Chlorozotocin. Mechanism of reduced bone marrow toxicity in mice.

Chlorozotocin is a chloroethyl nitrosourea with a glucose carrier that has curative activity for the murine L1210 leukemia, but is nonmyelosuppressive in mice. To determine the mechanism for this unique property of reduced bone marrow toxicity, comparative studies were conducted with chlorozotocin and CCNU, a myelotoxic chloroethyl nitrosourea. Suspensions of L1210 leukemia and murine bone marrow cells were incubated for 2 h with 0.1 mM [(14)C]-chloroethyl chlorozotocin or CCNU. Chlorozotocin demonstrated a fourfold increased covalent binding of the chloroethyl group to L1210 nuclei when compared to equimolar CCNU. Chlorozotocin alkylation of L1210 cells resulted in the binding of 57 pmol of [(14)C]ethyl group/mg of DNA, which represented a 2.3-fold increased alkylation when compared to CCNU. In marked contrast, the binding of the chloroethyl group to bone marrow nuclei was equivalent for both drugs. In addition, chlorozotocin alkylation of murine bone marrow DNA, 45 pmol of [(14)C]ethyl group/mg of DNA, was equivalent to that of CCNU. The ratio of L1210:bone marrow DNA alkylation was 1.3 for chlorozotocin compared to 0.6 for CCNU. The intracellular carbamoylation of L1210 and bone marrow protein by CCNU was 400- to 600-fold greater than that produced by chlorozotocin. After a 2-h exposure to 0.1, 0.05, or 0.01 mM drug, both chlorozotocin and CCNU produced a reduction in the cloning efficiency of L1210 cells that was dose dependent. However, chlorozotocin was significantly more cytotoxic than CCNU at all three molar concentrations (P < 0.01). Chlorozotocin, 0.1 mM, reduced L1210 DNA synthesis to 1% of control by 48 h, in contrast to 16% with equimolar CCNU (P < 0.01). In mice bearing 10(5) L1210 cells, chlorozotocin produced its optimal antitumor activity (332% increased life span [ILS]) at doses of 48-64 mumol/kg, with >50% indefinite survivors. In contrast, CCNU at the same molar doses resulted in only a 191% ILS; a CCNU dose of 128 mumol/kg was required for comparable optimal L1210 antitumor activity, 413% ILS. On a molar basis, the dose of chlorozotocin that produced optimal in vivo L1210 antitumor activity was one-third to one-half that of CCNU. Chlorozotocin, unlike CCNU, produced no murine bone marrow toxicity at its optimal therapeutic dose. This unique combination of antitumor activity without myelosuppression can be correlated with the advantageous ratio of L1210:bone marrow in vitro DNA alkylation by chlorozotocin (1.3) as compared to equimolar CCNU (0.6).

Streptozotocin diabetes. Correlation with extent of depression of pancreatic islet nicotinamide adenine dinucleotide.

The diabetogenic activity of streptozotocin has been correlated with a reduction in pyridine nucleotide synthesis in the mouse pancreatic islet. To determine the specificity of this reduction for diabetogenicity, a comparative study of streptozotocin, its cytotoxic moiety, 1-methyl-1-nitrosourea, and alloxan was performed. Streptozotocin administered intraperitoneally (i.p.) producd a dose-related reduction in islet NAD which was proportional to the degree of diabetogenicity. A diabetogenic dose, 200 mg/kg, attained a peak plasma N-nitroso intact streptozotocin concentration of 0.224 mumol/ml and reduced the mean islet NAD from a control of 0.78 to 0.15 pmol. At borderline, 150 mg/kg, and nondiabetogenic, 100 mg/kg, doses, plasma concentrations reached 0.161 and 0.136 mumol/ml, and NAD was 0.36 and 0.86 pmol/islet, respectively. 1-Methyl-1-nitrosourea, 100 mg/kg, attained a maximum N-nitroso intact 1-methyl-1-nitrosourea concentration of 0.162 mumol/ml and reduced the mean NAD to 0.58 pmol/islet, and was nondiabetogenic; 200 mg/kg attained a peak plasma concentration of 0.344 mumol/ml and depressed NAD to 0.38 pmol/islet, and was inconsistently diabetogenic. Islet NAD of 0.4 pmol/islet or greater is required for integrity of the beta cell. A diabetogenic dose of alloxan, 500 mg/kg, did not depress NAD, 0.85 pmol/islet, therefore confirming that its mechanism of diabetogenicity differs from that of streptozotocin. In vivo uptake of [methyl-(14)C]streptozotocin by islets was 3.8 times that of [methyl-(14)C]-1-methyl-1-nitrosourea, whereas uptake by the exocrine pancreas favored 1-methyl-1-nitrosourea over streptozotocin 2.4:1. The decreased islet uptake of 1-methyl-1-nitrosourea correlates with the 3.5 times increased molar dosage required to produce islet NAD depression comparable to that of streptozotocin, 150 mg/kg. These studies indicate that the glucose carrier of streptozotocin facilitates uptake of its cytotoxic group, 1-methyl-1-nitrosourea, into islets.

