Kinetics of hydrolysis in vitro of nornitrogen mustard,a metabolite of phosphoramide mustard and cyclophosphamide

A gas chromatographic (GC-ECD) method was developed for the determination of nornitrogen mustard (NOR) and its hydrolysis products. The method was based on derivatization by heptafluorobutyric anhydride. The structures of the derivatives of NOR were established by GC-MS. The method was used to characterize the rate of transformation of NOR and phosphoramide mustard (PAM), important metabolites of cyclophosphamide, into secondary products in vitro at 37° C and pH 7.4. The rate of disappearance of NOR had a half-life of 20 min. The half-life of appearance of N-(2-chloroethyl)-N-(2-hydroxy-ethyl)amine (NOR-OH) was 19 min. While most NOR appeared to be converted to NOR-OH, the yield of N,N-bis(2-hydroxyethyl)amine (NOR-OH-OH) was a small fraction of the starting material. The disappearance of NOR, when PAM was used as a starting material, had a half-life of 19 min; in these experiments NOR-OH and NOR-OH-OH were relatively much more abundant compared to when NOR was used as the starting material.Abbreviations NOR nornitrogen mustard - NOR-OH N-(2-chloroethyl)-N-(2-hydroxyethyl)amine - NOR-OH-OH, N N-bisN,-(2-hydroxyethyl)amine - PAM phosphoramide mustard - GC gaschromatography - GC-ECD gas chromatography-electron capture detection - GC-MS gas chromatography-mass spectroscopy - HFBA heptafluorobutyric anhydride     

Kinetics of phosphoramide mustard hydrolysis in aqueous solution

Hydrolysis of phosphoramide mustard was investigated using HPLC, 31P NMR, and GC-MS with specific deuterium labels. The hydrolysis of phosphoramide mustard in sodium phosphate buffers was found to follow apparent first-order kinetics. The rate of hydrolysis was temperature and pH dependent, being slower under acidic conditions. The hydrolysis was not catalyzed by hydroxyl ion, and its pH dependence appeared to be the result of a change in the mechanism of hydrolysis at different pH values. At a pH value approximately above the pKa of the phosphoramide mustard nitrogen, the major hydrolytic pathway of phosphoramide mustard was via the formation of the aziridinium ion, followed by nucleophilic attack. At pH values below its pKa, cleavage of the P-N bond predominated. At pH 7.4, the formation of an aziridinium ion was followed by a rapid hydrolysis to yield the monohydroxy and, subsequently, the dihydroxy products. The hydrolysis at this pH was adequately described by consecutive first-order kinetics. Seven species in the hydrolytic mixture have been identified as intact phosphoramide mustard, N-(2-chloroethyl)-N-(2-hydroxyethyl)phosphorodiamidic acid, N,N-bis-(2-hydroxyethyl)phosphorodiamidic acid, phosphoramidic acid, phosphoric acid, N,N-bis-(2-chloroethyl)amine, and N-(2-chloroethyl)-N-(2-hydroxyethyl)amine by GC-MS with the aid of deuterium labels. Phosphoramide mustard was found to be stabilized by chloride ion. The stabilization was linearly related to the chloride ion concentration, and the mechanism was found to be via the formation of phosphoramide mustard from the aziridinium and chloride ions. Phosphoramide mustard was significantly more stable in human plasma and in 5% human serum albumin as compared to aqueous buffers, an observation that may be important in vivo.     

Enzymatic detoxification of phosphoramide mustard by soluble fractions from rat organ tissues.

