Isolation and Identification of Metabolites of Porfiromycin Formed in the Presence of a Rat Liver Preparation

Isolation and Identification of Metabolites of Porfiromycin Formed in the Presence of a Rat Liver Preparation WENSHENG LANG, JOHN MAO, QIN WANG, CHUANSHENG NIU, TERRENCE W. DOYLE, BIJAN ALMASSIAN Department of Development, Vion Pharmaceuticals, Inc. Four Science Park, New Haven, Connecticut 06511

Received 18 June, 1999; revised 24 November 1999; accepted 29 November 1999

The isolation and identification of the major metabolites of porfiromycin formed in the presence of a rat liver preparation under aerobic conditions were performed with high-performance liquid chromatography and electrospray ionization mass spectrometry. Porfiromycin was extensively metabolized by the rat liver preparation in an aqueous 0.1 M potassium phosphate buffer (pH 7.4) containing an NADPH generating system at 37°C. A total of eight metabolites was identified as mitosene analogs. Of these, three primary metabolites are 2-methylamino-7-aminomitosene, 1,2-cis and 1,2-trans-1-hydroxy-2-methylamino-7-aminomitosene, which are consistent with those previously observed in hypoxia using purified rat liver NADPH-cytochrome c reductase. Interestingly, 2-methylamino-7-aminomitosene is a reactive metabolite, which undergoes further activation at the C-10 position by the loss of carbamic acid and then links with the 7-amino group of the primary metabolites to yield two dimeric adducts. In addition, three phosphate adducts, 10-decarbamoyl-2-methylamino-7-aminomitosene10-phosphate, 1,2-cis and 1,2-trans-2-methylamino-7-aminomitosene-1-phosphate, were also identified in the incubation system. The configurations of the diastereoisomeric metabolites were determined with 1HNMR and phosphatase digestion. On the basis of the metabolite profile, we propose in vitro metabolic pathways for porfiromycin. The findings provide direct evidence for understanding the reactive nature and hepatic metabolism of the drug currently in phase III clinical trials. © 2000 Wiley-Liss, Inc. and the ABSTRACT:

American Pharmaceutical Association J Pharm Sci 89:191–198, 2000

INTRODUCTION Porfiromycin (PM, Fig. 1), the N-methyl analog of mitomycin C (MC), is an alkylating agent capable of cross-linking two complementary DNA strands by way of the C-1 and C-10 positions on bioreductive activation.1–5 PM alkylation of DNA is believed to be the event relevant to cytotoxicity. Importantly, preferential toxicity of PM to hypoxic EMT6 tumor cells vs the oxygenated cells has been demonstrated in vitro and in vivo, suggesting that this agent may be potentially useful for

Correspondence to: B. Almassian. (E-mail: balmassi@ vionpharm.com) Journal of Pharmaceutical Sciences, Vol. 89, 191–198 (2000) © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association

the treatment of solid tumors.6–9 In addition, human phase I and phase II clinical trials have shown that PM is tolerated at a dose threefold higher than that of MC.10–15 With a broader therapeutic index, PM is potentially superior to MC in clinical applications for the treatment of solid tumors. At present, PM is being developed for the treatment of head and neck cancers as an adjunct to radiation therapy in phase III clinical trials. The bioreductive activation and in vitro metabolism of MC under hypoxic conditions have been extensively investigated.16–20 However, limited information on the metabolism and disposition of PM is available in the literature.21,22 Enzymatic activity that catalyzes the reductive activation of MC/PM has been demonstrated in the microsomes, cytosol, and nuclei of rat liver

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Figure 1. Structure of porfiromycin.

