Acyl derivatives of demethylpenclomedine, an antitumor-active, non-neurotoxic metabolites of penclomedine.

PURPOSE: The purpose of this investigation was to compare the antitumor activities of a series of acyl derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma metabolite of penclomedine (PEN) observed to be an active antitumor agent in vivo and non-neurotoxic in a rat model with that of DM-PEN. METHODS: Acyl derivatives were prepared from DM-PEN and evaluated in vivo against human MX-1 breast tumor xenografts implanted subcutaneously (s.c.) or intracerebrally (i.c.). Several derivatives were also evaluated against other human tumor xenografts and murine P388 leukemia cell lines. RESULTS: Several of the acyl derivatives were found to be superior to DM-PEN against MX-1, human ZR-75-1 breast tumor, human U251 CNS tumor and the P388 leukemia parent cell line and lines resistant to cyclophosphamide and carmustine. 4-Demethyl-4-methoxyacetylpenclomedine showed inferior activity to current clinical brain tumor drugs against a glioma cell line, superior activity to temozolomide and procarbazine against the derived mismatch repair-deficient cell line, and superior activity to cyclophosphamide and carmustine but inferior activity to temozolomide against two ependymoma cell lines, all of which were implanted s.c. CONCLUSION: Proposed mechanisms of activation and action of DM-PEN and the acyl derivatives support the potential clinical superiority of the acyl derivatives.

Synthesis and evaluation of several new (2-chloroethyl)nitrosocarbamates as potential anticancer agents.

Seven new (2-chloroethyl)nitrosocarbamates have been synthesized as potential anticancer alkylating agents. These compounds were designed with carrier moieties that would either act as prodrugs or confer water solubility. All compounds were screened in an in vitro panel of five human tumor cell lines: CAKI-1 (renal), DLD-1 (colon), NCI-H23 (lung), SK-MEL-28 (melanoma), and SNB-7 (CNS). Several agents showed good activity with IC(50) values in the range of 1-10 microg/mL against at least two of the cell lines. One compound, carbamic acid, (2-chloroethyl)nitroso-4-acetoxybenzyl ester (3), was selected for further study in vivo against intraperitoneally implanted P388 murine leukemia. In addition to the aforementioned compound, both carbamic acid, (2-chloroethyl)nitroso-4-nitrobenzyl ester (9) and carbamic acid, (2-chloroethyl)nitroso-2,3,4, 6-tetra-O-acetyl-1-alpha,beta-D-glucopyranose ester (24) were evaluated against subcutaneously implanted M5076 murine sarcoma in mice. None of these compounds were active in vivo.

DNA guanine-guanine crosslinking sequence specificity of isophosphoramide mustard, the alkylating metabolite of the clinical antitumor agent ifosfamide.

PURPOSE: The purpose of this investigation was to determine the base sequence specificity of isophosphoramide mustard (IPM), the alkylating metabolite of ifosfamide, by crosslinking of designed DNA oligomers in comparison with the clinical alkylating agents mechlorethamine (ME) (nitrogen mustard) and phosphoramide mustard (PM), the alkylating metabolite of cyclophosphamide. METHODS: IPM, as well as PM and ME were each reacted with three dodecameric duplexes, which were designed to detect interstrand crosslinking between guanines in 5'-GC-3' (I), 5'-GNC-3' (II) or 5'-GNNC-3' (III) sequences (N = A or T). RESULTS: All three agents preferentially react with 5'-GNC-3' target sequences. The 5'-GNNC-3' target sequence is less reactive by a factor of approximately 2.5- to 10-fold, while 5'-GC-3' is of even lower reactivity. CONCLUSION: These results indicate that all three agents show approximately equal preference for reaction with a 5'-GNC-3' target sequence in spite of the fact that IPM yields a 7-atom crosslink, while the other two agents yield 5-atom crosslinks.

Mutations induced by monofunctional and bifunctional phosphoramide mustards in supF tRNA gene.