Mutagenic activity of nitrosourea antitumor agents.

The relative mutagenic activities of chloroethyl-nitrosourea and methylnitrosourea antitumor agents in active clinical use were determined with the use of the Ames Salmonella typhimurium assay. The results indicated that the drugs induced base substitutions. 2-Deoxy-2-[[(methylnitrosoamino)carbonyl]amino]-D-glucopyranose (also called streptozotocin), a glucose-containing methylnitrosourea, was the most mutagenic of all compounds tested and showed at least a 250-fold increase in activity when compared to that of its chloroethyl analog, 2-[[[(2-chloroethyl) nitrosoamino]carbonyl]-amino]-2-deoxy-D-glucose (also called chlorozotocin). All nitrosoureas, with the exception of N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2-chloroethyl)-N-nitrosourea monohydrochloride, a pyrimidine chloroethyl analog, demonstrated an increase in mutagenicity after incubation with induced Sprague-Dawley rat liver microsomes. No correlation between in vitro chemical alkylating activity and mutagenic potential was observed. Mutagenic activity was not observed to be of predictive value for antitumor activity in the L1210 leukemia model system.

Antitumor activity and bone marrow toxicity of aminoglucose mustard anticancer agents in mice.

In previous structure-activity studies, we have demonstrated that attachment of a glucose molecule to the chloroethylnitrosourea cytotoxic group produces a compound with reduced murine bone marrow toxicity and retention of full antitumor activity. To further define this protective role conferred by the glucose moiety in bone marrow cells, we have replaced the nitrosourea cytotoxic group with another class of alkylating agent, a bifunctional nitrogen mustard. In a detailed structure-activity analysis, we have now characterized four analogues, with the mustard cytotoxic group positioned at carbon 2 [1,3,4,6-tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranos e (TGM)], carbon 6, or carbon 1 (D- and L-isomers) of the aminoglucose molecule. On a molar basis, TGM was most toxic to normal BALB/c X DBA/2 F1 mice, with a 10% lethal dose (LD10) of 3.8 mumol/kg. The D- and L-isomers of 2,3,4,6-tetra-O-acetyl-N,N-bis(2-chloroethyl)glucopyranosylamine (C-1) were the least toxic, with an LD10 of 73 mumol/kg for both. Optimal antitumor activity against the murine P388 leukemia (single i.p. administration of the LD10) did not differ significantly among the four analogues, with increased life span ranging from 83-86%. P388 antitumor activity for nitrogen mustard (HN2) was significantly less, 60% increased life span (P = 0.01), while p-di(2-chloroethyl)amino-L-phenylalanine produced an increased life span of greater than 101%. An LD10 of 6-bis-(2-chloroethyl) amino-6-deoxy-D-glucose (C-6) or TGM produced significantly less depression of WBC counts than did an equitoxic dose of the C-1 isomers, HN2, or p-di(2-chloroethyl)amino-L-phenylalanine. The mean nadir WBC count for C-6 equaled 86% of control, and for TGM, 80% of control. Consistent with this sparing effect on the peripheral WBC, C-6 and TGM produced significantly less in vivo murine bone marrow DNA synthesis depression, 77 and 64% of control, respectively, as compared to the depression nadir produced by HN2 (27% of control), the D-isomer of C-1 (17%), the L-isomer of C-1 (18%), and p-di(2-chloroethyl)amino-L-phenylalanine (2%). These structure-activity studies demonstrate that conjugation of the mustard cytotoxic group to carbon 6 or carbon 2 of glucose produces an analogue that retains P388 antitumor activity significantly greater than that of HN2, with a concomitant reduction in murine bone marrow toxicity.