cells, 16–19,21 as well as in other mammalian cells.20,22–27 Two major enzymes responsible for the reductive activation in the microsomal and cytosolic fractions are NADPH-cytochrome c (P450) reductase19–21 and NAD(P)H:quinone oxidoreductase (DT-diaphorase)20,23, respectively. The former exerts its activity only in hypoxia, whereas the latter shows similar activity under both aerobic and anaerobic conditions. Several primary metabolites of MC resulting from biotransformation at the C-1 reactive center by purified rat liver NADPH-cytochrome c reductase have been reported.17,19 As for PM, three similar (C-1) metabolites have been identified arising from the purified NADPH-cytochrome c reductase under hypoxic conditions.21 To date, no metabolites derived from biotransformation at C-10 of either MC or PM have been reported. In addition, significant differences between PM and MC in toxicity and tumor selectivity have been found.6–11,28 Therefore, investigation of the metabolism and detoxification of PM by well-oxygenated liver tissue is important for the drug. The complete characterization of the metabolites of PM formed in the presence of a liver preparation permits an understanding of its reactive nature and metabolic fate on biotransformation under aerobic conditions. In this article, we report the isolation and identification of the major metabolites resulting from biotransformation of PM at C-1 and C-10 by a rat liver preparation under aerobic conditions. Detailed analysis of the metabolite profile has allowed us to propose pathways for PM on biotransformation. The identification of three metabolites involving the C-10 functionality has led us to find a series of cysteine and mercapturic acid conjugates of PM in experimental animals and in humans. The in vivo results will be published elsewhere.

EXPERIMENTAL SECTION Materials PM was obtained from Sicor. S.p.A. (Milan, Italy). Glucose-6-phosphate disodium salt, glucose-6JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 2, FEBRUARY 2000

phosphate dehydrogenase, NADP, and alkaline phosphatase were purchased from Sigma Chemical Co. (St. Louis, MO). Magnesium chloride hexahydrate, EDTA disodium salt, potassium chloride, 37% hydrochloric acid, and ammonium acetate were purchased from J. T. Baker, Inc. (Phillisburg, NJ). Tris (hydroxymethyl)aminomethane was purchased from Aldrich Chemical Co. (Milwaukee, WI). HPLC-grade methanol and potassium phosphate monobasic were purchased from Sigma-Aldrich Chemical Co. and Fisher Scientific Co, (Pittsburgh, PA), respectively. Milli-Q Plus ultra pure water (Milli-Q water purification system, Millipore, Bedford, MA) was used throughout the study. Rat Liver Preparation Rat liver was prepared according to the method described by Curry and Whelpton.29 An adult Sprague-Dawley rat was killed with a blow to the head. The liver was removed immediately and minced into small pieces in ice-cold 0.01 M potassium phosphate buffer containing 1.15% potassium chloride (pH 7.6). The liver tissue was homogenized using a Potter-Elvehjem tissue grinder in 3 volumes of the ice-cold 0.01 M potassium phosphate buffer (pH 7.6). The homogenate was centrifuged at 10,000×g for 20 minutes at 4°C. Aliquots (1.5 mL) of the supernatant were stored at −80°C until used. The protein concentration of the rat liver preparation was determined by use of a modification of Lowry’s method.30 Incubation and Sample Preparation PM (5.8 mg) was incubated with rat liver preparation (26 mg protein) in a final volume of 10 mL of 0.1 M potassium phosphate buffer (pH 7.4) containing 2 mM NADP, 10 mM glucose-6phosphate, 10 unit/mL glucose-6-phosphate dehydrogenase, 1 mM EDTA, and 5 mM MgCl2 at 37°C. For optimization of incubation time, the enzymatic reaction was terminated at predefined incubation times (0–150 minutes) by transferring the incubation fluid (0.20 mL) into a microcentrifuge tube containing methanol (0.40 mL). The tubes were vortexed and then centrifuged at approximately 14,000×g for 10 minutes. The supernatant (0.25 mL) was transferred into an HPLC vial with 0.25 mL of 50 mM potassium phosphate buffer (pH 5.0). The resulting solution (50 ␮L) was injected onto HPLC. For isolation of metabo-

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lites, a separate incubation was conducted, and the reaction was terminated at 50 minutes by adding 2 volumes of methanol. After centrifugation, the supernatant was evaporated to dryness and the residue was reconstituted with the appropriate amount of water.

(10 mL) sequentially. The cartridge was washed with water (10 mL) and then eluted with 5 mL of methanol-water (1 : 1). The methanolic effluent was collected, and the solvent was removed under a stream of nitrogen. The samples were subjected to LC/MS and NMR analysis.

Phosphatase Digestion

LC/MS Analysis

The appropriate amount of each purified phosphate metabolite was incubated with 1 unit of alkaline phosphatase in a final volume of 0.20 mL of 20 mM Tris ⭈ HCl buffer (pH 8.0) as described by Bloch and Schlesinger31 with modification. The final digest was subjected to HPLC and LC/MS analysis.

