in vitro chromosomal aberration test, the in vivo unscheduled DNA synthesis test with rat liver cells was carried out using a single intravenous bolus injection according to the OECD guideline [17] as a follow-up assay according to the ICH-S2 guidelines. Hepatocytes were isolated at 2 h and 16 h after dosing, and the results were evaluated with the method described by Butterworth et al. [18] and Mirsalis et al. [19]. In addition, MP-124 and its 9 metabolites were assessed for their genotoxic potential using two in silico prediction systems, DEREK version DfW 9.0.0 and MultiCASE version 1.8.0.0. 2.6. Effect of nicotinic acid on micronucleus induction Male rats were pre-treated with nicotinic acid (NA) at 200 or 600 mg/kg/day or with PS as a reference control by continuous intravenous infusion for 3 days to increase the NAD level in the bone marrow cells (Fig. 1). After the pre-treatment, MP-124 dissolved in PS was administered by intravenous infusion at 1000 mg/kg/day for 24 h together with or without NA, followed by 24-h post treatment with NA or PS. Under the same treatment regimens with NA at 600 mg/kg/day or PS, two known genotoxicants, cyclophosphamide (CP) and nimustine (ACNU), were administered orally once at dose levels of 10 mg/kg and 6 mg/kg, respectively (Fig. 2). Bone marrow cells were collected at 24 h after the termination of MP-124 treatment or the administration of CP or ACNU. The numbers of MNIME were statistically tested by the method of Kastenbaum and Bowman to assess the difference between the reference control group (Group 1) and nicotinic acid treated group (Group 2), MP-124 or CP or ACNU treated group (Group 3), or positive control group (Group 6 in Fig. 1). The proportions of IME were analyzed with Student’s t-test to compare the reference control group (Group 1) with the MP-124 or CP or ACNU treated group (Group 3) or positive control group (Group 6 in Fig. 1). In addition, to assess the effect of NA treatment on the micronucleus induction by MP-124 or two genotoxicants CP or ACNU, Dunnett’s test was applied to the numbers of MNIME to compare Group 3 with MP-124 plus NA treated groups (Group 4 and 5 in Fig. 1), and Student’s t-test was applied to compare Group 3 with the two genotoxicants plus NA treated groups (Group 4 in Fig. 2). 2.7. Toxicokinetics Plasma concentrations of test chemicals were measured with LC–MS/MS (Agilent 1100 series [Agilent Technologies, Inc., California, US] and API 3000 or API 4000 [AB SCIEX, Massachusetts, US], Prominence series [Shimadzu Co., Kyoto, Japan] and API 4000) at 6 and 24 h after the initiation of treatment with MP-124, and at 0.5, 1, 2 and 4 h for CP or 0.25, 0.5, 1 and 2 h for ACNU after the dosing.

E. Yamamura et al. / Mutation Research 782 (2015) 1–8

3

Operation for infusion -7

1

2

3

4

5

6

Day

PS

Group 1 (n = 6)

NA (600 mg/kg/day)

Group 2 (n = 6)

MP-124 (1000 mg/kg/day)

PS

NA (200 mg/kg/day)

NA/MP-124

NA

Group 5 (n = 8)

NA (600 mg/kg/day)

NA/MP-124

NA

Group 6 (n = 6)

Not operated

CP 20 mg/kg, p.o.

Group 3 (n = 8)

PS

Group 4 (n = 8)

Sacrifice (sampling of bone marrow cells)

Fig. 1. Study design of investigational study for the mechanism of action of MP-124.

Operation for infusion -7

1

Group 1 (n = 6)

2

4

5

6

Day

PS

Group 2 (n = 6)

NA (600 mg/kg/day)

Group 3 (n = 8)

PS

Group 4 (n = 8)

3

NA (600 mg/kg/day)

Sacrifice (Sampling of bone marrow cells) CP or ACNU p.o. CP or ACNU p.o.

Fig. 2. Study design of supplemental studies for the mechanism of action of MP-124.