The relative mutagenicity, nature of the mutations and the sequence specificity of mutations induced by the bifunctional alkylating agent, phosphoramide mustard (PM) and a monofunctional derivative, dechloroethyl phosphoramide mustard (dePM), were analyzed by the Ames test and by an in vitro shuttle vector mutagenesis assay. Both PM and dePM increased the mutation frequency above background in either assay. However, on an equimolar basis, dePM was less mutagenic than PM. In the in vitro shuttle vector mutagenesis assay, sequencing demonstrated that about 40% of the mutant plasmids contained more than one mutation in the supF tRNA gene segment of the plasmid. About 70% of the mutations observed in dePM-treated plasmids were single base substitutions with A:T and G:C base pairs being mutated at equivalent rates. In contrast, only about 50% of the mutations observed in PM-treated plasmids were single base substitutions, 80% of which involved G:C base pairs. Single base deletions and insertions were found in approximately equal proportions with both compounds; however, these lesions were in greater abundance in PM-treated plasmids. Putative hot-spots for mutation in the supF tRNA gene included base pairs at position 102 and 110 for PM and positions 170 and 171 for dePM.

Phase I and pharmacologic study of penclomedine, a novel alkylating agent, in patients with solid tumors.

PURPOSE: To determine the maximum-tolerated dose (MTD), principal toxicities, and pharmacologic behavior of penclomedine, a novel alkylating agent. PATIENTS AND METHODS: Penclomedine (45 to 550 mg/ m2/d every 3 weeks) was administered as a 1- or 3-hour intravenous (IV) infusion for 5 consecutive days to patients with solid tumors. RESULTS: On a 1-hour dosing schedule, ataxia, vertigo, nystagmus, and a motor aphasia were the principal toxicities of penclomedine. These neurologic effects were dose-related, and evolved from complaints of somnolence and dizziness, to more pronounced signs and symptoms of cerebellar dysfunction. Up to and including doses of 415 mg/m2, these effects were well tolerated and resolved within 2 hours posttreatment. In contrast, both patients treated at the 550-mg/m2 dose level experienced a dose-limiting constellation of perinfusional aphasia and vertigo, with either ataxia of over 2 weeks' duration or recurrent dizziness. Prolongation of the infusion duration to 3 hours at this dose level resulted in less neurotoxicity; however, delayed trilineage hematologic toxicity precluded timely administration on this schedule. A statistically significant relationship was demonstrated between the development of ataxia and maximum plasma concentrations of penclomedine. CONCLUSION: Neurotoxicity was the dose-limiting toxicity (DLT) of penclomedine administered as a 1-hour infusion daily for 5 days every 3 weeks, and the recommended dose for further evaluations was 415 mg/m2. The nature of the principal toxicities and the lack of any detectible antitumor activity indicate that phase II evaluations of penclomedine on this administration schedule should be focused on specific disease settings, such as breast cancer and intracerebral tumors, in which antitumor activity has been demonstrated.

4-Demethylpenclomedine, an antitumor-active, potentially nonneurotoxic metabolite of penclomedine.

Penclomedine [3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl)pyridine], an antitumor agent, is currently in Phase I clinical trials and is believed to be a prodrug. In these studies, cerebellar effects have been dose limiting. Previous studies identified 4-demethylpenclomedine (4-DM-PEN) as the major plasma metabolite in rodents and humans. 4-DM-PEN was demonstrated to be an antitumor-active metabolite of penclomedine in vivo when evaluated against the penclomedine-sensitive MX-1 human breast tumor xenograft implanted either s.c. or intracerebrally and is believed to be on the metabolic activation pathway of penclomedine. Because earlier studies revealed an absence of neurotoxic cerebellar effects for 4-DM-PEN in contrast to penclomedine in a rat model, this metabolite may be a candidate for an alternative to penclomedine in the clinic for treatment of breast cancer or brain tumors, if the cerebellar effects of penclomedine preclude its further clinical development. Because neither penclomedine nor 4-DM-PEN were very active in vitro, the metabolism of penclomedine was also investigated using rat liver microsomes in an attempt to identify the ultimate active form of the drug. Metabolites and putative metabolites were prepared by chemical synthesis for antitumor evaluation in vitro and in vivo. A reductive metabolite, alpha,alpha-didechloro-PEN, was observed to be much more cytotoxic than penclomedine or 4-DM-PEN in vitro, but evaluation of this and the other metabolites and putative metabolites in vivo against the MX-1 tumor failed to identify any active metabolite among the structures evaluated other than 4-DM-PEN. The limited activity of 4-DM-PEN in vitro indicates that it, like penclomedine, is also a prodrug, demonstrating a need for additional studies on the metabolic activation of penclomedine to identify the ultimate active form of the drug.