Positive ion electrospray ionization (ESI)-mass spectra of metabolites were obtained on a Navigator single quadrapole mass spectrometer (Finnigan, San Jose, CA). MS operation parameters were as follows: capillary voltage, 3.6 kV; drying gas (nitrogen) flow rate, 350 L/hr; ion source temperature, 150°C; and mass scan speed, 400 mass units/sec. LC was performed on a Supelcosil LC-18 column (150 × 4.6 mm, 5 ␮m) with a Platinum C18 EPS guard column, 7.5 × 4.6 mm, 5 ␮m, (Alltech, San Jose, CA) at 40°C. Mobile phases were methanol (A) and 10 mM ammonium acetate (B). A linear gradient elution was carried out with 0% to 90% A within 30 minutes at a flow rate of 1.0 mL/min. The effluent from HPLC was introduced into the MS system at 0.25 mL/min by means of a 3 : 1 split.

HPLC Analysis HPLC analysis of metabolites was performed on a Hewlett Packard (HP) 1100 system. The HPLC system consisted of a quaternary pump G1311A, an autosampler G1313A, a diode array detector (DAD) G1315A, and a column thermostat G1316A. HP LC3D ChemStation software was used for system control and data processing. The chromatography was performed on a Supelcosil LC-18 column (150 × 4.6 mm, 5 ␮m) at 50°C. The mobile phases were methanol (A) and an aqueous 50 mM potassium phosphate buffer, pH 5.0, (B). Mobile phase A was linearly increased from 0% to 10% within 15 minutes, and then from 10% to 40% for 30 minutes at a flow rate of 1 mL/min. Detection wavelengths were set at 250 and 550 nm. Isolation of Metabolites Aliquots (100 ␮L) of the reconstituted enzymatic reaction mixture were repeatedly injected onto a Supelcosil LC-18-S column (250 × 4.6 mm, 5 ␮m) at 50°C. A linear gradient elution was conducted with 0% to 20% methanol in 50 mM phosphate buffer (pH 5.0) over 40 minutes at a flow rate of 1.5 mL/min. The effluent was collected in 0.5 mL/ vial intervals on a Gilson fraction collector Model FC204 (Gilson, Middleton, WI). The fractions containing the same chromatographic component were pooled. Solvent in each final fraction was removed under a stream of nitrogen. The residue was reconstituted with water (1 mL). The resulting solution was passed through a Waters SepPak Plus C18 cartridge, sorbent weight of 360 mg, preconditioned with methanol (5 mL) and water

1

HNMR Analysis

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HNMR spectra of metabolites in deuteromethanol were recorded on a Bruker AC300 NMR spectrometer at 300 MHz. Either internal tetramethylsilane or the residual methanol signal was used as a chemical shift reference. The following terminology is used: s, singlet; d, doublet; dd, doublet of doublets; q, quartet; m, multiplet; and J, coupling constant in hertz.

RESULTS AND DISCUSSION Isolation of Metabolites A total of eight major metabolites (M1 to M8) was isolated with reversed-phase HPLC after incubation of PM with a rat liver preparation in the presence of an NADPH-generating system and an aqueous 0.1 M potassium phosphate buffer (pH 7.4) at 37°C under aerobic conditions. A gradient elution was carried out to cover the broad polarity range of the metabolites. A representative chromatogram showing the metabolite profile is given in Figure 2. Good separation and sharp peaks for most of the metabolites were obtained on a Supelcosil LC-18-S column (250 × 4.6 mm) when an JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 2, FEBRUARY 2000

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Figure 2. Chromatogram of metabolites of porfiromycin (detection at 550 nm). Sample was prepared by incubation of PM with rat liver preparation in the presence of an NADPH-generating system and in 0.1 M phosphate buffer (pH 7.4) at 37°C for 50 min. Detailed chromatographic conditions are given in the Experimental Section.