2.8. Measurement of intracellular NAD levels in bone marrow Bone marrow cells were acidified with ice cold 3.3% percholic acid (Sigma–Aldrich Co.) for 30 min. After centrifugation, acidic supernatant was neutralized with 1 mol/L KOH in 0.03 mol/L potassium phosphate buffer. Then the precipitate was discarded after centrifugation and the supernatant was used for NAD+ analysis. Both samples and NAD+ standards were subjected to an enzyme cycling assay as described by Nisselbaum and Green [20] with slight modifications by using WST-8 (Dojindo laboratories, Kumamoto, Japan) instead of 3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl-2Htetrazolium bromide (MTT), and the absorbance of the colorometric product was measured at 450 nm with a microplate reader (Molecular Devices, USA). 3. Results 3.1. Standard battery for genotoxicity testing with MP-124 The reverse mutation test showed no increase in the number of revertant colonies up to 5000 ␮g/plate in any strains either in the absence or presence of S9 mix (Table 1). In the chromosomal aberration test using CHL/IU cells, a statistically significant

increase in the incidence of structural aberrations was noted at the following concentrations in the short-term treatment for 6 h: 200 (0.698), 400 (1.397) and 800 (2.794) ␮g/mL (mmol/L) in the absence of metabolic activation, and 400 (1.397) and 800 (2.794) ␮g/mL (mmol/L) in the presence of metabolic activation, and the increase was dose-related (Table 2). No significant increase in the incidence of cells with structural aberrations was noted in the continuous treatment for 24 h. There was no increase in the incidence of cells with numerical aberrations under any treatment conditions. In the bone marrow micronucleus test, MP-124 was administered to rats by a single intravenous bolus injection to the highest dose of 30 mg/kg, which was considered as the maximum tolerated dose (MTD) because animals in this dose group clearly showed toxic signs as hyperactivity, tachypnea and/or staggering gait, and animals died at 60 mg/kg in an independent study. No significant increase in the number of MNIME was noted in any test article groups when compared with the negative control group (Table 3). There was no significant difference in the ratio of IME to total erythrocytes in any test article groups when compared to the negative control group. MP-124 was further examined with the bone marrow micronucleus test using 24-h continuous intravenous infusion, which is the treatment intended as the clinical use of this compound. The

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E. Yamamura et al. / Mutation Research 782 (2015) 1–8

Table 1 Reverse mutation test of MP-124 in bacteria. Chemical

Dose

No. of revertants per platea

(␮g/plate)

