Synthesis and evaluation of novel spermidine derivatives as targeted cancer chemotherapeutic agents.

The utility of the spermidine moiety as the homing device for the selective delivery of chemotherapeutic and diagnostic agents into cancer cells was explored. Two spermidine analogs containing a cytotoxic agent were synthesized, N-[3,4-bis(benzyloxy)phenethyl]-N alpha-(3-amino-propyl)-L-ornithinamide trihydrochloride, 1a and N-[4-]bis(2-chloroethyl)amino]phenethyl]-N alpha-(3-aminopropyl)-L- ornithinamide tetrahydrochloride, 1b. These compounds were prepared from the fully protected spermidine molecule with a carboxyl group side chain, 8. The ability of the polyamine cytotoxic agents to inhibit B16-BL6 melanoma cell growth in culture was examined. The effects of pretreatment with DFMO on the activity of the synthesized compounds was also studied. The IC50 values of compounds 1a and 1b were on the same order of magnitude as the control compounds, N-acetyldopamine and chlorambucil, respectively. The inhibitory activities of compounds 1a and 1b were not enhanced by pretreatment with DFMO, suggesting that depletion of intracellular polyamines did not enhance the activity of these compounds.

Biliary excretion of a glutathione conjugate of busulfan and 1,4-diiodobutane in the rat.

gamma-Glutamyl-beta-(S-tetrahydrothiophenium)alanyl-glycine, the glutathione-sulfonium conjugate of busulfan and 1,4-diiodobutane, was identified in the bile of rats following intravenous administration of equimolar doses of either compound. The glutathione-sulfonium conjugate was synthesized from 1-bromo-4-chlorobutane and characterized by 1H and 13C NMR and FAB/MS. An HPLC method was developed to identify the conjugate from rat bile by pre-column fluorescent derivatization with o-phthalaldehyde. The biliary excretion of cyclic sulfonium conjugates was quantitated indirectly by measuring the release of tetrahydrothiophene (THT) after treatment of the bile with base. THT release was quantitative and was measured by gas chromatography. With busulfan, peak biliary concentrations of THT-releasing metabolite(s) were reached after 90 min and 26% of the dose of busulfan was recovered in the bile after 8 hr. When diiodobutane was administered, 21% of the dose was recovered, and the peak concentration was reached in 30 min. The decline in THT releasing metabolite(s) was more rapid with 1,4-diiodobutane, and THT was no longer measurable after 3.5 hr compared to 7.5 hr after busulfan administration. These data confirm that busulfan and other 1,4-disubstituted butanes are conjugated with glutathione in vivo.

Delivery of paraldehyde in 5% dextrose and 0.9% sodium chloride injections through polyvinyl chloride i.v. sets and burettes.

The delivery of paraldehyde in 5% dextrose injection and 0.9% sodium chloride injection was studied, and the potential interaction between paraldehyde and plastic i.v. containers and sets was evaluated. Paraldehyde was mixed with either 5% dextrose injection or 0.9% sodium chloride injection in polyvinyl chloride (PVC) bags to form a 4% solution. The bags were fitted with standard i.v. administration sets or burettes with administration sets. The solutions were allowed to drip through the i.v. sets for six hours at room temperature. Samples were taken from the i.v. bag or burette and from the distal end of the i.v. sets at zero, two, four, and six hours. Paraldehyde concentrations were measured using a stability-indicating gas chromatographic method, and the presence of plasticizers was detected by a scanning ultraviolet spectrophotometer. The cumulative amount of paraldehyde delivered at the end of the administration set at six hours was 84% for 5% dextrose solutions in burettes, and 89% or 90% for all other solutions and i.v. sets. An ultraviolet-light-absorbing substance appeared in some of the samples, although a relationship between the presence of this substance and type of solution, time of sampling, or site of sample did not emerge. Particulate matter appeared after two hours in all burettes. Approximately 10%-16% of paraldehyde in 5% dextrose or 0.9% sodium chloride injection is lost when delivered from PVC i.v. bags through standard i.v. administration sets and burettes over a six-hour period.(ABSTRACT TRUNCATED AT 250 WORDS)

The disposition and pharmacokinetics of ketoconazole in the rat.