aqueous 50 mM potassium phosphate buffer (pH 5.0) was used as an eluent. Poor resolutions between PM and its metabolites were observed if the pH of the phosphate buffer was adjusted to 7.0 (data not shown). For characterization, further separation of M7 from M8 was achieved on a Supelcosil LC-18 column (150 × 4.6 mm) under the same chromatographic conditions. A better resolution between M7 (Rt. 28.03) and M8 (Rt. 29.09 min) on this column than that on Supelcosil LC-18-S was obtained. Formation of Metabolites The formation of metabolites and the disappearance of PM at different incubation times were investigated over a period of 150 minutes with HPLC analysis. A relationship between incubation time and the relative amount of PM and its metabolites was generated. It was found that the peak area of PM was diminished by almost 80%, and the formation of metabolites reached a plateau at 30 minutes of incubation (Fig. 3). Most of the metabolites remained at high levels except for metabolite M-6, which declined after 30 minutes of incubation. The result indicates that M6 may serve as a reactive intermediate, which undergoes further metabolism. The further metabolism of M6 was confirmed by incubation of purified M6 with the rat liver preparation under the same conditions. In addition, three metabolite peaks (M1 to M3) were not observed when the incubation was carried out in an aqueous 0.1 M JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 2, FEBRUARY 2000

Figure 3. Porfiromycin metabolism and metabolite formation in the presence of a rat liver preparation were plotted with peak area (mAU) from HPLC vs incubation time (min).

Tris ⭈ HCl buffer (pH 7.4), indicating that the formation of these metabolites is phosphate dependent. Two phosphate metabolites similar to M1 and M3 have been identified for MC using purified enzyme under anaerobic conditions.19 No metabolites were observed when the incubation was conducted in an aqueous 0.1 M potassium phosphate buffer (pH 7.4) without rat liver protein over the same period of time. The rat liver 10,000×g supernatant contains both microsomal and cytosolic fractions. NADPH-cytochrome c reductase and NAD(P)H:quinone oxidoreductase may be involved in the catalysis of reductive metabolism of PM. Under aerobic conditions, NAD(P)H:quinone oxidoreductase may play a more important role in the metabolism of PM. Identification of Metabolites ESI-mass spectrometry primarily was used for the identification and characterization of PM metabolites. Ultraviolet (UV) spectra of the metabolites also were acquired on a diode array detector and proved to be useful for differentiation of the metabolites. The configurations of diastereoisomeric metabolites were determined using 1 HNMR spectra and by means of phosphatase digestion to convert the phosphate metabolites into corresponding alcohol derivatives. Representative UV spectra of PM and its metabolites are given in Figure 4. The UV spectrum of PM showed two absorption peaks at 365 and 215 nm, which differed from those of its metabolites. The metabolites of PM gave two types of UV absorption spectra. The metabolites showing the first

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Figure 4. UV spectra of PM and its metabolites. PM, porfiromycin. A representative UV spectrum (type I) for metabolites M1 to M6. A representative UV spectrum (type II) for metabolites M7 and M8.

type of UV spectrum included M1 to M6. The metabolites with the second type of UV spectrum were M7 and M8. The UV spectra of type I showed absorption peaks at 250, 310, 350 (sh), and 550 nm, whereas those of type II had four absorption bands at 248, 299, 366, and 552 nm. Identical UV

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profiles obtained within groups indicated that the metabolites share the same chromophore. The mass spectra of M1 and M3 were identical and showed a protonated molecular ion at m/z 415 and two major fragment ions at m/z 317 (MH-H3PO4)+ and 256 (MH-H2PO3-HOCONH2)+ in Figure 5(A). The MS fragmentation pattern indicated that the two metabolites were isomeric phosphate adducts and both retained a carbamate group. Examination of the 1HNMR spectrum of M1 revealed that the two C-10 methylene protons formed an AB quartet (J ⳱ 14.0 Hz) (Table 1). Downfield resonances of the C-1 proton of M1 were observed at ␦ 5.45 as a doublet of doublets (J ⳱ 7.3 and 2.0 Hz), indicating that the C-1 proton may be coupled to phosphorus and the C-2␣ proton. The configuration of the C-1 proton of M1 was assigned as ␤ based on the coupling constant to the C-2␣ proton. The 1HNMR spectrum of M3 was similar to that of M1 except that the C-1 proton appeared at ␦ 5.73 as a doublet of doublets (J ⳱ 7.8 and 5.4 Hz). The configuration of the C-1 proton for M3 was assigned as ␣. For further structure confirmation, phosphatase digestion after LC/MS analysis was performed. The