TA100

TA1535

WP2 uvrA-

TA98

TA1537

−

0 78.1 156 313 625 1250 2500 5000 0.01 0.02 0.1 0.5 80

104 ± 3 NT 100 ± 6 116 ± 5 124 ± 3 102 ± 6 101 ± 0 61 ± 5b 493 ± 12 NT NT NT NT

12 ± 2 15 ± 1 15 ± 3 17 ± 4 18 ± 1 19 ± 3 10 ± 1b 0 ± 0b NT NT NT 353 ± 25 NT

23 ± 3 NT 22 ± 1 21 ± 4 21 ± 6 21 ± 3 17 ± 2 18 ± 2 NT 357 ± 7 NT NT NT

24 ± 1 NT 18 ± 4 18 ± 4 19 ± 2 16 ± 3 15 ± 1 8 ± 3b NT NT 432 ± 19 NT NT

6±2 6±2 4±1 7±3 6±4 8±1 3 ± 1b 0 ± 0b NT NT NT NT 604 ± 27

+

0 156 313 625 1250 2500 5000 0.5 1 2 10

119 ± 6 118 ± 6 122 ± 5 131 ± 6 125 ± 2 127 ± 6 135 ± 8 NT 1165 ± 42 NT NT

11 ± 2 15 ± 4 17 ± 2 17 ± 4 18 ± 2 16 ± 4 18 ± 2 NT NT 318 ± 12 NT

21 ± 1 23 ± 5 19 ± 1 23 ± 6 21 ± 1 18 ± 2 19 ± 1 NT NT NT 131 ± 8

28 ± 4 25 ± 3 20 ± 1 24 ± 3 24 ± 4 21±1 19 ± 4 773 ± 24 NT NT NT

10 ± 2 9±2 11 ± 3 9±1 8±2 11 ± 1 9±1 NT NT 262 ± 12 NT

S9 mix

PS MP-124

AF-2

NaN3 9AA PS MP-124

2AA

PS, physiological saline; AF-2, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; NaN3 , sodium azide; 9AA, 9-aminoacridine hydrochloride hydrate; 2AA, 2-aminoanthracene; NT, not tested. a Mean ± SD from three plates. b Antibacterial activity was noted.

highest dose of 1000 mg/kg/day was selected as MTD based on the independent study results, i.e., rats died at 1500 mg/kg/day, but not at 1000 mg/kg/day. A statistically significant increase in the number of MNIME was noted at 1000 mg/kg/day but not at 250 and 500 mg/kg/day (Table 4). No significant decrease in the IME%

was noted in any dose groups. The toxicokinetics was examined separately under the same experimental conditions (Table 5). The increase in systemic exposure was proportional across the dose range. The plasma concentration of MP-124 reached a steady state level generally by 6 h after the start of infusion. The concentration

Table 2 Chromosomal aberration test of MP-124 using CHL/IU cells. Chemical

S9 mix

Timea (h)

Concentration (␮g/mL)b

Relative cell count (%)

Mitotic index (%)

No. of cells observedc

Cells with structural aberrations excluding gaps (%)

Cells with numerical aberrations (%)d

PS MP-124

−

6–18

0 50 (0.175) 100 (0.349) 200 (0.698) 400 (1.397) 800 (2.794) 0.15

100 91.8 91.8 72.6 63 46.6 83.6

100 101.5 96.9 75.4 44.6 18.5 43.1

200 200 200 200 200 200 200

0.5 1 3 4.5* 9.5* 37.0* 25.5

0.0 0.0 0.5 0.5 0.5 0.0 0.0

+

6–18

0 50 (0.175) 100 (0.349) 200 (0.698) 400 (1.397) 800 (2.794) 20

100 94.4 92.1 77.5 70.8 49.4 69.7

100 100 87.3 78.4 52 44.1 42.4

200 200 200 200 200 200 200

0.5 0 1 1.5 4.0* 16.0* 25.5

0.0 0.5 0.5 0.5 0.5 1.5 0.0

−

24–0

0 12.5 (0.044) 25 (0.087) 50 (0.175) 100 (0.349) 200 (0.698) 0.05

100 96.7 91.1 73.3 53.3 16.7 38.9

100 79.7 71.9 26.6 3.1 1.6 71.9

200 200 200 200 TOX TOX 200

0.5 1 3 3.5 – – 19.5

0.0 0.5 1 0.5 – – 0.0

MMC PS MP-124

B[a]P PS MP-124

MMC

PS, physiological saline; MMC, mitomycin C; B[a]P, benzo[a]pyrene; TOX, not observed due to cytotoxicity. a Treatment-recovery time. b Values in parentheses represent millimolar concentration. c One hundred cells were observed from each of 2 plates. d Numerical aberrations include polyploidy and endoreduplication. * Significantly different from negative control (p < 0.05, chi-square test).

E. Yamamura et al. / Mutation Research 782 (2015) 1–8

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Table 3 Micronucleus test of MP-124 following i.v. bolus injection to male rats. Chemical

Timea (h)

No. of animals (%)

Dose (mg/kg)

MNIMEb (%)

IMEc

PS

24 48 24 48 24 48 24 48 24

0 0 7.5 7.5 15 15 30 30 20

6 6 6 6 6 6 6 6 6

0.13 ± 0.07 0.14 ± 0.08 0.17 ± 0.08 0.14 ± 0.09 0.13 ± 0.06 0.17 ± 0.08 0.22 ± 0.09 0.22 ± 0.12 2.47 ± 0.68*