Enzymatic degradation of phosphoramide mustard (PM), the ultimate cytotoxic metabolite of cyclophosphamide (CP), by the cleavage of the phosphorous-nitrogen (P-N) bond was investigated in vitro using 65,000g-soluble fractions from rat organ tissues. In the presence of physiologic bicarbonate in vivo, the P-N bond cleavage of PM was previously found to form 3-(2-chloroethyl)-1,3-oxazolidin-2-one (CNM), which is devoid of antitumor activity. This product was thus quantitated in the present studies using GC/MS and a deuterium-labeled analog (CNM-d8) as the internal standard. Under the experimental conditions, CNM was found to be enzymatically produced from PM in soluble fractions of rat liver, kidney, spleen, and intestine. Mean KM and Vmax values in soluble fractions of rat liver at pH 6.7 and 37 degrees C were determined to be 1.65 +/- 0.536 mM (N = 7) and 6.38 +/- 1.37 microM/min (N = 6), respectively. The enzymic activity of the rat liver soluble fraction was significantly reduced following boiling at 100 degrees C for 5 min. No CNM production was detected from PM incubated in plasma. The P-N bond cleavage for CP and for two other metabolites, 4-ketocyclophosphamide and alcophosphamide, was also investigated using soluble fractions from rat liver similar to that for PM. None of these compounds has been found to form CNM, however, indicating enzyme specificity for the P-N bond in PM. This enzyme probably resembles the previously described phosphamidases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkylation of guanosine by phosphoramide mustard, chloromethine hydrochloride and chlorambucil

Guanosine was reacted in vitro with phosphoramide mustard, chloromethine hydrochloride, and chlorambucil. The products were isolated by HPLC and characterized by UV and fluorescence spectroscopy, and C-8 tritium exchange. The primary products were 7-alkylguanosines according to such evidence. Phosphoramide mustard had 1/10 of the apparent alkylation activity of two other mustards. The primary 7-alkylguanosines were unstable at pH 7.4 and 37 degrees; t1/2 were 3 min. for chloromethine hydrochloride, 2.7 hrs for chlorambucil and 3.0 hrs for phosphoramide mustard. Both dechlorination at the unbound arm of the mustard and imidazole ring opening og guanosine appeared to account for such instability.     

Glutathione conjugation with phosphoramide mustard and cyclophosphamide. A mechanistic study using tandem mass spectrometry.

The conjugations of cyclophosphamide and of phosphoramide mustard with glutathione are shown to be catalyzed by hepatic cytosolic glutathione-S-transferases. Cyclophosphamide conjugation is also catalyzed by microsomal glutathione-S-transferases, both in intact microsomes and after solubilization and immobilization. Deuterium isotope labels are used to test whether chloride is directly displaced by glutathione in the enzyme-catalyzed conjugations, or whether conjugation takes place via symmetrical cyclic aziridinium ions. Tandem mass spectrometry with high energy collisional activation is shown to provide reliable analysis of the isotope-labeling patterns in the conjugated products. This experiment leads to the conclusion that the aziridinium ion is opened in the conjugation of phosphoramide mustard in both the enzyme-catalyzed and the chemical reactions. Cyclophosphamide, on the other hand, is shown to be conjugated through direct displacement of chloride.     

Enhancement of the embryotoxicity of acrolein, but not phosphoramide mustard, by glutathione depletion in rat embryos in vitro

The intracellular thiol glutathione is known to protect cells against the toxicity of certain drugs and reactive intermediates. In this study, the role of glutathione in protecting the embryo against two embryolethal and teratogenic metabolites of cyclophosphamide, and anticancer drug, was assessed in vitro using the rat whole embryo culture system. Day 10.5 rat embryos were cultured in rat serum medium containing phosphoramide mustard (1, 10, or 25 microM) or acrolein (10, 25, 50 or 100 microM), with and without buthionine sulfoximine (10 or 100 microM), a compound which depletes glutathione by inhibiting its synthesis. After 45 hr, embryos were assessed for viability, malformations, growth and development, and the glutathione content of embryos exposed to buthionine sulfoximine alone was assayed. The glutathione levels of the embryos and their yolk sacs were decreased significantly by 100 microM buthionine sulfoximine, whereas 10 microM buthionine sulfoximine decreased glutathione levels in the yolk sacs only. Phosphoramide mustard alone, at concentrations of 10 and 25 microM, did not produce embryo deaths but did cause malformations and growth retardation in 100% of the exposed embryos. The addition of buthionine sulfoximine (100 microM) had no effect on the teratogenicity or growth-retarding effects of phosphoramide mustard. Acrolein alone produced a 25 and 48% incidence of embryo deaths at 50 and 100 microM, respectively, and a 46% incidence of embryo malformations, as well as significant growth retardation, among the surviving embryos at 100 microM. Buthionine sulfoximine (10 or 100 microM) significantly enhanced the embryotoxic effects of acrolein. The addition of 10 microM buthionine sulfoximine resulted in 100% embryolethality at 100 microM acrolein; this buthionine sulfoximine concentration decreased the EC50 values for embryo deaths and malformations to 50% of those for acrolein alone. The addition of 100 microM butionine sulfoximine significantly potentiated the embryolethality of acrolein at 25, 50 and 100 microM; the combination of 100 microM acrolein plus 100 microM buthionine sulfoximine was 100% embryolethal. The incidence of embryo malformations was enhanced significantly at 10 and 25 microM acrolein by 100 microM buthionine sulfoximine. The EC50 values for embryo deaths and malformations were decreased to 50 and 20%, respectively, of those values for acrolein alone. Both butionine sulfoximine concentrations produced significant growth retardation at all acrolein concentrations compared to either acrolein or buthionine sulfoximine alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