Figure 5. Representative ESI-mass spectra of porfiromycin metabolites. (A) phosphate adduct M1; (B) metabolite M4; (C) metabolite M6; (D) metabolite M7. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 2, FEBRUARY 2000

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Table I. Position 1␣ 1␤ 2␣ 3␣ 3␤ 10 6 CH3 N−CH3

1

HNMR Data of Four Isomeric Metabolites M1

M3

M4

M5

— 5.45 dd (J ⳱ 7.3, 2.0) 4.04 m 4.48 dd (J ⳱ 13.0, 5.9) 4.08 dd (J ⳱ 15.8, 2.8) 5.23 1/2ABq (J ⳱ 14.0); 5.31 1/2ABq (J ⳱ 14.0) 1.80 s 2.49 s

5.73 dd (J ⳱ 7.8, 5.4) — 3.90 m 4.56 dd (J ⳱ 12.6, 7.3) 4.01 dd (J ⳱ 12.6, 7.5) 5.17 1/2ABq (J ⳱ 13.9); 5.34 1/2ABq (J ⳱ 14.0) 1.80 s 2.60 s

— 4.95 d (J ⳱ 2.0) 3.60 m 4.48 dd (J ⳱ 13.3, 6.3) 4.01 dd (J ⳱ 13.4, 3.2) 5.17 1/2ABq (J ⳱ 12.7); 5.24 1/2ABq (J ⳱ 12.7) 1.80 s 2.46 s

5.10 d (J ⳱ 5.1) — 3.66 m 4.55 dd (J ⳱ 12.0, 7.1) 3.76 dd (J ⳱ 12.0, 8.8) 5.18 1/2ABq (J ⳱ 12.9); 5.26 1/2ABq (J ⳱ 12.9) 1.80 s 2.50 s

results indicated that M1 was converted into corresponding alcohol derivative M4, and M3 into M5. Thus, M1 and M3 were identified as 1,2-trans and 1,2-cis-2-methylamino-7-aminomitosene-1phosphate, respectively. The mass spectrum of M2 demonstrated a protonated molecular ion at m/z 356 and a corresponding potassium adduct ion at m/z 394 (data not shown). Major fragment ions at m/z 258 (MHH 3 PO 4 ) + , 275 (MH-H 2 PO 3 ) + , and 290 (MHH3PO4+MeOH)+ were observed, indicating that M2 may also be a phosphate adduct. The observation of the characteristic fragment ion at m/z 258 for M2 in place of the ion at m/z 256 for M1 and M3 suggested that a protonation was occurring at the C-1 position. The mechanism of C-1 protonation by means of an internal redox reaction has previously proposed by Tomasz and Lipman.17 The MS data also demonstrated that M2 was devoid of the carbamate group. The structure of M2 was assigned as 10-decarbamoyl-2methylamino-7-aminomitosene-10-phosphate. The ESI-mass spectra of metabolites M4 and M5 were similar. A representative MS spectrum for these metabolites is given in Figure 5(B). The MS spectrum showed quasi-molecular ions at m/z 335 (MH)+, 357 (MNa)+, 367 (MH+MeOH)+, and 373 (MK)+. A series of fragment ions at m/z 317 (MH-H2O)+, 274 (MH-HOCONH2)+, 256 (MHH 2 O-HOCONH 2 ) + , and 246 (MH-HOCONH 2 CO)+ were also observed. The integration of the 1 HNMR spectra of M4 and M5 indicated that there was a single C-1 proton in the molecules. The downfield C-1 proton signal of M4 was located at ␦ 4.95 as a doublet with a coupling constant of 2.0 Hz, whereas the NMR resonances of the C-1 proton of M5 appeared at ␦ 5.10 as a doublet (J ⳱ 5.1 Hz) (Table 1). We assigned the C-1 proton as ␤ for M4 and ␣ for M5. Thus, metaboJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 2, FEBRUARY 2000