50.10 ± 2.20 46.58 ± 3.32 48.40 ± 4.70 45.05 ± 4.87 46.78 ± 2.97 45.32 ± 4.00 49.23 ± 4.42 45.86 ± 3.44 36.93 ± 1.24#

MP-124 MP-124 MP-124 CP

PS, physiological saline; CP, cyclophosphamide monohydrate. a Sampling time after the termination of treatment. b Incidence of micronucleated immature erythrocytes. c Ratio of immature erythrocytes to total erythrocytes. * Significantly different from negative control (p < 0.05, Kastenbaum and Bowman’s method). # Significantly different from negative control (p < 0.05, Student’s t test). Table 4 Micronucleus test of MP-124 following 24-h continuous i.v. infusion to male rats. Chemical

Timea (h)

No. of animals (%)

Dose (mg/kg)

MNIMEb (%)

IMEc

PS MP-124 MP-124 MP-124 CP

24 24 24 24 24

0 250 500 1000 20

6 6 6 6 6

0.18 ± 0.04 0.22 ± 0.11 0.28 ± 0.17 1.03 ± 0.32* 2.03 ± 0.37*

40.40 ± 1.72 41.13 ± 2.78 39.33 ± 2.21 38.40 ± 2.67 32.62 ± 2.80#

PS, physiological saline; CP, cyclophosphamide monohydrate. a Sampling time after the termination of treatment. b Incidence of micronucleated immature erythrocytes. c Ratio of immature erythrocytes to total erythrocytes. * Significantly different from negative control (p < 0.05, Kastenbaum and Bowman’s method). # Significantly different from negative control (p < 0.01, Student’s t test). Table 5 Plasma concentrations of MP-124 during 24 h of i.v. infusion to male rats. Dose (mg/kg/day)

Number of animals

1000 1500

4 4

a b

Mean concentrations (ng/mL)

Concentration at steady state (ng/mL)

1h

3h

6h

24 h

3424 8159

5002 9214a

5868 12087a

6905 16164b

6386 14098

Mean of 3 animals because one animal died. Mean of 2 animal because two animals died.

at steady state at 1000 mg/kg/day was approximately 6400 ng/mL. Based on this value, the level at 250 mg/kg/day was estimated to be about 1500 ng/mL. 3.2. Follow-up genotoxicity tests with MP-124 The in vivo UDS test in rat hepatocytes was conducted by a single intravenous bolus injection with the highest dose of 30 mg/kg. The number of net grains and the incidence of cells in repair in the MP-124-treated groups did not differ from those in negative control groups (Table 6). The structural alerts in chemical structure of MP-124 and all metabolites identified in the in vitro and/or in vivo test systems were examined with the in silico genotoxicity analyses using DEREK (version DfW 9.0.0) and MultiCASE (version 1.8.0.0), and there were no alerts for genotoxicity in DEREK and no biophores for bacterial mutagenicity in MultiCASE (data not shown).

MP-124 at 1000 mg/kg/day significantly increased the incidence of MNIME compared to the reference control. In contrast, this increase was significantly suppressed by the co-treatment with NA at 200 mg/kg/day and 600 mg/kg/day (p < 0.01, Dunnet’s test) (Table 7-2, Fig. 3). Co-treatment with NA did not affect the plasma concentration of MP-124. In two separate micronucleus tests using the known DNA-reactive genotoxicants CP and ACNU (Table 8), oral administration of both chemicals significantly increased the incidence of MNIME compared to the vehicle control. The increases were not reduced by co-treatment with NA, while the amount of NAD in the bone marrow cells of rats treated with NA was approximately 2-fold higher than that of the reference control group. Interestingly, micronucleus induction by ACNU increased slightly but significantly under the condition of co-treatment with NA. NA treatment had no or little effect, if any, on the plasma exposure levels of CP and ACNU. 4. Discussion

3.3. Effects of nicotinic acid on micronucleus induction The amount of NAD in the bone marrow cells of rats treated with 200 or 600 mg/kg/day of NA was approximately 2-fold higher than that of the non-treated rats (Tables 7-1 and 7-2). NA did not increase the incidence of MNIME at 600 mg/kg/day (Table 7-1).