The pharmacokinetics of cyclophosphamide, phosphoramide mustard and nor-nitrogen mustard studied by gas chromatography in patients receiving cyclophosphamide therapy

1 Simple, accurate and specific gas-chromatographic methods for the estimation of derivatized phosphoramide and non-nitrogen mustards utilizing alkali-flame ionization detection are described.

2 The pharmacokinetics in plasma of cyclophosphamide, phosphoramide mustard, nor-nitrogen mustard and nitrobenzyl pyridine alkylating activity were investigated following administration of cyclophosphamide by intravenous and oral routes to patients with malignant disease

3 The mean T_½ for cyclophosphamide was 8.88 h (s.d. 1.25 h) and the apparent volume of distribution (V_dβ) was 0.74 l kg^-1 (s.d. 0.16 l kg^-1).

4 The decline in plasma concentration of phosphoramide mustard was biphasic, the longer T_½ being 8.68 h (s.d. 2.50 h). This was not significantly different from that of cyclophosphamide. This could indicate that the true biological T_½ for phosphoramide mustard is identical with or shorter than that of cyclophosphamide.

5 The plasma concentrations of phosphoramide mustard following cyclophosphamide doses of known therapeutic efficacy are probably insufficient to produce important cytotoxic effects. This suggests that if phosphoramide mustard is the major alkylating metabolite derived from cyclophosphamide, it is transported in the blood in precursor form.

6 The mean plasma T_½ of nor-nitrogen mustard was 3.31 h (s.d. 1.60 h) which was significantly different from that of cyclophosphamide.

7 The mean plasma T_½ of the nitrobenzylpyridine alkylating activity was 9.81 h (s.d. 4.18 h) and did not significantly differ from that of cyclophosphamide. Although the area under the plasma alkylating activity concentration, time curve is related to the T_½ of cyclophosphamide, the alkylating activity does not reflect the concentrations of the two plasma metabolites measured.

     

Synthesis of phosphoramide mustard analogues belonging to the L-sugar series

During the past decades numerous cyclophoshamide (mustard) derivatives of nucleosides and aminodeoxy sugars have been prepared for investigating their antitumor activities. The cyclophosphamide analogues of aminotrideoxy hexoses belonging to the D-series of sugars have been prepared by Monneret et al. The present paper reports the synthesis of the new phosphoramide mustards 16-17 from 12 and 15 (belonging to the L-sugar series). First compound 10 was synthesized from the L-rhamnose (9). Methyl 3-azido-2,3,6,-trideoxy-alpha-L-ribo-hexopyranoside (11) was obtained by the replacement of the 3-O-p-toluene-sulfonyl group of 10 with sodium azide. Methyl 3-azido-2,3,6,-trideoxy-alpha-L-arabino-hexopyranoside (14) was synthesized by rign opening of 13 with sodium azide. The corresponding amino sugars (12, 15) were obtained by catalytic hydrogenation (over palladium on carbon) of 11 and 14. Our compounds 12 and 15 were transformed into the cyclophosphamide derivatives 16a,b-17a,b upon treatment with bis(2-chloroethyl)phoshoramidic dichloride in the presence of triethylamine (36 h, r.t.). The approximately 1:1 mixtures of isomers (due to the different steric position of the P=O group) could be readily separated by chromatography. The 1H NMR assignments of compounds 16a, 16b, 17a and 17b, were based on one-dimensional selective decoupling experiments or two-dimensional chemical shift-correlated spectroscopy (COSY-60). The assignment of configuration to the isomeric phosphoramidates was based on the magnetic anisotropy of the P=O bond. The distinctly different chemical shift patterns of sugar protons observed for the two isomers allowed the unambiguous assignment of the P=O stereochemistry. The compounds 16a,b-17a,b (mixture of isomers) were tested for inhibitory activity using L1210 and HT29 cell lines.     

Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro

Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM (≥ 3 μM) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation (γH2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1 μM PM induced significant losses of primordial and small primary follicles, respectively. PM-induced γH2AX was observed predominantly in oocytes, in which foci of γH2AX staining increased in a concentration-dependent manner and peaked 18-24 h after exposure to 3-10 μM PM. Numbers of oocytes with ≥ 5 γH2AX foci were significantly increased both 1 and 8 days after exposure to ≥ 1 μM PM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers (≥ 30 μM) and increased γH2AX foci (≥ 3 μM) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.     

Chemically stable, lipophilic prodrugs of phosphoramide mustard as potential anticancer agents

Benzyl phosphoramide mustard (3), 2,4-difluorobenzyl phosphoramide mustard (4), and methyl phosphoramide mustard (5) were examined as lipophilic, chemically stable prodrugs of phosphoramide mustard (2). These phosphorodiamidic esters are designed to undergo biotransformation by hepatic microsomal enzymes to produce 2. The rate of formation of alkylating species, viz., 2, from these prodrugs and their in vitro cytotoxicity toward mouse embryo Balb/c 3T3 cells were comparable to or better than that of cyclophosphamide (1). Preliminary antitumor screening against L1210 leukemia in mice, however, suggests that these prodrugs are devoid of any significant antitumor activity in vivo.     

Critical exposure level of cadmium for elevated urinary metallothionein--an occupational population study in China

Cadmium is a well-known nephrotoxic agent with extremely long biological half-time of 15-30 years in humans. To prevent nephrotoxicity induced by cadmium, it is necessary to identify specific and sensitive biomarkers of cadmium exposure and renal damage, and to define critical exposure levels related to minimal nephrotoxicity in humans. In this study, urinary cadmium (UCd) and blood cadmium (BCd) were used as cadmium exposure indicators, urinary beta(2)-microglobulin (UB2M), N-acetyl-beta-D-glucosaminidase (UNAG) and albumin (UALB) were applied as the effect biomarkers of tubular and glomerular dysfunction. The relationship between urinary metallothionein (UMT) and cadmium exposure biomarkers as well as effect biomarkers was examined. Significant correlations were found between the UMT and BCd, and UCd. At the same time, UB2M, UALB and UNAG showed positive correlation with UMT as well. According to this result, cadmium-exposed individuals with renal dysfunction excreted more metallothionein than those without. Dose-response relationships between UCd and urinary indicators of renal dysfunction were studied. The critical concentration of UCd was quantitatively estimated by the benchmark dose (BMD) method. The lower confidence limit of the BMD-10 (BMDL) of UCd (3.1 microg/g Cr) related to increased excretion of urinary metallothionein was slightly higher than that for UNAG (2.7 microg/g Cr), but lower than those of UB2M (3.4 microg/g Cr) and UALB (4.2 microg/g Cr). The results demonstrate that UMT may be used as a sensitive biomarker of renal tubular dysfunction in cadmium-exposed populations.     

