Red blood cell polyamine levels and host toxicity during continuous alpha-difluoromethylornithine infusion

The dose effects of continuous alpha-difluoromethylornithine (DFMO) infusion on red blood cell (RBC) polyamine levels, host toxicity and tumor growth were determined. Male rats with and without a transplantable methylcholanthrene-induced sarcoma received intravenously either 0.45% NaCl or DFMO at 500 mg, 1,000 mg, or 2,000 mg/kg body wt/day for 6 or 12 days. Dose-related inhibition of tumor growth was noted after the 12-day treatment. There were no changes in host carcass weight, food intake, plasma albumin, hematocrit or white blood cell counts. Platelet suppression was associated with the 1,000- and 2,000-mg doses with the 12-day treatment. Morphometry of the small intestine revealed mild but significant shortening of villi in the duodenum and jejunum at the 2,000-mg dose, but none of the animals developed diarrhea. The 500-mg DFMO dose reduced the rate of tumor growth without inducing platelet suppression or altering intestinal morphology. A decrease in RBC putrescine levels was noted at all doses. RBC spermidine levels increased with the 500-mg dose. RBC spermine levels were higher at all doses compared with controls. These results suggest that thrombocytopenia is the major dose-limiting side-effect of continuous DFMO infusion but does not occur at a dose of 500 mg/kg body wt/day.     

Effect of intravenous alpha-difluoromethylornithine on the polyamine levels of normal tissue and a transplantable fibrosarcoma

The effect of a continuous i.v. infusion of alpha-difluoromethylornithine (DFMO) on the polyamine metabolism of tumor and normal host tissue was determined. Non-tumor-bearing Fischer 344 rats or rats bearing a transplantable fibrosarcoma received continuous infusions of DFMO through a central venous catheter at three dose levels. Treatment with DFMO resulted in a time- and dose-dependent, cytostatic effect on the growth of the tumor. In fibrosarcoma-bearing rats the tumor putrescine levels were reduced after 6 and 12 days of DFMO treatment. Tumor spermidine levels were consistently reduced after 6 and 12 days of treatment with the reduction being dose dependent. The decrease in tumor ornithine decarboxylase activity was dose dependent. Erythrocyte putrescine levels were decreased in tumor- and non-tumor-bearing rats, suggesting that DFMO reduces the tumor contribution to the erythrocyte pool. Erythrocyte spermidine levels of fibrosarcoma- and non-tumor-bearing rats were elevated at the lower DFMO doses administered for 12 days but returned to normal as the dose was increased. Erythrocyte spermine levels were elevated in both groups of rats at all DFMO doses. Although normal host tissue weights were not affected by treatment with DFMO, the putrescine and spermidine levels of liver, spleen, and kidney and ornithine decarboxylase activity of the liver and kidney were decreased. These data demonstrate that i.v. DFMO has a cytostatic effect toward a rapidly growing fibrosarcoma associated with the depletion of both tumor putrescine and spermidine levels.     

Effects of alpha-difluoromethylornithine on the growth of experimental Wilms' tumor and renal adenocarcinoma

Because polyamines are essential for cellular growth and differentiation, and because human renal carcinomas have spermidine levels that are higher than those in normal renal tissue, effects of 2-difluoromethylornithine (DFMO) on the growth of experimental renal tumors were investigated. DFMO is a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme controlling polyamine biosynthesis. DFMO (2%) in drinking water was administered to BALB/c mice with intrarenal transplants of a renal adenocarcinoma cell suspension and to Wistar/Furth rats with s.c. transplants of a Wilms' tumor. At 28 days, renal carcinomas in DFMO-fed mice weighed 72% less than those in control animals (p less than 0.001). Wilms' tumor weight was not affected by DFMO feeding. DFMO caused 72 to 75% inactivation of ornithine decarboxylase activity and reduced putrescine levels in renal carcinoma and Wilms' tumor, reduced spermidine levels in Wilms' tumor, and apparently raised spermine levels in the latter as a consequence. DNA content was not affected by DFMO feeding. The mean number of lung metastases in DFMO-fed, renal carcinoma-bearing mice was 0.1 and in controls was 1.4 (p less than 0.001). DFMO feeding increased survival of mice bearing renal carcinomas by 3.0 +/- 0.8 (S.E.) days (p less than 0.05), i.e., from 30.5 +/- 0.8 days to 33.5 +/- 1.2 days. DFMO did not affect the growth of Wilms' tumor; however, in renal adenocarcinoma, it reduced growth, prevented lung metastases, and increased survival.     