Quantification of 4-hydroxyifosfamide in plasma of ifosfamide-treated mice.

PURPOSE: Ifosfamide is becoming an important clinical anticancer drug. Meaningful pharmacology studies require quantification of its activated and active metabolites, 4-hydroxyifosfamide (HOIfos) and isophosphoramide mustard (IPM), respectively. METHODS: Current methodology for quantifying the unstable HOIfos in biological fluids consists of trapping acrolein as it is produced during the decomposition of this metabolite. However, unlike cyclophosphamide, ifosfamide is extensively metabolized to two dechloroethylated metabolites, which are susceptible to 4-hydroxylation and similarly are capable of yielding acrolein upon decomposition. Because the current method has the potential to yield higher than actual values for HOIfos, it was compared with an HOIfos-specific method that traps the first stage degradation product of HOIfos, aldoifosfamide, as its semicarbazone, and depends on the use of radiolabeled drug for quantification. Six experiments in mice were conducted with blood collection 15 or 30 min after drug treatment followed by determination of HOIfos in plasma by the two methods. RESULTS: Comparison of plasma levels of HOIfos determined by the two methods indicated only minor differences between the two. CONCLUSION: These results suggest the possibility that the nonspecific method may be acceptable as a first estimation of levels of this metabolite in biological fluids until the development of a specific method that does not require radiolabeled drug, such as high-performance liquid chromatography or gas chromatography/mass spectrometry, has been developed.

Tissue and tumor distribution of C-penclomedine in rats.

Penclomedine, a lipophilic alpha-picoline derivative, is undergoing clinical development presently because of its pronounced antitumor activity against intracerebral (i.c.) tumor xenografts. Penclomedine may be metabolized in vivo to a more potent compound. Although it may be useful in the treatment of brain tumors, the drug has caused significant neurotoxicity in early clinical trials. The possibility that antitumor activity and neurotoxicity may be mediated by different mechanisms prompted a study assessing the differential distribution of penclomedine and penclomedine metabolites to brain and i.c.-implanted tumors in rats. In the present study, quantitative autoradiographic analysis demonstrated a homogenous distribution of 14C-penclomedine in all organs within 1 h of administration. Levels of 14C-penclomedine in both i.c. and s.c. tumors were three times higher than in normal brain tissue. High-performance liquid chromatography combined with gas chromatography and mass spectrophotometry demonstrated that two metabolites, O-demethyl penclomedine and penclomic acid, were responsible for most of the plasma radioactivity. Penclomic acid was also the most common urinary metabolite of penclomedine. In liver samples, although a large number of metabolite peaks were detected, no parent compound could be identified. However, in tumors and all other tissues, penclomedine was the main compound detected. The finding of penclomedine in normal brain tissue indicates not only that this drug may be useful in tumors with normal blood-brain barrier function, but also that it may be directly neurotoxic.

Antitumor activity of halogen analogs of phosphoramide, isophosphoramide, and triphosphoramide mustards, the cytotoxic metabolites of cyclophosphamide, ifosfamide, and trofosfamide.

A series of halogen analogs of phosphoramide mustard, isophosphoramide mustard, and triphosphoramide mustard, the cytotoxic metabolites of the antitumor drugs cyclophosphamide, ifosfamide, and trofosfamide, respectively, was evaluated in vitro against human tumor cell lines and in vivo against experimental tumors to investigate the effect of replacement of chlorine with bromine or fluorine on the antitumor activity of the parent phosphoramide mustards. In the experimental tumors L1210 leukemia, B16 melanoma, mammary adenocarcinoma 16/C, and ovarian sarcoma M5076, the antitumor activity of the analogs was observed to be generally comparable with that of the parent mustards when chlorine was replaced by bromine but uniformly lower when chlorine was replaced by fluorine. Furthermore, the monobromo analog of isophosphoramide mustard displayed equal or somewhat greater activity in comparison with cyclophosphamide when evaluated against subcutaneously implanted L1210 leukemia with intraperitoneal drug treatment and against mammary adenocarcinoma 16/C.

Metabolism and disposition of a thiazolobenzimidazole active against human immunodeficiency virus-1.