The disposition, biliary excretion, and pharmacokinetics of ketoconazole in Sprague-Dawley rats were determined after intravenous administration. Greater than 80% of the radioactivity after a 5 mg/kg iv dose of 3H-ketoconazole was excreted in the feces. Urinary excretion was essentially complete after 48 hr; however, fecal excretion was prolonged over a 7-day period. Biliary excretion of radioactivity averaged 54.3 +/- 18.0% of the dose over a 7.5-8-hr period in pentobarbital-anesthesized rats. The possibility of enterohepatic recirculation was examined using a linked rat technique. Less than 2% of the radioactivity was found in the recipient bile over 9-12 hr. In eight male rats, the plasma pharmacokinetics of ketoconazole, as determined by an HPLC assay with fluorescence detection, were as follows: VD = 655 +/- 91 ml/kg, Cl = 14.4 +/- 5.1 ml/min/kg, and t 1/2 = 35.0 +/- 12.3 min. Three of the rats were given an additional oral dose to determine absolute bioavailability. The time to peak was 30-60 min, and the bioavailability was 35.8 +/- 3.55%. Previous studies have indicated that ketoconazole is well absorbed in rats; therefore, the poor bioavailability is probably due to first pass metabolism. The prolonged fecal excretion of radioactivity from an intravenous dose was probably caused by slow elimination of ketoconazole metabolites.

Stereochemical aspects of the glutathione S-transferase-catalyzed conjugations of alkyl halides.

(R,S)-2-iodooctane and (R,S)-2-bromooctane were found to be substrates for the glutathione S-transferases from rat liver. The conjugation reactions of the enantiomeric 2-halooctanes and glutathione were found to proceed with inversion of configuration at the chiral carbon of the substrate. Selective titration of the free cysteine residues of the glutathione S-transferases provided no observable effect on the stereochemical course of these conjugation reactions. No evidence for substrate stereoselectivity was observed. The diastereomeric S-(2-octyl)glutathiones were produced in approximately equal amounts from racemic 2-halooctane substrates. With S-(+)-2-iodooctane as the electrophilic substrate, a biphasic double reciprocal plot of glutathione concentration vs. initial velocity of product formation was observed suggesting complex kinetics. The S-2-octylglutathione diastereomers were found to be potent inhibitors of the glutathione S-transferase-catalyzed conjugation of 1-chloro-2,4-dinitrobenzene. These results provide support for a single displacement mechanism for the conjugation of 2-halooctanes and glutathione catalyzed by the glutathione S-transferases with product inhibition at low glutathione concentrations.

Glutathione S-transferases catalyzed conjugation of 1,4-disubstituted butanes with glutathione in vitro.

Rat liver glutathione S-transferases catalyzed the conjugation of 1,4-diiodobutane with glutathione in vitro. The reaction followed saturation kinetics and was dependent on the concentration of the enzyme, substrate and glutathione in the incubation media. S-Benzylglutathione inhibited the enzymatic conversion of 1,4-diiodobutane to product. The cyclic sulfonium compound, gamma-glutamyl-beta-(S-tetrahydrothiophenium) alanyl-glycine was identified as the product of this conjugation reaction. This product was stable under physiological conditions in presence of rat liver cytosol but rapidly and quantitatively decomposed at pH greater than or equal to 12 to give tetrahydrothiophene.

Circadian rhythmicity of polyamine urinary excretion.

Polyamines are small, highly charged, organic cations of possible regulatory importance in RNA-dependent protein synthesis, the production of which reflects cellular growth and division. The cytokinetics of normal cell populations is circadian rhythmic. This is reflected by a circadian rhythmicity undescribed previously in urinary monoacetylputrescine and the ratio of N1-acetylspermidine to N8-acetylspermidine in healthy individuals. Patients harboring advanced cancers sometimes excrete abnormal quantities of certain acetylated polyamines, and their urine samples may exhibit an abnormally high ratio of N1-acetylspermidine to N8-acetylspermidine. Changes in polyamine production and excretion associated with cancer may be best perceived by rhythmometric analysis of carefully timed samplings.

Stereochemical aspects of conjugation reactions catalyzed by rat liver glutathione S-transferase isozymes.