A comparison of the biological and biochemical properties of 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea and 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose.

1-4-Amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) is a water-soluble nitrosourea that has produced delayed hematological toxicity in man during Phase 1 clinical trials. ACNU has in vitro alkylating activy 40% less than that of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin) but shares the property of negligible carbamoylating activity with the latter compounds. ACNU is highly active against murine L1210 leukemia. However, the maximum therapeutic dose, 30 mg/kg (a dose lethal to 10% of the animals), produced a 64% reduction in peripheral WBC and an 85% decrease in circulating neutrophils in normal mice. This was correlated with a 76% inhibition of normal mouse bone marrow DNA synthesis within 24 hr after treatment, followed by full recovery within 48 hr after treatment, followed by full recovery within 48 hr. In contrast, DNA synthesis in L1210 cells was suppressed to less than 10% of control for a minimum of 72 hr. While ACNU, a pyrimidine analog, possesses many of the chemical properties of chlorozotocin, it does not share with the latter compound its reduced myelotoxicity at therapeutic doses. The glucose carrier of the chlorozotocin molecule appears to impart the selective sparing of normal bone marrow.

Chlorozotocin, 2-(3-(2-chloroethyl)-3-nitrosoureido)-D-glucopyranose, an antitumor agent with modified bone marrow toxicity.

Chlorozotocin, 2-(3-(2-chloroethyl)-3-nitrosoureido)-D-glucopyranose, is a newly synthesized, water-soluble nitrosourea antitumor agent that is active against L1210 leukemia in mice. A 701% and a 401% increase in life-span were attained with a dose that was lethal to 10% of the animals (15 to 20 mg/kg, i.p.) in mice treated on Day 2 or Day 6 of L1210 tumor growth, respectivley. Sixity % of Day 2-treated mice and 30% of Day 6-treated mice survived for 90 days. At the maximally effective dose against L1210, chlorozotocin produced no significant depression in normal bone marrow DNA synthesis nor in peripheral neutrophil count, in contrast to a sustained greater than 90% inhibition in L1210 ascites cell DNA synthesis. If the antitumor activity and reduced bone marrow toxicity of chlorozotocin are confirmed in man the use of this compound would facilitate treatment of patients with neoplastic disease who have preexisting abnormal bone marrow function or would allow for the more effective use of a nitrosourea agent in combination with anticancer agents possessing more potent myelosuppressive properties.

Doxorubicin-induced chronic cardiotoxicity and its protection by liposomal administration.

The chronic cardiotoxicity of doxorubicin as a free drug or entrapped in positive and negative liposomes was morphologically evaluated in mice treated seven times i.v. at a dose of 4 mg/kg. Liposomes were composed of phosphatidylcholine, cholesterol, and stearylamine (positive charge) or phosphatidylserine (negative charge). Administration of free doxorubicin caused a pattern of cardiac damage characterized by loss of myofiber elements, mitochondrial damage, nuclear abnormalities, swollen and distended sarcoplasmic reticulum leading to vacuolization, and increasing myeloid body accumulation. Cardiac tissues of mice treated with doxorubicin entrapped in negatively charged liposomes demonstrated pronounced loss of filaments, enlarged mitochondria, disruptive loss of crests, and expanded nuclear membrane. However, electron microscopic examination of the cardiac muscles of mice treated with positive liposomes demonstrated a significant protection from drug-induced toxicity, with only minor loss of parallel fibrillar arrangement and myofilaments in limited focal areas. The majority of the tissue demonstrated normal vasculature and intercalation of myocytes as compared to control groups. The mean qualitative and quantitative scores of toxic lesions for free doxorubicin and entrapped in negative liposomes are 2.7 and 2.23, respectively. However, the mean score for the group of mice treated with positive liposomes is only 1.12, showing a better than 2-fold scoring protection of both the extent and severity of cardiac lesions.