pH-dependent toxicity of sulphur mustard in vitro

The dependence of sulphur mustard (HD) toxicity on intracellular (pH(i)) and extracellular pH was examined in CHO-K1 cells. HD produced an immediate and significant concentration-dependent decline in cytosolic pH, and also inhibited the mechanisms responsible for restoring pH(i) to physiological values. The concentration-response of HD-induced cytosolic acidification, closely paralleled the acidification of the extracellular buffer through HD hydrolysis. A viability study was carried out in order to assess the importance of HD-induced cytosolic acidification. Cultures were exposed to HD for 1 h in media that were adjusted through a pH range (pH 5.0-10), and the 24 h LC(50) values were assessed using the viability indicator dye alamarBlue. The toxicity of HD was found to be dependent on extracellular pH, with a greater than eight-fold increase in LD(50) obtained in cultures treated with HD at pH 9.5, compared to those treated at pH 5.0. Assays of apoptotic cell death, including morphology, soluble DNA, caspase-3 activity and TUNEL also showed that as pH was increased, much greater HD concentrations were required to cause cell death. The modest decline in HD half-life measured in buffers of increasing pH, did not account for the protective effects of basic pH. The early event(s) that HD initiates to eventually culminate in cell death are not known. However, based on the data obtained in this study, we propose that HD causes an extracellular acidification through chemical hydrolysis and that this, in both a concentration and temporally related fashion, results in cytosolic acidification. Furthermore, HD also acts to poison the antiporter systems responsible for maintaining physiological pH(i), so that the cells are unable to recover from this insult. It is this irreversible decline in pH(i) that initiates the cascade of events that results in HD-induced cell death.     

Observations on the effects of cyclophosphamide,phosphoramide mustard and some activated oxazaphosphorines on murine L1210 leukemia

Summary The L1210 tumor system was used in vitro and in vivo in comparative studies with activated cyclophosphamide analogs, cyclophosphamide and phosphoramide mustard. All the above compounds gave substantial cell kills (5 logs) of L1210 in vivo at doses that were non-toxic, but slight differences were noted. ASTA Z 7557 had a slight advantage in cure rate over cyclophosphamide when these drugs were given i.v. or i.p. to early tumor (i.p.). However, cyclophosphamide had the advantage in cure rate when drug administration was i.v. to advanced tumor. At equimolar concentrations in vitro ASTA Z 7557 was more cytotoxic than either phosphoramide mustard or acrolein. In vivo, the activated cyclophosphamide derivatives caused some unusual toxicities at therapeutic doses that were not seen with cyclophosphamide. The toxicities manifested as spastic responses and acute deaths on rapid i.v. or i.p. injections and as chronic liver atrophies and fibrosis with i.p. treatment.     

Covalent binding of 2-phenylpropionyl-S-acyl-CoA thioester to tissue proteins in vitro.

In this study, we investigated the possible involvement of acyl-CoA, reactive intermediary metabolites of 2-arylpropionic acids (profens), in protein adduct formation in rat liver homogenate and in human serum albumin (HSA) in buffer. (RS)-[1-14C]-2-Phenylpropionic acid (14C-2-PPA, 1 mM) was incubated with rat liver homogenate (1.5 mg/ml) in the presence of cofactors of acyl-CoA formation (Mg2+, ATP, and CoA). Aliquots of the incubation mixture were analyzed for covalent binding and acyl-CoA formation over a 3-h period. High-performance liquid chromatographic analysis of the products from such incubations showed the presence of 2-phenylpropionyl-S-acyl-CoA (2-PPA-CoA), which was confirmed by coelution with authentic 2-PPA-CoA, as well as by mass spectrometry. In the same incubations, 2-PPA was shown to bind covalently to hepatic proteins in a time- and ATP-dependent fashion. Inhibition of 2-PPA-CoA formation by acyl-CoA synthetase inhibitors, such as palmitic acid, lauric acid, octanoic acid, and ibuprofen, markedly decreased the extent of covalent binding of 2-PPA to hepatic proteins. Results from these in vitro studies strongly suggest that acyl-CoA thioester derivatives are chemically reactive and are able to bind covalently to tissue proteins in vitro, and, therefore, may contribute significantly to covalent adduct formation of profen drugs in vivo.     

