Dose-dependent pharmacokinetics of N-5-dimethyl-9-[(2-methoxy-4-methylsulphonylamino)phenylamino]-4-acridinecarboxamide (CI-921) in rabbits

Summary N-5-dimethyl-9-[(2-methoxy-4-methylsulphonylamino)phenylamino]-4-acridinecarboxamide (CI-921), which is an analogue of amsacrine, has entered phase 1 clinical trials as an antitumour drug. The plasma pharmacokinetics of CI-921 has been studied in six rabbits after short i. v. infusions of 6.35, 12.7 and 25.4 mol/kg. Total plasma concentrations of CI-921 were determined by a high-performance liquid chromatography method for up to 12 h post infusion. Comparison of pharmacokinetic parameters for each rabbit by within-subject analysis of variance indicated that with a four-fold increase in the dose from 6.35 to 25.4 mol/kg there was a 44% increase in the area under the concentration-time curve normalised to dose (P(0.001) and a 43% increase in the elimination half-life (P(0.005), and a 30% decrease in the total plasma clearance (P(0.001). Dose had no effect on the end of infusion concentration normalised to dose, or on the steadystate volume of distribution. These results indicate that CI-921 experiences dose-dependent elimination kinetics in the rabbit.This study was supported by a grant from the Medical Research Council of New Zealand     

The clinical pharmacokinetics of N-5-dimethyl-9-[(2-methoxy-4-methyl-sulfonylamino)phenylamino]-4-acridinecarboxamide (CI-921) in a phase 1 trial

Summary The pharmacokinetics of CI-921 were studied after 65 infusions over a 20-fold dose range (13–270 mg/m² per day) in 16 patients during a phase 1 trial. CI-921 was given by a 15 min infusion on three consecutive days.Plasma samples were collected after the first and third infusions, and urine, at 6 h intervals throughout the 3 days. CI-921 concentrations were measured by an HPLC method. Maximum plasma concentrations ranged from 3–86 mol/l.The plasma concentration-time disposition curves were mainly biphasic over the 24-h postinfusion period. There was no significant difference by the paired t-test between the C_max, AUC,CL, V_ss, MRT, t_1/2, or t_1/2 calculated for the first and third infusions. The means (range) of model-independent pharmacokinetic parameters were: CL, 158 (94–290) ml/h per kg; V_ss, 319 (219–614) ml/kg; MRT, 2.1 (1.1–3.5) h; t_1/2, 0.5 (0.2–1.1) h; and t_1/2, 2.6 (1.1–5.0) h. There was a strong linear correlation between the dose and the AUC and C_max,suggesting linear kinetics over this dose range. A very small amount (<1%) of the total dose was excreted as unchanged CI-921 in the urine, mostly in the 12-h postinfusion period.     

Synthesis, antitumor activity, and DNA binding properties of a new derivative of amsacrine, N-5-dimethyl-9-[(2-methoxy-4-methylsulfonylamino) phenylamino]-4-acridinecarboxamide

The 4-(N-methylcarboxamido)-5-methyl derivative of amsacrine (NSC 249 992) has been synthesized as part of a program aimed at optimizing solid tumor activity in this series. Physicochemical studies of this analogue (N-5-dimethyl-9-[(2-methoxy-4-methylsulfonylamino) phenylamino]-4-acridinecarboxamide; NSC 343 499) indicate a slightly increased lipophilicity (estimated log p = 1.10), a decreased acridine base strength (pKa 6.40), and a 16-fold-higher association constant for double-stranded calf thymus DNA (Ka 2.1 X 10(6) M-1 at 0.01 ionic strength). Like amsacrine, the drug binds to DNA by intercalation. Inhibition of cell growth has been monitored by continuous drug exposure assays with a variety of rodent and human cell lines. The concentration for 50% inhibition varied from 6.7 nM (T-47D, a human breast carcinoma line) to 800 nM (P388/ADR, a murine cell line resistant to Adriamycin). N-5-Dimethyl-9-[(2-methoxy-4-methylsulfonylamino) phenylamino]-4-acridinecarboxamide was cytotoxic at growth-inhibitory concentrations and also induced cell cycle arrest in the G2 phase. It was active against P388 leukemia following administration by p.o., i.v., or i.p. routes, and it was superior to amsacrine, daunorubicin, and Adriamycin. It was curative towards i.v.-injected Lewis lung tumor in a proportion of animals when treatment was started on Day 1 or Day 5 after tumor inoculation. It also produced highly significant life extensions against advanced tumors (treatment starting Day 9 after i.v. inoculation or on Day 8 after s.c. inoculation) and was comparable to cyclophosphamide in its effectiveness. It is a candidate drug for clinical trial.     