Substrate enantioselectivity in the conjugation of phenethyl halides catalyzed by the glutathione S-transferases was studied with partially purified isozymes from rat liver. All of the isozymes tested possessed measurable activity with phenethyl chloride. Tranferase A was the most active isozyme tested. Each of the isozymes demonstrated a high degree of substrate enantioselectivity, with transferase A being the most enantioselective isozyme. The enantioselectivity was determined by high-pressure liquid chromatographic analysis of the enzymatically formed diastereomeric products. The effect of limiting glutathione concentrations on the stereochemical outcome of the transferase A catalyzed conjugation of the chiral substrate, (S)-phenethyl chloride (4 mM), was investigated. The stereochemical course of the enzymatic reaction was not significantly altered at glutathione concentrations as low as 25 microM. The major product of conjugation had the opposite stereochemistry at the benzylic carbon, indicating that the reaction proceeded primarily with inversion of configuration. The glutathione conjugates, S-[(R)-1-phenylethyl]glutathione, S-[(S)-1-phenylethyl]glutathione, S-benzylglutathione, and S-methylglutathione were studied as inhibitors of the transferase A catalyzed conjugation of 1-chloro-2,4-dinitrobenzene. The order of the inhibitory potency was S-[(S)-1-phenylethyl]glutathione = S-benzylglutathione greater than S-[(R)-1-phenylethyl]glutathione greater than S-methylglutathione. This represented the first demonstration of the stereoselective product inhibition of the glutathione S-transferases.

Urinary excretion of monoacetyl polyamines in patients with non-Hodgkin's lymphoma.

The pretreatment concentrations of polyamines were determined in the 24-hr urine of 14 patients with widespread non-Hodgkin's lymphoma. In ten of 14 patients, the ratio of N1-acetylspermidine to N8-acetylspermidine was significantly higher than the mean for normal subjects. These results confirm our previous observations that the urinary excretion of N1-acetylspermidine is increased in some patients with lymphoma and suggest that the determination of urinary acetyl polyamines may be useful in conjunction with other procedures in the diagnosis of lymphoma. The ratio of N1-acetylspermidine to N8-acetylspermidine in the postchemotherapy 24-hr urine was 25 in one patient who had diffuse histiocytic lymphoma. This is the highest ratio ever reported. The patient responded well to chemotherapy, and rapid lysis of lymphoma lesion was observed. The potential utility of the rapid increase in the ratio of N1-acetylspermidine to N8-acetylspermidine as a criterion of tumor lysis is of interest and is currently under further investigation.

Inhibitors of polyamine biosynthesis. 9. Effects of S-adenosyl-L-methionine analogues on mammalian aminopropyltransferases in vitro and polyamine biosynthesis in transformed lymphocytes.

Seven analogues of S-adenosyl-L-methionine were studied as inhibitors or substrates for mammalian spermidine and spermine synthases. One of these, S-(5'-deoxy-5'-adenosyl)-(+/-)-1-methyl-3-(methylthio)propylamine (5), showed a unique spectrum of activities on the polyamine biosynthesis enzymes. It was an inhibitor of S-adenosyl-L-methionine decarboxylase from rat liver and spermine synthase from bovine brain and rat ventral prostate. This compound was a substrate for the spermidine synthases from bovine brain and rat ventral prostate but not a substrate for the spermine synthases from these same sources. At concentrations of 0.2 mM and higher, compound 5 blocked the increases in polyamine levels and in [3H]thymidine incorporation induced by concanavalin A in cultured mouse lymphocytes. At approximately a 0.5 mM concentration of 5, the cellular polyamine levels and the rate of thymidine incorporation were similar to those of the unstimulated lymphocytes. Lower concentrations of 5 (0.02-0.1 mM) produced a dose-dependent increase in thymidine incorporation. A dose-dependent decrease in the cellular polyamine levels was observed in the range of 0.05-0.5 mM of the inhibitor. These results suggest that the effects of 5 on transformed lymphocytes are complex and may not be solely due to the inhibition of polyamine biosynthesis by this compound.

Determination of monoacetyldiamines and -polyamines in urine by high-performance liquid chromatography.