lites M4 and M5 were identified as 1,2-trans and 1,2-cis-1-hydroxy-2-methylamino-7aminomitosene, respectively. The mass spectrum of M6 demonstrated quasimolecular ions at m/z 319 (MH)+, 341 (MNa)+, 351 (MH+MeOH)+, and 357 (MK)+ in Figure 5(C). One major fragment ion appeared at m/z 258 (MH-HOCONH2)+, indicating the loss of carbamic acid and that M6 was a C-1 proton adduct. Examination of the 1HNMR spectrum of M6 indicated that (i) two doublets of doublets, corresponding to two C-1 methylene protons, were located upfield at ␦ 2.73 and 3.13, respectively; and (ii) one singlet at ␦ 5.14 was observed for two C-10 protons instead of an AB quartet as in M4 and M5. The NMR data were similar to those previously reported for 2, 7-diaminomitosene, a metabolite of MC identified in rat liver microsomes.17 M6 was identified as 2-methylamino-7aminomitosene. Two unresolved peaks corresponding to metabolites M7 and M8 were observed at retention times of 26.5 and 26.8 minutes, respectively (Fig. 2). The UV spectra of M7 and M8 were identical and different from those of type I (Fig. 4), indicating that an alteration of the 7-aminomitosene conjugation system had occurred. The mass spectrum of M7 showed the presence of quasimolecular ions at m/z 593 (MH)+, 615 (MNa)+, and 631 (MK)+ in Figure 5(D). The observation of a base peak at m/z 532 (MH-HOCONH2)+ demonstrated the loss of carbamic acid. The 1HNMR spectrum of M7 consisted of two sets of resonances. The first set of signals was similar to those observed for M5. The other set of signals was comparable to M6 except for the two C-10 methylene protons. The combined MS, 1HNMR, and UV data have allowed us to assign this compound as a dimeric adduct linked at the C-10 po-

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Scheme 1. Possible metabolic pathways of porfiromycin in a rat liver preparation.

sition of 10-decarbamoyl M6 with the 7-amino group of M5. The mass spectrum of M8 indicated the presence of quasi-molecular ions at m/z 577 (MH)+, 599 (MNa)+, and 615 (MK)+ and a base peak at m/z 516 (MH-HOCONH2)+ (data not shown). M8 is believed to be a dimer of M6. In summary, a total of eight metabolites (M1– M8) were identified in the in vitro system. The metabolites were rapidly produced in the presence of a rat liver 10,000×g supernatant under aerobic conditions. The requirement of anaerobic conditions for metabolism of MC or PM by rat liver NADPH-cytochrome c reductase or microsomes has been reported previously.17,19,21 Our results imply that some enzymes present in rat liver cytosolic fraction may play an important role in biotransformation of PM. The metabolite patterns strongly support the proposal by Moore32 that bioreductive activation of PM results in the formation of a reactive intermediate. On the basis of our findings, possible metabolic pathways for PM are proposed and summarized in Scheme 1. The in vitro metabolites were generated by means of nucleophilic and electrophilic additions at the C-1 and C-10 reactive centers of the reactive intermediate by the agents available in the aqueous medium. Water and phosphate anion acted as nucleophiles and attached to the C-1 reactive center to yield a pair of 1-hydroxymitosene diastereomers (M4 and M5) and a pair of 1-phosphate mitosenes (M1 and M3), respectively. Metabolite M6 is believed to be the product of C-1 protonation of the intermediate through an internal redox reaction.17 The identification of these primary metabolites provides a good basis for understanding the bifunctional nature of the C-1 reactive center of the intermediate. Of the five metabolites, M6 is a unique reactive metabolite, which undergoes further metabolism at the C-10 position by nucleophilic substitution of the carbamate group to

yield M2, M7, and M8. No further metabolism at the C-10 position of M1, M3, M4, and M5 was observed in the incubation system. Moreover, the identification of the C-10 dimeric adducts (M7 and M8), which may be considered as DNA-like reaction products, provides a molecular basis for recognizing the potential reactivity of the C-10 reactive center with the amino groups of base residues of DNA strands.

ACKNOWLEDGMENTS The authors wish to thank Dr. Zhemin Gu of XenoBiotic Laboratories, Inc. for his experimental effort and helpful discussion on LC/MS analysis of the metabolites. We also extend our appreciation to Dr. Yang Xu of Yale University for her critical review of this manuscript.

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