Individual and combined effects of alpha-difluoromethylornithine and ovariectomy on the growth and polyamine milieu of experimental breast cancer in rats

Despite considerable evidence suggesting a critical role of polyamines in the hormonal control of breast cancer growth in vitro, their role in in vivo tumor growth is not established. In these experiments, we evaluated the individual and combined effects of the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) and ovariectomy on the growth and cellular levels of ornithine decarboxylase (ODC) and polyamines of N-nitrosomethylurea-induced rat mammary tumors. Despite a similar suppressive effect on ODC activity, the two treatments had a different effect on polyamine levels. As expected, DFMO selectively suppressed putrescine, whereas spermidine and spermine levels were minimally or not affected at all. Since quantitatively putrescine contributes the least to overall polyamine pools, the DFMO effect on this latter parameter was modest. In contrast, ovariectomy, by suppressing the more abundant spermidine and spermine, produced a more profound suppression of total polyamine pools. This finding is in agreement with the notion that hormones not only control ODC activity, but also other enzymes involved in the synthesis of the distal polyamines. Ovariectomy was also more potent than DFMO administration in inhibiting N-nitrosomethylurea-induced mammary tumor growth. No major additive/synergistic effects were observed between DFMO and ovariectomy on tumor growth and cellular levels of ODC activity and polyamines. DFMO administration lowered the tumor level of progesterone receptors and appeared to potentiate the suppressive effect of ovariectomy. In contrast, neither treatment, alone or in combination, altered tumor levels of estrogen receptors. DFMO administration did not affect circulating levels of estradiol and prolactin or uterine and ovarian weights, thus suggesting that its effects were not indirectly mediated through alterations of the endocrine milieu of the host.     

Differential effect of alpha-difluoromethylornithine on the in vivo uptake of 14C-labeled polyamines and methylglyoxal bis(guanylhydrazone) by a rat prostate-derived tumor

The uptake of exogenously administered radiolabeled polyamines by a rat prostate-derived tumor line, the Dunning R3327 MAT-Lu, and various normal tissues was studied. Pretreatment of tumor cells in vitro with alpha-difluoromethylornithine (DFMO), a polyamine synthesis inhibitor, resulted in a markedly enhanced uptake of both [14C]putrescine and [14 C]spermidine. The in vitro uptake of [14C]putrescine by these cells was effectively inhibited by unlabeled spermine, spermidine, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1,4-diaminopentane, and 1,4-diaminobutane, but less effectively by 1,4-diamino-2,3-butene and 1,4-diamino-2,3-butyne. The diamines, 1,3-diaminopropane and 1,2-diaminoethane, were ineffective in inhibiting [14C]putrescine uptake in vitro into the R3327 MAT-Lu cell line. When tumor-bearing animals were pretreated with DFMO or with DFMO and 5-alpha-dihydrotestosterone propionate, the tumor and prostate uptake of [14C]putrescine and [14C]-cadaverine was enhanced but not substantially increased in other tissues. In contrast to the in vitro results, spermidine and spermine were not enhanced substantially by DFMO pretreatment into any tissue, and their uptake into the tumor actually decreased. Ethylenediamine, which does not utilize the polyamine transport system, did not have its uptake increased into any tissue following DFMO pretreatment. The chemotherapeutic agent, methylglyoxal bis(guanylhydrazone), which utilizes the polyamine transport system for uptake into cells, exhibited uptake behavior different from that of the polyamines. Thus, methylglyoxal bis(guanylhydrazone) uptake into the tumor was not significantly increased or decreased by DFMO or by DFMO + 5-alpha-dihydrotestosterone propionate pretreatment, and only the ventral, but not the dorsal-lateral, lobe of the prostate showed increased uptake of methylglyoxal bis(guanylhydrazone) following DFMO + 5-alpha-dihydrotestosterone propionate pretreatment.     

Inhibition of mouse skin tumor promotion and of promoter-stimulated epidermal polyamine biosynthesis by alpha-difluoromethylornithine

Application of the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin leads to a manifold induction of ornithine decarboxylase (ODC) activity within 5 hr and an increased accumulation of putrescine. The relevance of these TPA-induced changes to the mechanism of tumor promotion was investigated using alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO applied to mouse skin (0.3 mg in 0.2 ml of solvent) or administered in the drinking water (1%) in conjunction with skin tumor promotion by TPA inhibited the formation of mouse skin papillomas by 50 and 90%, respectively. TPA-induced ODC activity and the accumulation of putrescine were almost completely inhibited. DFMO given in the drinking water decreased spermidine levels, but DFMO treatment by any route did not alter the spermine levels of mouse epidermis. DFMO decreased TPA-induced hyperplasia by 25 to 40%, and the TPA-caused increases in DNA synthesis and mitotic index were inhibited by 60 and 50%, respectively. Therefore, in mouse epidermis, enhanced cell proliferation can be dissociated from ODC induction and the accumulation of putrescine. At the tested dose levels and routes of administration, DFMO did not inhibit the inflammatory response to TPA in several tissues. These results provide evidence for an essential role of ODC induction and the accumulation of putrescine in tumor promotion by TPA and add strength to the proposal that DFMO may be a promising drug for the prevention and treatment of cancer in human beings.     