Biological and biochemical properties of 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea (NSC D 254157), a nitrosourea with reduced bone marrow toxicity.

1-(2-Chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea (GANU), a water-soluble nitrosourea, differs from 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin) by the placement of the cytotoxic group on C-1 of glucose. Its biological and biochemical properties are compared with those of chlorozotocin. At a 10% lethal dose (10 mg/kg i.p.), GANU demonstrates minimal myelosuppression. This dose failed to depress normal bone marrow DNA synthesis, in contrast to a 96% inhibition in L1210 DNA synthesis. In L1210 cell suspension, equimolar doses of GANU and chlorozotocin produced equivalent degrees of inhibition in DNA synthesis. GANU has significant L1210 activity in BALB/c X DBA/2 F1 mice treated on Day 2 of tumor growth. A 117% increased life-span and 15% 45-day survivors are atained with 15 mg/kg i.p., a 50% lethal dose. However, in concurrent studies using randomly selected littermate groups of mice, GANU proved less active than chlorozotocin which produced a 306% increased life-span (15 mg/kg i.p.). GANU and chlorozotocin have similar in vitro alkylating activity but the in vitro carbamoylating activity of GANU is sevenfold that of chlorozotocin. On a molar basis, the lethal toxicity of GANU is twice that of chlorozotocin. The significant carbamoylating activity of GANU may contribute to its greater toxicity and therefore limit the mumoles of alkylating agent that can be administered to the tumor. These structure-activity studies further confirm that the addition of a glucose carrier to a cytotoxic nitrosourea moiety can selectively reduce bone marrow toxicity while retaining antitumor activity.

Pharmacological, toxicological, and therapeutic evaluation in mice of doxorubicin entrapped in cardiolipin liposomes.

Doxorubicin possesses high affinity for binding to cardiolipin. We have utilized these properties in preparing stable liposomes of doxorubicin and cardiolipin with a net positive charge. Doxorubicin liposomes were formed by using 11.2 mumol of drug, 5.6 mumol of cardiolipin, 28.5 mumol of phosphatidylcholine, 19.5 mumol of cholesterol, and 11.1 mumol of stearylamine. These liposomes were sonicated for 90 min at 37 degrees followed by extensive dialysis against buffer. The pharmacological, toxicological, and therapeutic effects of doxorubicin entrapped in cardiolipin liposomes were compared with those of free doxorubicin in mice. At a dose of 4 mg/kg i.v., the peak cardiac concentration was achieved in 30 min following free doxorubicin administration, the value being 8.1 micrograms/g. The peak cardiac concentration with doxorubicin in cardiolipin liposomes was obtained at 5 min with a value of 2.8 micrograms/g of tissue. The cardiac concentration X time values for free doxorubicin for the 24-hr period of observation were 55.1 micrograms X hr/g, whereas it was only 7.8 micrograms X hr/g with the drug entrapped in cardiolipin liposomes. Compared to free drug, the liposomal entrapped doxorubicin significantly reduced the histopathological lesions in cardiac tissue of mice at a dose of 15 mg/kg as determined by electron microscopy. The nadir of peripheral white blood cell counts in mice with free drug, 6 mg/kg, was observed on Day 3 which was 50% of control, whereas with liposomal encapsulated drug it was reduced only 23% on Day 7. Doxorubicin in cardiolipin liposomes demonstrated enhanced chemotherapeutic potential against murine ascitic P388 leukemia with a 144% increased life span compared to 55% increased life span with free drug at a dose of 7.5 mg/kg on Days 1, 3, and 7. We conclude that doxorubicin liposomes developed in these studies possess improved therapeutic action as demonstrated by their ability to reduce the toxicity of the drug substantially.