The effect of buthionine sulphoximine,cimetidine and phenobarbitone on the disposition of amsacrine in the rabbit

Summary Evidence suggests that the main elimination pathway for amsacrine is hepatic oxidation to the quinone diimine derivative followed by conjugation with glutathione (GSH) and excretion in the bile. If this is so, amsacrine elimination should be susceptible to induction by phenobarbitone (PB) and inhibition by cimetidine (CT) and perhaps by buthionine sulphoximine (BSO), a specific depleter of tissue GSH. This study was carried out in groups of six rabbits. Each rabbit acted as its own control and received pretreatment with saline or PB, CT, or BSO, followed by an amsacrine infusion. Blood (8x3 mL) was collected up to 12 h and total plasma amsacrine concentrations determined by HPLC. PB pretreatment resulted in a significant increase in amsacrine's Cl (mean 46%, range 25%–70%) and also in the Vd (mean 58%, range 25%–117%), but had no effect on t_1/2, t_1/2 or MRT_ni. In addition, there was no change in the plasma protein binding of amsacrine after PB pretreatment. CT pretreatment had the opposite effect, resulting in a significant decrease in amsacrine's Cl (mean 33%, range 21%–38%) and a decrease in Vd, although this latter decrease was not significant at the 5% level. As with PB, the time parameters were not significantly changed. BSO pretreatment resulted in a significantly reduced Cl (mean 22%, range 15%–30%), no effect on Vd or on t_1/2, but significantly prolonged t_1/2 and MRT_ni. BSO pretreatment was also associated with a significant reduction in red blood cell GSH concentration. These results are consistent with the involvement of the hepatic mixed function oxidase system and GSH status in the elimination of amsacrine in the rabbit.The work described in this paper was supported by a grant from the Medical Research Council of New Zealand     

Phase I trial of the amsacrine analogue 9-[( 2-methoxy-4-[(methylsulfonyl)amino]-phenyl]amino)-N,5-dimethyl-4- acridinecarboxamide (CI-921)

CI-921, an analogue of amsacrine with superior activity against in vivo and in vitro experimental tumor models, has been studied in 16 patients with solid tumors refractory to chemotherapy or for which conventional therapy does not exist. Thirty-nine cycles were given and doses escalated from 39 to 810 mg/m2. This total dose was divided over 3 consecutive days and administered by 15-min infusion each day, repeated three times weekly. Neutropenia (Eastern Cooperative Oncology Group) Grade greater than or equal to 3 occurred at Day 8 (range, 7-13) in 10/13 courses at 648 mg/m2 and in 2/2 courses at 810 mg/m2 with recovery in 10 (range, 4-20) days. At 810 mg/m2 Grade 2 mucositis and phlebitis were noted. Mild nausea and venous irritation occurred in some patients at doses greater than or equal to 288 mg/m2. No objective response was seen. Pharmacokinetics were evaluated following 65 infusions on Days 1 and 3 with plasma concentrations of CI-921 measured by high performance liquid chromatography. Peak plasma concentrations ranged from 3.36 to 85.6 mumol/liter and were significantly correlated with dose. Mean (range) model-independent pharmacokinetic parameters were: distribution half-life, 0.46 h (0.24-1.08); elimination half-life, 2.63 h (1.08-4.98); mean residence time, 2.0 h (1.05-3.35); plasma clearance, 158 ml/h/kg (95-290); and steady-state volume of distribution, 319 ml/kg (219-614) with no significant difference between Day 1 and 3. Toxicity as defined by absolute granulocyte count nadir was significantly correlated with dose, area under concentration-time curve, and peak plasma concentration. The recommended dose for Phase II studies in this schedule is 648 mg/m2 (216 mg/m2 daily for 3 days) repeated every three weeks.     

Comparison of the pharmacokinetics and protein binding of the anticancer drug,amsacrine and a new analogue,N-5-dimethyl-9-[(2-methoxy-4-methylsulfonylamino)phenyl-amino]-4-acridinecarboxamide in rabbits