This study was undertaken to evaluate the disposition of the thiazolobenzimidazole, 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-a]benzimidazole (TZB), which has promising antiviral activity. For mice, the maximum tolerated intravenous dose of TZB was 50 mg/kg. An HPLC procedure developed for TZB was used to determine the distribution of the drug. TZB showed no measurable binding to plasma proteins. With intravenous dosing, the kinetic values for TZB in plasma and in each of five tissues were similar in that there was an initial, short alpha-phase (1.8-7.2 min) and a longer beta phase (38-68 min). The concentrations in liver were higher than those in plasma and other tissues. For mice dosed subcutaneously with TZB, the AUC value for plasma was considerably lower than that for mice dosed intravenously; mice dosed intraperitoneally had higher plasma levels of the drug than after oral or subcutaneous dosing. No intact drug could be detected in the plasma of mice dosed topically. After intravenous, oral, or subcutaneous dosing, urinary excretion of intact TZB was < 2% of the dose. Of several vehicles tested in an attempt to increase the plasma levels of unchanged TZB in mice dosed orally, 40% hydroxypropyl beta-cyclodextrin was most effective. Two metabolites present in plasma and urine of mice were tentatively identified as the axial and equatorial sulfoxide isomers of TZB; a third, minor metabolite, was tentatively designated as the sulfone. Although the compound has activity against HIV-1, its low solubility and extensive metabolism reduce its potential for clinical use.

32P-postlabeling of acrolein-deoxyguanosine adducts in DNA after nuclease P1 digestion.

In order to study the relationship between the level of acrolein-DNA adducts and their biological effects, sensitive methods are needed to quantitate DNA adducts. 32P-postlabeling is one such method that has been widely used and we have adapted the technique to detect acrolein-deoxyguanosine adducts. Adducts formed by the reaction of acrolein and deoxyguanosine-3'-monophosphate were isolated by HPLC. Based on their UV spectra and cochromatography with standards after dephosphorylation with acid phosphatase, these adducts were identified as the nucleotide equivalents of cyclic 1,N2-propanodeoxyguanosine adducts formed by acrolein that have been described by Chung et al. [15]. As nucleotides, the adducts were good substrates for polynucleotide kinase-mediated transfer of phosphate from ATP and were able to be detected by 32P-postlabeling. These adducts were resistant to the activity of nuclease P1 and dinucleoside monophosphates in the form d(G*pN) where G* is the acrolein-guanine adduct also resisted digestion by nuclease P1. Digestion of DNA by nuclease P1 and acid phosphatase resulted in the conversion of normal nucleotides to nucleosides and selective enrichment of the adducts as dinucleoside monophosphates. Using nuclease P1/acid phosphatase digestion, followed by 32P-postlabeling and TLC separation, levels of the two adducts in acrolein-treated DNA were found to be about 6185 and 19,222 nmol/mol.

Lack of ranitidine effects on cyclophosphamide bone marrow toxicity or metabolism: a placebo-controlled clinical trial.

We previously reported that cimetidine but not ranitidine significantly enhances cyclophosphamide-induced bone marrow toxic effects and the appearance of cyclophosphamide alkylating species in a murine leukemia mouse model, and we advised caution in the use of cimetidine with microsomally metabolized anticancer drugs. Both drugs have been used for the treatment of gastric complications of chemotherapy. Using a randomized, double-blind, crossover study design, we have now evaluated the potential interaction of ranitidine with cyclophosphamide in seven cancer patients, who received two courses of cyclophosphamide, one with ranitidine and one with placebo. Four patients received ranitidine in the first course, and three received placebo. Ranitidine or placebo was started 3 days before a single dose of cyclophosphamide and given for 17 consecutive days. Ranitidine or placebo was given orally (300 mg/d), and cyclophosphamide (600 mg/m2) was given intravenously with [3H]cyclophosphamide (1000 muCi). Cyclophosphamide treatment was repeated at 4 weeks plus or minus 4 days. Blood samples were collected at intervals from 5 minutes to 24 hours after cyclophosphamide treatment and analyzed by thin-layer chromatography and radioassay for the drug and its metabolites. On days 0, 7, 14, and 21 after cyclophosphamide administration, complete blood cell counts, white blood cell differential counts, platelet counts, and SMA-17 were determined. The differences in mean nadir white blood cell counts, granulocyte counts, hemoglobin levels, and hematocrit values during ranitidine versus placebo treatment were not statistically significant. In a statistical but not a clinical sense, mean nadir platelet counts were significantly lower with ranitidine. There was a statistically significant increase in area under the curve for drug concentration in plasma x time (AUC) with ranitidine as well as a statistically significant decrease in the total-body clearance rate of the cyclophosphamide molecule. However, the effect on AUC for the major oncolytic metabolites 4-hydroxycyclophosphamide and phosphoramide mustard was not statistically significant. The lack of toxicologic or metabolic interaction between ranitidine and cyclophosphamide suggests that ranitidine can be used safely with cyclophosphamide.