6-[Bis(2-chloroethyl)amino]-6-deoxygalactopyranose hydrochloride (C6-galactose mustard), a new alkylating agent with reduced bone marrow toxicity.

We have previously reported that chloroethyl nitrosourea and nitrogen mustard bone marrow toxicity can be selectively reduced by placement of the cytotoxic group on specific positions of a glucose molecule. We have now synthesized and evaluated a new drug in which the mustard cytotoxic group is attached to the carbon-6 position of galactose (C6-GLM). C6-GLM, administered i.p. as a single 10% lethal dose of 15.5 mg/kg, produced a 121% increase in life span (ILS) in mice bearing the ascitic P388 leukemia, compared to a 60% ILS with a 10% lethal dose of nitrogen mustard (P less than 0.01). A single p.o. dose of C6-GLM, 16 mg/kg, produced an ILS of 58%. Against i.p.-implanted B-16 melanoma, i.p. C6-GLM produced a 56% ILS compared to 30% with an equitoxic dose of nitrogen mustard (P less than 0.01). The activity of the two drugs for Ehrlich ascites was comparable, with 60% survivors with the galactose mustard. A single 10% lethal dose of C6-GLM reduced the white blood cells to 74% of control; circulating granulocytes remained at 91% of initial values. With nitrogen mustard, the nadir white blood cell count was 57% of control with an absolute granulocyte count of 70% of initial values (P less than 0.01). The toxicity of melphalan was considerably greater, with a lower and more protracted while blood cell nadir and an absolute neutrophil count nadir of 49% of control. These findings paralleled the relative decrements in bone marrow DNA synthesis produced by the three drugs. Measurement of human bone marrow granulocyte-macrophage colony-forming units, following in vitro exposure to graded concentrations of the three mustards, confirmed the bone marrow sparing properties of C6-GLM. At the highest concentration, 1 X 10(-2) mM, the latter drug produced only a 33% reduction in colonies compared to a 75% reduction with nitrogen mustard and a virtual elimination of activity of colony-forming units with melphalan. The demonstration of antitumor activity, at least equivalent to nitrogen mustard, without the necessity of significant bone marrow toxicity supports the development of C6-GLM for clinical trials in humans.

Biological and biochemical properties of the 2-hydroxyl metabolites of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

The lethal and bone marrow toxicity and antitumor activity of the cis- and trans-2-hydroxylated metabolites of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) have been correlated with their relative in vitro alkylating and carbamoylating activities. Both the isomers have considerably greater alkylating activity and shorter chemical half-lives than the parent compound and on a molar basis have greater antitumor activity against i.p. L1210 leukemia. However, in terms of molar doses resulting in the death of 10% of normal mice, the cis- and trans-2 isomers were 2- and 3-fold more toxic than was CCNU in normal mice. In comparing the antitumor activity produced by a maximum nonlethal dose for each compound, we found that the trans isomer had activity identical to that of CCNU (413 and 410% increased life span compared to control), and the cis isomer had considerably less (152%). Like chlorozotocin, both isomers possess low carbamoylating activity and increased water solubility, two features that have been considered possible contributors to the bone marrow-sparing character of chlorozotocin. However, in the murine model the human bone marrow colony formation (CFU-C) assay neither hydroxylated metabolite of CCNU was associated with reduced myelotoxicity.

Pharmacological disposition of 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea in mice.