Summary Amsacrine (NSC 249992) is a new anticancer drug which, although effective for the treatment of various disseminated tumors, has shown disappointing activity against most solid tumors. A new analogue, N-5-dimethyl-9-[(2-methoxy-4-methylsulfonylamino)phenylamino]-4-acridine-carboxamide (CI-921, NSC 343499) has been identified, which might offer a broader clinical antitumor spectrum. This analogue is more lipophilic (0.5 log p units) and is also a considerable weaker base (pKa 6.40) than amsacrine (pKa 7.43). This study compared the pharmacokinetics of total and unbound amsacrine and CI-921 in plasma after equimolar dose infusions (12.7 mol/kg) in a balanced crossover design in six rabbits. Drug concentrations were determined by high-pressure liquid chromatography and the unbound fraction by equilibrium dialysis. Three fold higher total plasma concentrations were achieved with CI-921 than with amsacrine. However, the unbound fraction was significantly less for CI-921 (0.33%±0.04) than for amsacrine (2.78%±0.53). There was no significant difference between distribution and elimination half-life and mean residence time, but the apparent volume of distribution (means, 121 vs 45 l/kg) and clearance (means, 46.6 vs 16.3 l h^-1 kg^-1) of unbound CI-921 were threefold greater than the corresponding parameters for unbound amsacrine. We suggest that despite higher binding in plasma, the greater distribution or tissue uptake of CI-921 may be partly responsible for its greater anticancer activity in vivo.The work described in this paper was supported by a grant from the Medical Research Council of New Zealand. It was reported on at the 18th Annual Meeting of the Australasian Society of Clinical and Experimental Pharmacologists, Melbourne, Australia, December 1984     

Disposition of amsacrine and its analogue 9-([2-methoxy-4-[(methylsulfonyl)amino]phenyl]amino)-N,5-dimethyl-4- acridinecarboxamide (CI-921) in plasma, liver, and Lewis lung tumors in mice

9-([2-Methoxy-4-[(methylsulfonyl)amino]phenyl]amino)-N,5-dimethyl-4- acridinecarboxamide (CI-921), an analogue of the clinical antileukemia drug amsacrine with improved solid tumor activity in mice, is currently being evaluated in patients. In order to determine whether CI-921 possesses any advantages over amsacrine in terms of tissue delivery, the pharmacokinetics of amsacrine and CI-921 were determined following i.v. injection in male B6D2F1 mice. Plasma kinetics in normal mice were measured following administration of 14.4, 28.9, and 57.7 mumol/kg. The kinetics in s.c. Lewis lung tumors, and in plasma and livers of normal and tumor-bearing mice were measured following administration of 57.7 mumol/kg. CI-921 and amsacrine were quantitated by high-performance liquid chromatography after extraction from plasma and from liver and tumor homogenates. In experiments with appropriate 3H-labeled compounds, both total and covalently bound radioactivity (determined after precipitation and washing with acetonitrile) were measured in plasma and in liver homogenates. Over this dose range, nonlinear kinetics were observed in plasma for unchanged CI-921 and amsacrine, and a reasonable fit was obtained with Michaelis-Menten kinetics to a one-compartment model for CI-921 (Km 3.7 mumol/liter; Vmax 18 mumol/h/kg; V ss 3.3 liter/kg) and a two-compartment model for amsacrine (Km 3.6 mumol/liter; Vmax 76 mumol/h/kg; Vss 4.8 liter/kg). The area under the concentration-time curve (AUC) for plasma following a dose of 57.7 mumol/kg was 31 mumol.h/liter for CI-921 and 6.3 mumol.h/liter for amsacrine. However, equilibrium dialysis measurements indicated high plasma protein binding with free drug fractions for CI-921 and amsacrine of 0.63 and 6.7%, respectively. In the liver, unchanged drug concentrations and total radioactivity for both compounds were approximately 10-fold those in plasma, and the tissue half-life of CI-921 was approximately 4-fold longer for CI-921 than for amsacrine. Plasma and liver kinetics in mice with s.c. Lewis lung tumors were similar to those in normal mice. Tumor half-lives of unchanged CI-921 and amsacrine were 3.9 and 2.7 h, respectively, considerably longer than those for plasma (1.2 and 0.30 h respectively) or liver (1.2 and 0.28 h, respectively). Tumor AUC values for CI-921 and amsacrine were 68 and 37 mumol.h/liter, respectively, as compared to the calculated AUC values for free drug in plasma of 0.19 and 0.42 mumol.h/liter, respectively. It is concluded that the uptake into tumors from the plasma free drug fraction is more efficient for CI-921 than for amsacrine.     