Identification of 7-(2-hydroxyethyl)guanine as a product of alkylation of calf thymus DNA with clomesone.

Evidence at the molecular level is presented in support of alkylation of O6-guanine moieties of DNA as the mechanism of cytotoxicity of Clomesone to HT-29 cells and consists in the isolation and identification of a product resulting from alkylation of calf thymus DNA with Clomesone, followed by depurination to yield 7-(2-hydroxyethyl)guanine, whose formation is reasonably explained by O6-guanine chloroethylation followed by intramolecular alkylation at N7 of guanine and subsequent hydrolysis to the hydroxyethylguanine.

Disposition and metabolism of carbovir in mice dosed intravenously or orally.

To determine the disposition of carbovir and [3H]carbovir in mice, HPLC and thin-layer chromatographic assays were developed and mice were dosed iv and by gavage. Carbovir had no lethal effect at iv doses up to 500 mg/kg and was stable for 24 hr in mouse plasma at temperatures ranging from 0-37 degrees C. Binding to plasma proteins was minimal. Following an iv dose of 500 mg/kg of carbovir or [3H] carbovir, elimination phases with half-lives of 26-37 min (alpha) and 206-330 min (beta) were observed for plasma. For mice dosed with 27 mg/kg of [3H]carbovir, however, only a single phase with a half-life of 17 min was noted. Of several tissues examined, kidney contained the highest concentration of radioactivity. For the high dose, 19.0 +/- 2.6% was excreted in the urine in 24 hr as unchanged carbovir and 42.2 +/- 2.4% as metabolites; for the low dose, 54.5 +/- 6.1% was excreted as carbovir and 26.5 +/- 5.0% as metabolites. When mice were dosed orally with 500 mg/kg, plasma concentrations of carbovir were low. The initial plasma half-life for carbovir was 69 min; the terminal half-life was 822 min. Urinary excretion of unchanged carbovir was 21.3 +/- 7.1%. These results indicate that clearance of high doses of carbovir is limited and that its absorption is poor after oral dosing.

32P-postlabeling analysis of binding of the cyclophosphamide metabolite, acrolein, to DNA.

The cyclophosphamide metabolite, acrolein, was reacted with 2'-deoxyguanosine-3'-monophosphate, and two adducts were detected by high performance liquid chromatography and 32P-postlabeling assay. These adducts were resistant to dephosphorylation by nuclease P1 and could be isolated and detected from calf thymus DNA that had been reacted in vitro with acrolein. A combination of HPLC purification and enzymatic digestion of normal nucleotides by nuclease P1 allowed for the detection of these adducts in hepatic DNA from mice treated with cyclophosphamide. The level of the two adducts in the hepatic DNA, as determined by 32P-postlabeling, was one adduct per 2.7-4.1 x 10(7) normal nucleotides.

The effect of cimetidine on cyclophosphamide metabolism in rabbits.