1-(2-Chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU; NSC 95466) is a lipid-soluble nitrosourea that is presently in clinical trial. We have studied the pharmacological disposition of [ethyl-14C]PCNU in mice using an i.v. drug dose of 20 mg/kg/animal. Disappearance of total radioactivity from plasma was biphasic with mean half-lives of the two exponential phases of 21.7 min and 27.4 hr, respectively. The plasma half-life of intact drug was 29 min, and levels of intact drug, as measured by thin-layer chromatography, fell below detectable levels by 4 hr. The area under the plasma concentration-time curve for intact drug was 32.72 nmol X hr/ml. Computer analysis of the data for total radioactivity (PCNU equivalents), based upon an open two-compartment model, yielded values of the pharmacokinetic parameters K12, K21, and K10 of 1.49 hr-1, 0.25 hr-1, and 0.19 hr-1, respectively. The highest peak organ level of drug was 168.9 nmol of PCNU equivalents per g tissue in the liver 1 hr after drug administration. Maximum levels in kidney, lungs, heart, and spleen were observed at 5 min, with values of 119.5, 115.4, 80.3, and 66.7 nmol of PCNU equivalents per g of tissue, respectively. A high peak drug level in brain (50.6 nmol/g) agreed with the prediction that PCNU can cross the blood-brain barrier. The levels of intact drug relative to total radioactivity at 30 min were 60% in brain, 55% in heart, and 48% in spleen. The concurrent value in liver was 7% of the total radioactivity, suggesting that metabolism or decomposition of PCNU occurs in this organ. The principal excretory route of [ethyl-14C]PCNU was urinary, with a cumulative excretion of 62% in the first 24 hr.

Repair of DNA alkylation induced in L1210 leukemia and murine bone marrow by three chloroethylnitrosoureas.

The removal of DNA adducts is an essential step of DNA repair following exposure to chloroethylnitrosoureas. Adduct removal was evaluated in both L1210 and murine bone marrow DNA for lesions induced by three chloroethylnitrosoureas. 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea, a marrow-toxic agent with high carbamoylating activity, was not removed in either system for at least 6 to 12 hr. These results were compared with those obtained with two glucose-linked chloroethylnitrosoureas, chlorozotocin and 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea. Both of these agents have low marrow toxicity at therapeutic doses. Chlorozotocin, which has very low chemical carbamoylating activity, was found to permit approximately 40% removal of drug-derived DNA adducts in both systems within the first 6 hr and approximately 50% by 18 hr. The second glucose-linked analog, 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, has relatively high carbamoylating activity and was found to inhibit early removal of DNA adducts as effectively as does 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. It would thus appear that the selective marrow-sparing property of the sugar-linked chloroethylnitrosoureas is not dependent upon carbamoylation-mediated differences in the rate and extent of DNA adduct removal. In view of the comparable therapeutic activity of the three drugs for L1210 leukemia, therapeutic efficacy does not appear to be impaired by the increased rate of adduct removal observed with chlorozotocin in this system.

Sensitivity of human and murine hematopoietic precursor cells to 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose and 1,3-bis(2-chloroethyl)-1-nitrosourea.

The sensitivity of mouse and human bone marrow hematopoietic precursor cells [colony-forming units committed to granulocyte-macrophage differentiatin (CFU-C)] was determined after in vitro incubation with chlorozotocin (2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose), or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with the use of drug concentrations achieved during clinical administration. Chlorozotocin, at a concentration of 1 X 10(-4) M, did not decrease mouse CFU-C below the control of 44 colonies/10(5) nucleated cells; 5 X 10(-4) M produced a 70% reduction in CFU-C, and 1 X 10(-3) M chlorozotocin eliminated all colony formation. In contrast, BCNU at 1 X 10(-4) M resulted in a 55% reduction in CFU-C, and at 5 X 10(-4) M it eliminated all colony formation. For human marrow the threshold concentration for chlorozotocin toxicity was 1 X 10(4) M, which resulted in 25% reduction in CFU-C as compared to the control of 32 colonies/10(5) nucleated cells. In contrast, BCNU at 5 X 10(-5) M decreased human CFU-C to 47% of control, and at 1 X 10(-4) M it eliminated all colony formation. Twenty-four hr after in vitro exposure to 1 X 10(-4) M chlorozotocin, there was no reduction in human bone marrow DNA synthesis in contrast to a 42% reduction with 1 X 10(-4) M BCNU. The plasma concentration of drugs following a therapeutic dose in patients was measured and was found to correspond to the concentration range used in the in vitro studies of marrow toxicity.