Effects of a new amsacrine derivative, N-5-dimethyl-9-(2-methoxy-4-methylsulfonylamino)phenylamino-4- acridinecarboxamide, on cultured mammalian cells

The effects of N-5-dimethyl-9-(2-methoxy-4-methylsulfonylamino)-phenylamino-4- acridinecarboxamide (CI-921; NSC 343499), a lipophilic and water-soluble derivative of amsacrine (NSC 249992), on cell viability, growth, clonogenicity, and progression through the cell cycle were investigated in suspension cultures of Friend erythroleukemic cells and in in suspension cultures of Friend erythroleukemic cells and in adherent cultures of Chinese hamster ovary cells. CI-921 was less toxic toward stationary than toward exponentially growing Chinese hamster ovary cells; colony formation was inhibited by 50% following a 1-h pulse of 190 versus 80 nM CI-921, respectively. Cell viability was unaffected in Friend erythroleukemic cell cultures at concentrations up to 50 nM, although growth was inhibited by 50% following 24 h of continuous exposure to 9.5 nM or a 1 h pulse of 67.5 nM CI-921. Constant exposure of Friend erythroleukemic cells to 10 nM CI-921 slowed proliferation and resulted in prolongation of cell transit through late S and G2 phases. Higher drug concentrations (50 nM) caused a complete cessation of growth marked by greatly suppressed cell transit through S phase and an irreversible block in G2 phase, about 30 min prior to division. In such cases, unbalanced growth was observed with total RNA and protein content of drug-treated cells increasing by 74 and 34%, respectively. Pulse exposure of cells to CI-921 resulted in transient accumulations of cells in S and/or G2 phase depending upon dose. The cell cycle distribution of stationary cultures treated for 1 h with drug and replated at a low cell density were identical to that of controls. Binding of the drug affected the sensitivity of DNA in situ to acid denaturing conditions which provides additional evidence that CI-921 binds to DNA by intercalation.     

Biotransformation of the bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide in mice

The biotransformation of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), a potent urinary bladder carcinogen, was studied in mice. About 82% of radioactivity was excreted as 14CO2 within 36 hr after intragastric administration of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-[14C]formamide, suggesting its deformylation to 2-amino-4-(5-nitro-2-furyl)thiazole ( ANFT ). The latter was formed in vitro as a product following incubation of FANFT with mouse liver homogenates. Chromatographic analysis of mouse urine obtained 24 hr after the i.p. administration of N-[4-(5-nitro-2-furyl)-[2-14C]thiazolyl]formamide revealed excretion of ANFT and unmetabolized FANFT, suggesting the prevalence of the deformylation reaction in vivo. In addition, at least two more metabolites were present in urine. One of these metabolites exhibited chromatographic properties similar to those exhibited by a compound derived from the in vitro nitroreduction of ANFT . This metabolite was isolated from urine of FANFT-fed animals and from in vitro enzymatic reduction of ANFT with mouse liver homogenates. The isolated products had chromatographic and spectral properties and a mass spectral fragmentation pattern similar to that of a compound obtained by catalytic reduction of ANFT with palladium and activated carbon. Spectroscopic analyses established the structural identity of the chemical reduction product as 1-[4-(2-aminothiazolyl)]-3-cyano-1-propanone ( ATCP ). Since the chromatographic properties of the enzymatically derived product and the urinary metabolite were identical to those of a compound obtained by chemical reduction, they must be structurally the same and thus correspond to ATCP . About 5% of the urinary metabolites of FANFT is ATCP , and thus ATCP is quantitatively a minor excretory product. ATCP was far less active than was ANFT of FANFT in the Ames mutagenicity assay with Salmonella typhimurium TA.     

Pharmacological disposition of 1,4-dihydroxy-5-8-bis[[2[(2-hydroxyethal)amino] ethyl]amino]-9,10-anthracenedione dihydrochloride in the dog

Summary DHAQ, a new antitumor agent, has been selected for clinical trial on the basis of its activity against a number of transplantable rodent tumors. In anticipation of the clinical trial of this agent, the pharmacology of DHAQ was studied in beagles by high-pressure liquid chromatographic and radiochemical techniques that are specific for the unchanged drug. ¹⁴C-DHAQ was administred IV to beagles at a dose of 5 mg/kg, 100–125 Ci total. With a maximal plasma concentration of 75 = 2.7 ng/ml, DHAQ was eliminated from the plasma with a half-life of 28.1 h during the terminal phase. The total clearance of DHAQ was 10.1±0.4 mg/kg/min, while the apparent volume of distribution was 26.6±4.9 l/kg. In 48 h 2.4%±0.6% of the dose was excreted in the urine and 3.0%±0.1% in the bile as the unchanged drug. At autopsy performed 5 h after dosing, the highest percentage of the administered DHAQ was in the liver (49.7%±2.7%), followed by the small intestine (7.1%±0.7%), kidneys (2.7%±0.1%), lung (1.9%±0.3%), spleen (1.6%±0.3%), and stomach (1.3%±0.1%). The heart, large intestine, pancreas, gallbladder, urinary bladder, and brain each retained less than 1% of the dose. However, 24 h after dosing 10.6% of the drug was detected in the liver and 2.9% in the small intestine. In terms of the percentage of the dose, the distribution of DHAQ in the other organs either remained unchanged or increased slightly. In concentrations varying from 10 ng/ml to 10 g/ml the drug was 70%–80% bound to plasma protein. DHAQ was metabolized to two unidentified metabolites. Thus, this drug appeared to be cleared from the plasma of beagle dogs chiefly by tissue binding, leading to possible persistence of the drug in certain body compartments.     