Covalent binding of DBCP to proteins in vitro

[¹⁴C] 1,2-Dibromo-3-chloropropane (DBCP) was incubated with rat liver 9000 × g supernatant in order to investigate the covalent binding of DBCP to proteins. Radioactivity incorporated into proteins was dependent on the microsomal oxidase system, but was not prevented by sufficient puromycin to inhibit protein synthesis. From these results DBCP was considered to bind to proteins covalently after activation by microsomal oxidase.     

Covalent cross-linking of proteins by carbon disulfide.

Carbon disulfide is known to react with amino groups of proteins to generate dithiocarbamates (2). We observed covalent cross-linking of dithiocarbamate-derivatized proteins under physiological conditions which may occur through several mechanisms. Evidence for the structure of these covalent bridges and the reactive intermediate was obtained using 13C NMR spectroscopy in conjunction with specific isotopic labeling. On incubation at 37 degrees C oxidative coupling of dithiocarbamates generated bis(thiocarbamoyl) disulfides (3) which were reduced by cysteine. In addition, an electrophilic isothiocyanate (4) was generated from decomposition of the dithiocarbamate. Nucleophilic addition of sulfhydryl and amine moieties to the isothiocyanate produced dithiocarbamate ester (5) and thiourea linkages (6), respectively. Evidence for the presence of inter- and intramolecular cross-links was obtained using denaturing polyacrylamide gel electrophoresis under reducing conditions. The formation of isothiocyanate in neutral solution, through elimination of sulfhydryl ion, was correlated with increased pKa values of the parent amine of amino acids. Dithiocarbamates derived from terminal amino groups of proteins did not appear to generate isothiocyanate or form thiourea or dithiocarbamate ester. Both the thiourea and the dithiocarbamate ester were stable at reduced pH, whereas in alkaline media the thiourea was stable but dithiocarbamate ester was hydrolyzed. Although the disulfide and ester linkages were formed more rapidly than the thiourea, generation of the latter appeared to be irreversible, leading to its gradual accumulation over a longer period of time. Generation of isothiocyanate by CS2-derived dithiocarbamates and subsequent covalent cross-linking of proteins may provide a molecular mechanism for CS2-induced axonopathy.     

Covalent modification of lipids and proteins in rat hepatocytes and in vitro by thioacetamide metabolites

Thioacetamide (TA) is a well-known hepatotoxin in rats. Acute doses cause centrilobular necrosis and hyperbilirubinemia while chronic administration leads to biliary hyperplasia and cholangiocarcinoma. Its acute toxicity requires its oxidation to a stable S-oxide (TASO) that is oxidized further to a highly reactive S,S-dioxide (TASO(2)). To explore possible parallels among the metabolism, covalent binding, and toxicity of TA and thiobenzamide (TB), we exposed freshly isolated rat hepatocytes to [(14)C]-TASO or [(13)C(2)D(3)]-TASO. TLC analysis of the cellular lipids showed a single major spot of radioactivity that mass spectral analysis showed to consist of N-acetimidoyl PE lipids having the same side chain composition as the PE fraction from untreated cells; no carbons or hydrogens from TASO were incorporated into the fatty acyl chains. Many cellular proteins contained N-acetyl- or N-acetimidoyl lysine residues in a 3:1 ratio (details to be reported separately). We also oxidized TASO with hydrogen peroxide in the presence of dipalmitoyl phosphatidylenthanolamine (DPPE) or lysozyme. Lysozyme was covalently modified at five of its six lysine side chains; only acetamide-type adducts were formed. DPPE in liposomes also gave only amide-type adducts, even when the reaction was carried out in tetrahydrofuran with only 10% water added. The exclusive formation of N-acetimidoyl PE in hepatocytes means that the concentration or activity of water must be extremely low in the region where TASO(2) is formed, whereas at least some of the TASO(2) can hydrolyze to acetylsulfinic acid before it reacts with cellular proteins. The requirement for two sequential oxidations to produce a reactive metabolite is unusual, but it is even more unusual that a reactive metabolite would react with water to form a new compound that retains a high degree of chemical reactivity toward biological nucleophiles. The possible contribution of lipid modification to the hepatotoxicity of TA/TASO remains to be determined.