Prevention of chronic doxorubicin cardiotoxicity in beagles by liposomal encapsulation.

Antitumor drugs such as doxorubicin have been encapsulated into liposomes as a means of enhancing activity and reducing toxicity. The present study was initiated to determine whether chronically administered liposome-encapsulated doxorubicin would be less toxic than the free drug. Doxorubicin was prepared in positively charged cardiolipin liposomes, and 1.75 mg/kg was given i.v. to each of five beagles. A second group received the free drug at 1.75 mg/kg. Additional animals received i.v. injections of either doxorubicin-free liposomes or 0.9% NaCl solution. All substances were given at 3-week intervals, and the experiment ended 1 week after the seventh injection (total dose, 12.25 mg/kg). A temporary reduction in food consumption was noted during the first few days after the administration of either form of doxorubicin. The effect was more severe in the dogs given free doxorubicin, and body weight decreased significantly only in this group of animals. Three dogs given free doxorubicin died or were killed before the end of the study because they were in poor condition. Lesions consisting mainly of vacuolization and myofibrillar loss were noted in the hearts of all five dogs given free doxorubicin. The severity of the lesions ranged from 2 to 4 (average, 3.4). In contrast, no abnormalities were found in any of the hearts from dogs given the liposomal doxorubicin. The most obvious general toxic effect caused by administration of free doxorubicin was alopecia, which was entirely prevented when doxorubicin was encapsulated into liposomes. At the dosage regimen utilized, liposomal doxorubicin and free doxorubicin exerted comparable degrees of bone marrow suppression. Thus, liposomal encapsulation of doxorubicin decreased cardiac and other toxic effects elicited by free doxorubicin. Whether this advantage can be translated into effective antineoplastic activity will need further evaluation.

Nitrosourea interaction with chromatin and effect on poly(adenosine diphosphate ribose) polymerase activity.

Poly(adenosine diphosphate ribose) polymerase, a chromatin-bound enzyme, was stimulated 150 to 200% after treatment of HeLa cells with methylnitrosourea (MNU). In contrast, a slight inhibitory effect on enzyme activity was observed after treatment of cells with various concentrations of chloroethylnitrosoureas. To define precisely the differential effects of nitrosoureas on the enzyme activity, their interactions with chromatin substructure were studied. A nonrandom, in vivo alkylation of chromatin DNA by equimolar concentrations of MNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was revealed by digestion of nuclei from drug-treated cells with micrococcal nuclease and DNase I. [methyl-14C]MNU interacted preferentially with the more accessible regions of chromatin, the internucleosome linkers, whereas, the [chloroethyl-14C]CCNU alkylated the nucleosomal core DNA to a greater extent. These two drugs also differed in their extent of covalent modification of histone and nonhistone chromosomal protein. The binding of MNU to histones was greater than of CCNU. CCNU mainly affected nonhistone proteins. This difference in the reactivity of methyl and chloroethyl nitrosoureas with chromatin may relate to their differential effect on poly(adenosine diphosphate ribose) polymerase activity, as well as to their carcinogenic and antitumor properties.

Phase I trial and clinical pharmacology of 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea.

1-(2-Chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (NSC 95466) is a lipid-soluble nitrosourea that is presently undergoing clinical evaluation. In this Phase I study, the toxicity of this drug was examined after administration of the drug to cancer patients on 3 successive days every 6 to 8 weeks. Clinical pharmacology was studied using 1-[chloroethyl-14C](2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea. The dose-limiting toxicity was myelosuppression. The maximal tolerated dose was 105 mg/sq m, which produced a median platelet nadir of 40,000/microliter on Day 32 and a median white blood cell count nadir of 2200/microliter on Day 42. Progressive anemia was also observed. There was no evidence of acute or chronic hepatic, renal, or pulmonary damage. One patient with a metastatic hypernephroma exhibited a partial clinical remission. Plasma disappearance of the drug following bolus administration was biphasic, with an initial half-life of 18 to 25 min and a second half-life of 9 hr. Clearance of intact drug coincided largely with the initial disappearance phase of total radioactivity. Entry of radioactivity into the cerebrospinal fluid was observed. Approximately 35% of plasma radioactivity was protein bound, the major binder being albumin. Drug excretion was predominantly renal, and biliary elimination was only minor.