A procedure is described for the determination of monoacetylputrescine, N1-acetyl-spermidine and N8-acetylspermidine in human urine. The procedure is based on the high-performance liquid chromatographic separation of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivatives of these amines using two different chromatographic modes. Monoacetyl-1,6-diaminohexane was used as an internal standard. The amines were extracted from urine using a silica gel cartridge. The dansyl monoacetylpolyamines were separated from the mixture of dansyl derivatives of urinary amines on a bonded-phase CN column using a programmed solvent gradient elution. The dansyl acetylpolyamines were rechromatographed on a silica gel column. This chromatographic procedure was used for the determination of the concentration of N1-acetylspermidine, N8-acetylspermidine and monoacetylputrescine in the urine of healthy volunteers and cancer patients.

Inhibitors of polyamine biosynthesis VII: Evaluation of pyruvate derivatives as inhibitors of S-adenosyl-L-methionine decarboxylase.

The mechanism of the enzymatic decarboxylation of S-adenosyl-L-methionine catalyzed by S-adenosyl-L-methionine decarboxylase and its inhibition by methylglyoxal bis(guanylhydrazone) were investigated. The results indicate that the carbonyl group of the pyruvate cofactor does not form an azomethine bond with an amino group of the enzyme protein. The substrate and/or product forms an azomethine bond with the pyruvate cofactor, which can be reduced efficiently with sodium cyanoborohydride. Methylglyoxal bis(guanylhydrazone) appears to interfere with the formation of the enzyme--substrate complex by competing with the substrate for binding with the active enzyme site. The dimethylaminoethylhydrazone, semicarbazone, and guanylhydrazone derivatives of pyruvic acid, ethyl pyruvate, pyruvic acid amide, and pyruvyl glycineamide were synthesized. None of these compounds had significant inhibitory activity on the enzymatic decarboxylation of S-adenosyl-L-methionine by S-adenosyl-L-methionine decarboxylase from rat liver in vitro. These results indicate that the structural requirements for binding of methylglyoxal bis(guanylhydrazone) to the enzyme are strict and that structural modifications of this compound result in a dramatic loss of activity.

Polyamine metabolism III: urinary acetyl polyamines in human cancer.

Polyamine levels were determined by high-pressure liquid chromatography in the unhydrolyzed 24-hr urine obtained from 15 cancer patients and nine normal subjects. N1-Acetylspermine, N4-acetylspermine, N4-acetylspermidine, and N-(3-aminopropyl)acetamide were not detected in any samples. N1-Acetylspermidine, N8-acetylspermidine, acetylputrescine, and acetylcadaverine were present in all samples. Furthermore, acetylspermidine and acetylputrescine were excreted in much greater quantities than the respective amines in the urine of both cancer patients and normal subjects. The levels of N1-acetylspermidine were considerably higher than the levels of N8-acetylspermidine in the 24-hr urine of cancer patients, resulting in a ratio of N1- to N8-acetylspermidine in the urine of cancer patients significantly higher than that in normal subjects. The urinary levels of acetylputrescine were also significantly higher in cancer patients. The urinary polyamines in 13 of the 15 cancer patients were outside the 95% confidence limits of the normal mean for the ratio of N1- to N8-acetylspermidine and acetylputrescine. All cancer patients showed values outside the 95% confidence limits of the mean for either of these two parameters. Diurnal variation was observed in the urinary excretion of the acetyl polyamines but not for the free amines in two normal subjects.

Inhibitors of polyamine biosynthesis VI: 2,5-Diamino-2-(cyanomethyl) pentanoic acid, a potential irreversible inhibitor of ornithine decarboxylase.

(+/-)-2,5-Diamino-2-)cyanomethyl)pentanoic acid was obtained by the reaction of chloracetonitrile with the anion obtained by treatment of 3-(benzylideneamino)-2-piperidinone with sodium hydride, followed by hydrolysis in the presence of trifluoroacetic anhydride. The target compound was isolated as the monohydrochloride salt of the lactam. The compound was synthesized as a potential irreversible inhibitor of the enzyme L-ornithine decarboxylase by the mechanism generally known as suicide or Kcat inhibition. The synthesized compound produced no inhibition of the enzyme ornithine decarboxylase obtained from rat prostate gland. The inactivity of the target compound is attributed to the hydrophilicity of the cyanomethyl group.