Based on the results of standard battery of genotoxicity testing and follow-up genotoxicity studies, MP-124 and its metabolites themselves are considered to lack direct DNA-reactive genotoxicity. Although MP-124 induced structural chromosomal aberrations in CHL/IU cells at 200 ␮g/mL or more in the short-term treatment

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E. Yamamura et al. / Mutation Research 782 (2015) 1–8

Table 6 In vivo UDS test of MP-124 by i.v. bolus injection using male rat hepatocytes. Chemical

Dose (mg/kg)

No. of animals (%)

Cell viabilitya (%)

Net grainsb (%)

Incidence of cells in repairb

2-h time point PS PS MP-124 DMN

0 15 30 20

3 3 3 3

89.8 ± 2.0 89.8 ± 2.0 85.8 ± 2.4 80.9 ± 1.7

−0.67 ± 0.47 −0.90 ± 0.08 −0.48 ± 0.23 15.82 ± 0.48

3.7 ± 2.1 3.3 ± 0.6 7.3 ± 1.5 97.3 ± 1.2

16-h time point PS MP-124 MP-124 2-AAF

0 15 30 20

3 3 3 3

77.3 ± 2.1 76.9 ± 2.4 75.6 ± 3.6 69.3 ± 4.5

−0.73 ± 0.22 −0.90 ± 0.30 −0.63 ± 0.45 10.20 ± 0.39

1.0 ± 1.7 3.7 ± 0.6 4.3 ± 1.2 87.7 ± 1.5

PS, physiological saline; DMN, dimethylnitrosamine; 2-AAF, 2-acetylaminofluorene. (No.of viable cells) a × 100. [No.of total cells(viable cells+dead cells)] b

Calculated from 100 cells in each animal.

Table 7-1 Micronucleus induction by MP-124 or nicotinic acid in male rats. Group

Chemical

Dose (mg/kg/day)

NA (mg/kg/day)

MNIME (%)a (Mean ± SD)

1 2 3 6

PS PS MP-124 CP

0 0 1000 20 mg/kg p.o.

0 600 0

0.15 0.15 0.79 2.43

± 0.08 ± 0.08 ± 0.12** ± 0.36**

IME (%)b (Mean ± SD)

NAD (pmol/106 cells)

58.4 ± 1.6 58.8 ± 1.1 59.4 ± 2.3 49.9 ± 5.4#

53.89 ± 11.7 117.22 ± 16.8 55.31 ± 11.0 NE

Plasma concentration of MP-124 (ng/mL, Mean ± SD) 6h

24 h

– – 4626.8 ± 730.16 –

– – 4972.7 ± 682.91 –

PS, physiological saline; CP, cyclophosphamide monohydrate; NA, nicototinic acid. a Incidence of micronucleated immature erythrocytes. b Ratio of immature erythrocytes to total erythrocytes. ** Significantly different from Group 1 (p < 0.01, Kastenbaum and Bowman’s method). # Significantly different from Group 1 (p < 0.05, Student’s t test).

Table 7-2 Effects of nicotinic acid on micronucleus induction by MP-124 in male rats. Group

Chemical

Dose (mg/kg/day)

NA (mg/kg/day)

MNIME (%)a (Mean ± SD)

IME (%)b (Mean ± SD)

NAD (pmol/106 cells)

3 4 5

MP-124 MP-124 MP-124

1000 1000 1000

0 200 600

0.79 ± 0.12 0.26 ± 0.10** 0.29 ± 0.08**

59.4 ± 2.3 59.3 ± 1.6 59.6 ± 2.5

55.31 ± 11.0 115.72 ± 20.9 131.67 ± 26.6

Plasma concentration of MP-124 (ng/mL, Mean ± SD) 6h

24 h

4626.8 ± 730.16 4568.3 ± 332.89 3968.4 ± 1351.48

4972.7 ± 682.91 5410.5 ± 671.93 4365.1 ± 1153.80

NA, nicotinic acid. a Incidence of micronucleated immature erythrocytes. b Ratio of immature erythrocytes to total erythrocytes. ** Significantly different from Group 3 (p < 0.01, Dunnett’s test).