Influence of hydrocortisone on the binding of nitrosoureas to nuclear chromatin subfractions.

The effects of steroid-induced modifications of chromatin structure on the extent and sites of chloroethylnitrosourea binding to chromatin were studied using log-phase HeLa cells. The cells were exposed to 0.1 to 2.0 microM hydrocortisone for 22 hr; this resulted in depressed DNA synthesis while transcriptional activity was stimulated. Hydrocortisone had no effect upon cellular or nuclear uptake of the two nitrosoureas under study, 0.6 mM chlorozotocin or 1-(2-chloroethyl-3-cyclohexyl-1-nitrosourea). Both drugs were found to alkylate transcriptional chromatin preferentially, as demonstrated by DNase II and DNase I digestion. This alkylation was stimulated 2-fold by the same micromolar concentrations of hydrocortisone, 0.1 to 2.0 microM, which stimulated transcription. The extent of nuclear RNA alkylation, determined using RNase T2 as a probe, was found to contribute less than 20% of total chromatin alkylation and was unaffected by steroid pretreatment. Instead, the increased alkylation within these chromatin subfractions was attributed to a steroid-induced alteration of chromatin structure. Electron microscopic examination of HeLa nuclear morphology revealed a hydrocortisone-induced disaggregation of nuclear membrane-associated heterochromatin resulting in a more heterogeneous, less condensed distribution of chromatin. Such data are consistent with a relaxation of the supercoiled chromatin structure, resulting in increased transcription and increased accessibility of potential target sites for nitrosourea alkylation.

Mechanisms of sensitivity and resistance of murine tumors to 5-fluorouracil.

The biochemical basis for the resistance of murine leukemia P388 to 5-fluorouracil (FUra) was systematically investigated by examining the transport and metabolism of FUra, or its anabolites, as well as the inhibition of enzymes and processes known to be affected by the drug. Of these parameters, only three were found to be altered significantly in the resistant line: (a) the enzyme required for the phosphorylation of uridine 5'-monophosphate to uridine 5'-diphosphate was present at a significantly lower specific activity in the resistant line than in its sensitive counterpart; (b) the rates of generation and persistance of 5-fluoro-2'-deoxyuridine 5'-monophosphate were significantly lower and shorter in the variant; and (c) there was a 1.6- and 3-fold decrease in the incorporation of FUra into polyadenylic acid-containing RNA and polyadenylic acid-lacking RNA, respectively, in resistant versus sensitive cells. Taken together, these findings suggest a dual mechanism for resistance to FUra in these leukemic cells, namely, a depressed capacity to generate di- and triphosphates of the riboside and deoxyriboside of the drug leading to lower pools of the proximate antimetabolite, fluorouridine 5'-triphosphate, and accelerated excretion of 5-fluoro-2'-deoxyuridine 5'-monophosphate, so that thymidylate synthetase is perturbed in a less than lethal way.

5-Fluorouracil, mitomycin, and doxorubicin (FAM) in carcinoma of the biliary tract.

A phase II trial of the regimen 5-fluorouracil, doxorubicin, and mitomycin C (FAM) was conducted in 17 patients with advanced or recurrent biliary tract cancer. Among 14 patients with measurable disease, 31% achieved a partial response. An additional seven patients evidenced stabilization of disease for periods of three to 18+ months. One patient with advanced but unmeasurable tumor has survived 72 months after 12 cycles of treatment. There was no serious life-threatening toxicity, and with appropriate dosage adjustment, the drug was administered to patients with mild hepatic insufficiency. We recommend the initiation of a larger controlled trial and a potential application with radiotherapy for patients with less advanced disease.