Six female rabbits were given 20 mg/kg cyclophosphamide (containing 100 microCi [3H-chloroethyl]-cyclophosphamide) alone or 1 h following 100 mg/kg cimetidine. Serial plasma and urine specimens were collected and levels of cyclophosphamide and its metabolites (4-hydroxycyclophosphamide, 4-ketocyclophosphamide, phosphoramide mustard, and carboxyphosphamide) were measured. 4-Ketocyclophosphamide was the major metabolite present in rabbit plasma and urine, with lesser amounts of 4-hydroxycyclophosphamide, carboxyphosphamide, and phosphoramide mustard also being identified. Cimetidine pretreatment resulted in prolongation of cyclophosphamide's half-life from 24.3 +/- 7.3 to 33.5 +/- 9.5 min (mean +/- SD; P = 0.036) but did not significantly alter the AUC0-8 h for the latter drug. Cimetidine pretreatment resulted in a significantly greater AUC0-8 h for 4-hydroxycyclophosphamide (189.4 +/- 77 vs 364.6 +/- 126.7 mumol min/l-1; P = 0.016) as compared with control values. A higher AUC0-8 h value for phosphoramide mustard (53.7 +/- 69.2 vs 95.7 +/- 34.7 mumol min/l-1) was also observed after cimetidine dosing but the difference was not significant (P = 0.21). Kinetics of 4-ketocyclophosphamide and carboxyphosphamide were not significantly affected by cimetidine treatment. Cimetidine was added to hepatic microsomes isolated from phenobarbital-treated rabbits; it did not inhibit cyclophosphamide's metabolism in vitro, suggesting that its in vivo effect may be mediated through mechanisms other than cytochrome P-450 inhibition. Cimetidine pretreatment increases exposure to cyclophosphamide and its major activated metabolite, 4-hydroxycyclophosphamide. Potentiation rather than inhibition of cyclophosphamide's pharmacodynamic effect is to be predicted when cimetidine is given concomitantly with the former. Alterations in hepatic blood flow or mechanisms other than microsomal inhibition by cimetidine may explain this potentiation.

Formation of a phosphoramide mustard-nucleotide adduct that is not by alkylation at the N7 position of guanine.

The reaction of 2'-deoxyguanosine 3'-monophosphate with phosphoramide mustard resulted in the formation of several adducts. One of these adducts was formed by linking phosphoramide mustard to the phosphate group of 2'-deoxyguanosine 3'-monophosphate rather than by the generally accepted mechanism involving alkylation at the N7 position of guanine. This adduct served as an acceptor for the transfer of 32p from [gamma 32P]ATP by polynucleotide kinase and thus could be detected by the sensitive 32p-postlabeling assay.

Induction of sister-chromatid exchanges in L1210 leukemia cells by new antitumor 2-haloethyl(methylsulfonyl) methanesulfonate compounds.

The antitumor 2-halo(chloro, bromo, and fluoro)-ethyl(methylsulfonyl) methanesulfonates, ethyl(methylsulfonyl) methanesulfonate, and chlorozotocin, a 2-chloroethylnitrosourea, were evaluated for their potential to induce SCEs in L1210 cells. The results indicate that all the compounds induced approximately 2-fold or greater increases in SCEs in a dose-related manner. 2-Chloroethyl(methylsulfonyl) methanesulfonate, a DNA-interacting agent and a drug selected for clinical trials, exhibited the highest SCE increase in these cells.

The effect of "activated" cyclophosphamide on rat granulosa cells in vitro.

We investigated the mechanism of cyclophosphamide (CTX)-induced ovarian toxicity by studying the effect of an activated form, 4-hydroperoxycyclophosphamide (PCTX), on rat granulosa cells in vitro. Cells were obtained from PMSG-primed immature rats and incubated with PCTX at concentrations of 1, 10, 100, and 500 micrograms/mL. Ovine LH (10 ng/mL) was added in selected tubes. Cell viability before and after seven hours incubation was determined. Progesterone and prostaglandin E accumulation were measured by radioimmunoassay. Granulosa cell viability was significantly decreased at PCTX concentrations of 10 micrograms/mL or higher in a dose-related manner. PCTX at concentrations of 100 micrograms/mL and 500 micrograms/mL significantly decreased basal and LH-induced progesterone and prostaglandin E accumulation. The above findings demonstrate that cyclophosphamide metabolites decrease granulosa cell survival and function in vitro. These direct effects suggest a possible mechanism for CTX-induced premature ovarian failure.

Lipid peroxidation induced by cyclophosphamide.

Intraperitoneal administration of a single dose of cyclophosphamide (CP) to rats was found to produce hepatic glutathione depletion and to enhance NADPH-mediated lipid peroxidation in the 15,000 x g supernatant fraction of the liver. These effects were associated with CP in a dose- and a time-dependent manner. The data suggest that the glutathione depletion is, at least in part, responsible for the enhancement in lipid peroxidation induced by CP.