MNIE (%)

3

2

p<0.01 (Dunnett’s test)

1

0

Group

1

2

3

4

5

MP-124 (mg/kg/day)

0

0

1000

1000

1000

Nicotinic acid (mg/kg/day)

0

600

0

200

600

6

CP 20 mg/kg

Fig. 3. Effects of nicotinic acid on the micronucleus induction by MP-124.

0

E. Yamamura et al. / Mutation Research 782 (2015) 1–8

7

Table 8 Effects of nicotinic acid treatment on micronucleus induction by cyclophosphamide monohydrate or nimustine in male rats. Group

Chemical

Dose (mg/kg)

NA (mg/kg/day)

MNIME (%)a (Mean ± SD)

IME (%)b (Mean ± SD)

NAD (pmol/106 cells)

TK parameters of CP or ACNU

Tmax (h)

Cmax (␮g/mL)

AUCc (ug h/mL)

Experiment with cyclophosphamide monohydrate 1 PS 0 0 2 PS 0 600 3 CP 10 0 4 CP 10 600

0.13 ± 0.10 0.13 ± 0.11 1.89 ± 0.61** 2.07 ± 0.58**

62.4 ± 6.7 56.6 ± 12.6 57.0 ± 9.6 60.6 ± 6.9

54.69 ± 7.00 116.34 ± 13.70 65.75 ± 7.91 132.86 ± 18.48

– – 0.5 0.5

– – 3.71 4.93

– – 7.63 8.04

Experiment with nimustine 1 PS 0 2 PS 0 3 ACNU 6 4 ACNU 6

0.15 ± 0.13 0.13 ± 0.06 1.90 ± 0.41* 2.48 ± 0.50#

68.3 ± 4.6 61.3 ± 5.3 62.9 ± 2.5$ 57.2 ± 6.2†

43.07 ± 6.10 91.95 ± 14.45 50.45 ± 7.73 99.46 ± 10.97

– – 0.25 0.25

– – 228 251

– – 127 169

0 600 0 600

PS, physiological saline; CP, cyclophosphamide monohydrate; ACNU, nimustine; NA, nicotinic acid; Tmax, the time to reach maximum concentration following dosing; Cmax, the maximum plasma concentration; AUC, the area under the plasma concentration-time curve. a Incidence of micronucleated immature erythrocytes. b Ratio of immature erythrocytes to total erythrocytes. c Values represent AUC0-4h for CP and AUC0-2h for ACNU. ** Significantly different from Group 1 (p < 0.01, Kastenbaum and Bowman’s method). * Significantly different from Group 5 (p < 0.05, Kastenbaum and Bowman’s method). # Significantly different from Group 7 (p < 0.05, Student’s t test). $ Significantly different from Group 5 (p < 0.05, Student’s t test). † Significantly different from Group 7 (p < 0.05, Student’s t test).