Metabolism of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide by prostaglandin endoperoxide synthetase

Cooxidative metabolism of the urinary bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) was examined using solubilized and particulate microsomal preparations from the rabbit renal inner medulla and the ram seminal vesicle. Metabolism was measured by the rate of decrease in absorbance at 400 nm. In these soluble and particulate preparations, FANFT metabolism was observed only in the presence of specific fatty acids. These fatty acids are substrates for prostaglandin endoperoxide synthetase. Structurally dissimilar inhibitors of prostaglandin endoperoxide synthetase such as indomethacin, aspirin, 5,8,11,14-eicosatetraynoic acid, ethoxyquin, and meclofenamic acid specifically inhibited FANFT metabolism. Other inhibitor and substrate specificity studies suggest that FANFT was not metabolized by nitroreductase, xanthine oxidase, lipoxygenase, lipid peroxidation, or mixed-function oxidases. In addition, the lack of detectable 2-amino-4-(5-nitro-2-furyl)thiazole formation suggests that arylformamidase was not participating in FANFT metabolism measured in these experiments. The data indicate that prostaglandin endoperoxide synthetase can mediate FANFT metabolism by a cooxidative process.     

Effect of p-hydroxyacetanilide, sodium sulfate, and L-methionine on the leukemogenicity of N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide

Dietary administration of N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide to mice for 14 weeks followed by 16 weeks of control diet resulted in a high incidence of lymphocytic leukemia and a low incidence of forestomach squamous cell papillomas. The coadministration of p-hydroxyacetanilide at a dose of 1.0% with either 250 or 500 ppm of N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide resulted in inhibition of leukemogenesis, whereas when p-hydroxyacetanilide was coadministered with 1000 ppm of N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide the leukemia incidence was not significantly reduced, but the latent period was prolonged. When sodium sulfate was administered with p-hydroxyacetanilide and N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide, leukemogenesis was partially restored. L-Methionine, fed in place of sodium sulfate, unblocked leukemogenicity inhibition by p-hydroxyacetanilide. None of these chemicals, p-hydroxyacetanilide, sodium sulfate, or L-methionine, significantly affected the incidence of forestomach papillomas induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide, although tumor incidences in all groups were low. p-Hydroxyacetanilide and sodium sulfate had no significant effect on the high incidence of stomach tumors induced by formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl]hydrazide or bladder tumors induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide.     

Metabolic activation of the carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide by prostaglandin H synthase

It has been demonstrated that N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide(NFTA), when fed with the diet, causes transitional carcinomasin rats. An important step in the mechanism of NFTA-inducedcarcinogenesis is endogenous metabolic activation to an ultimatecarcinogen. We have proposed that the enzyme complex prostaglandinH synthase (PHS) is involved in the activation of certain renaland urinary tract carcinogens. This proposal was assessed byexamining the activation of the 5-nitrofuran renal carcinogenNFTA and its deacetylated analogue 2-amino-4-(5-nitro-2-furyl)thiazole(ANFT) by PHS. Ram seminal vesicular and rabbit renal innermedullary microsomes were used as a source of PHS. Both NFTAand ANFT were activated by PHS to bind microsomal protein. Bothmicrosomal preparations activated ANFT to bind DNA. However,only ram seminal vesicular microsomes activated NFTA to bindDNA. The rate of ANFT binding to macromolecules was considerablygreater than NFTA with both microsomal preparations. Althoughactivated ANFT was shown to bind several different homopolynucleotides,a preference for binding polyguanylic acid was demonstrated.Glutathione inhibition of carcinogen binding to macromoleculeswas shown to be due to the formation of a thioether conjugate.Deacetylation of NFTA was demonstrated in both tissues withdeacetylation significantly exceeding acetylation of ANFT toNFTA in the kidney. Thus, renal PHS activation of both NFTAand ANFT was demonstrated with the rate of ANFT activation beingconsiderably greater than NFTA. The conversion of NFTA to ANFTby intact tissue suggests that ANFT may contribute to NFTA renalcarclnogenesis.