condition (but not up to 50 ␮g/mL in the continuous treatment condition), the positive concentrations of MP-124 are approximately 30-fold higher than the positive concentration at steady state in the rat micronucleus test of infusion treatment with MP-124. The continuous treatment with MP-124 in rats increased MNIME in the bone marrow, and the increase was clearly reduced with the co-treatment with NA which caused increased NAD levels in the bone marrow cells. Since, increased MNIME after treatment with CP and ACNU failed to be reduced under the same treatment conditions with NA, it is considered that increased NAD levels cannot inhibit MNIME induced by the treatment with DNA-reactive genotoxicants. Those results strongly indicate that increased MNIME in rats continuously treated with MP-124 is due to the inhibition of PARP-1 enzyme in the bone marrow. This is consistent with the known activity of MP-124 as a competitive inhibitor of PARP, which requires NAD as a substrate for generating the poly-ADPribose. Spronck et al. report that rats fed with a niacin deficient diet showed an increase of micronucleated polychromatic erythrocytes (MNPCE) and sister chromatid exchanges (SCE) in the bone marrow [21]. As niacin is a precursor of NAD, the deficiency reduced intracellular NAD level and resulted in an almost complete (80%) loss of basal poly (ADP-ribosyl) ation in the rat bone marrow cells [6]. These reports demonstrate that decreased PARP activity would increase the spontaneous frequency of MNPCE and SCE, and thus it is expected that treatment with the PARP-1 inhibitor MP-124 would increase the spontaneous frequency of MNIME. It has been reported that a number of indirect mechanisms lead to genotoxicity. Some of these mechanisms involve non-DNA targets such as enzymes of DNA synthesis and repair, imbalance of DNA precursors, energy depletion, production of active oxygen species, lipid peroxidation, nuclease release from lysosomes, ionic imbalance, etc. [22–24]. Severe disturbance of those non-DNA targets may indirectly lead to DNA damage. PARP-1 is an essential enzyme for maintaining genomic integrity, and is responsible for post-translational modification of proteins in response to numerous endogeneous and genotoxic agents. It plays a role in the early events following DNA damage, and influences the effectiveness of cellular DNA repair process by recruiting DNA repair enzymes to repair single-strand breaks [25]. Consequently, inhibition of this enzyme can lead indirectly to DNA damage in exposed cell populations due to an effect upon DNA repair. In general, chemical genotoxicity related to non-DNA targets is assumed to have a

threshold for DNA and/or chromosomal damage. In such cases, in which a genotoxic endpoint is increased due to an indirect mode of action that does not involve direct reactivity with DNA, it is generally accepted that there is a threshold level of exposure below which the effect is not expected to occur and that safe exposure levels can be defined for which an adequate margin of exposure exists between clinical exposure levels and the NOEL for the observed effect [26–28]. For example, topoisomerase inhibitors are expected to have a threshold and the evidence has been shown by Lynch et al. [29]. The results with MP-124 indicate that the observed genotoxic effects have a threshold. The primary structure of PARP-1 is highly conserved in eukaryotes (homology at the level of amino acid sequence: human and mouse, 92% [30]; human and chicken, 87% [31]) with the catalytic domain showing high degree of homology between different species. In addition, the active site, known as “the PARP signature” and formed by a sequence of 50 amino acids, shows 100% homology between human and mouse [32]. These results suggest the inhibition effect of MP-124 on PARP is expected to occur at the concentration with not much difference between species. In fact, there is no species difference for the effective plasma concentrations at steady state in rats (126 ng/mL at 1 mg/kg/h) and monkeys (98.1 ng/mL at 0.3 mg/kg/h) [13] in pharmacological animal models. In consideration of above discussions, we hypothesize that the plasma concentration at steady state of 1500 ng/mL of MP-124 would be a threshold for genotoxicity in humans, which is equivalent to the NOAEL of 250 mg/kg/day based on the results of rat micronucleus test with the infusion treatment of MP-124. In pharmacological rat stroke models, MP-124 exhibited neuroprotective effects at 24 mg/kg/day or more [12], doses significantly lower than the NOAEL, and the pharmacological effect of MP-124 in humans is also expected to have effect at lower concentrations than the anticipated threshold levels of genotoxicity. In addition, cells in cerebral ischemic areas use excessive intracellular NAD and result in extreme NAD-depletion because PARP enzymes are more activated in such cells than normal cells. Since MP-124 inhibits PARP-1 competitively against intracellular NAD, it is speculated that NAD-depleting cells in cerebral ischemic areas would be more susceptible to the pharmacological effects of MP-124 than normal cells. From this point of view, the pharmacological effect is expected for cells in cerebral ischemic areas at lower concentrations of MP124 than those showing genotoxicity in normal cells. Moreover, the

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