Microbial flora in the gastrointestinal tract abolishes cytostatic effects of alpha-difluoromethylornithine in vivo

Although treatment with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) leads to depletion of intracellular polyamines and to related growth inhibition in vitro, its cytostatic effects in vivo are disappointing. This may be due to abolition of DFMO-induced growth inhibition by polyamines released during normal body cell turnover, to dietary polyamines, or to putrescine synthesized by the microbial flora in the GI tract. We studied selectively (aerobic) and totally (aerobic + anaerobic) GI tract-decontaminated LI210-bearing mice fed with 3 types of diet differing in their polyamine and carbohydrate residue contents and treated with combinations of intraperitoneal DFMO and oral deuterium-labelled putrescine. Our data show that, irrespective of diet type, total decontamination markedly potentiates the moderate tumor growth inhibition that is caused by DFMO alone. During total decontamination, growth-inhibited L1210 cells accumulate in the G0/G1 phase of the cell cycle. Although orally administered deuterium-labelled putrescine gave rise to deuterium labelling of L1210 putrescine, spermidine and spermine, the polyamine levels in our diets played only a minor role.     

Putrescine-dependent invasive capacity of rat ascites hepatoma cells.

The effects of inhibitors of polyamine synthesis on the invasive capacity of rat ascites hepatoma (LC-AH) cells were examined by in vitro assay of penetration of the LC-AH cells through a monolayer of calf pulmonary arterial endothelial (CPAE) cells. Pretreatment of LC-AH cells with alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, before seeding them onto a CPAE cell monolayer and culturing them for 24 h in the absence of DFMO decreased the number of penetrating tumor cells time and dose dependently (about 35% of the maximal inhibition) without affecting their viability or proliferative activity. DFMO treatment caused a marked decrease in the intracellular level of putrescine but not of spermidine or spermine. The DFMO-induced decreases in invasive capacity and putrescine level were almost completely reversed by the addition of putrescine to the medium during pretreatment with DFMO or invasion assay but were not affected by exogenous spermidine or spermine. No change in the invasive capacity was observed when the CPAE cells were treated with DFMO and the LC-AH cells with methylglyoxal-bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, which depressed the spermidine and spermine levels but increased the putrescine level in the LC-AH cells. These results suggest that intracellular putrescine modulates the in vitro invasive capacity of LC-AH cells.     

Effects of alpha-difluoromethylornithine and 5-fluorouracil on the proliferation of a human colon adenocarcinoma cell line

Because alpha-difluoromethylornithine (DFMO) reduces the incidence of experimental colon cancers, inhibits the growth of human lung cancer cells and human leukemia cells in culture, and in combination with methylglyoxal (bis)guanylhydrazone induces remission in children with leukemia, its effectiveness against a human colon adenocarcinoma cell line (Colo 205) was tested alone and in combination with 5-fluorouracil (5-FU). Both DFMO (2 X 10(-4) M) and 5-FU (10(-6) M) inhibited Colo 205 cell proliferation. Above 5 X 10(-4) M DFMO (p less than 0.001) and at 10(-4) M 5-FU (p less than 0.001), Colo 205 growth was completely inhibited. Although DFMO did not sensitize Colo 205 cells to a noninhibitory concentration of 5-FU, the effectiveness of inhibitory concentrations of 5-FU and DFMO in reducing Colo 205 cell growth was additive. DFMO (2 X 10(-4) M) caused 89 to 93% inhibition of ornithine decarboxylase activity (p less than 0.001) and reduced levels of putrescine (93%; p less than 0.01) and spermidine (57%; p less than 0.02). Growth rate and the intracellular putrescine and spermidine contents were restored by 10(-6) M putrescine. DFMO could be an effective chemotherapeutic agent against human colonic cancer because of its effects at such unusually low concentrations in vitro.     

Properties of L1210 cells resistant to alpha-difluoromethylornithine

L1210 cells were selected for resistance to the ornithine decarboxylase (ODC) inhibitor, alpha-difluoromethylornithine. When grown in the absence of the inhibitor, these cells possessed very high ornithine decarboxylase levels. These represented about 1 part in 300 of the soluble protein, which is several hundred times greater than the maximal value found in the original L1210 cells. The resistant cells contained at least 100-fold higher levels of ODC mRNA but the half-life of ODC (about 45 min) was not altered significantly. The resistant cells had much higher putrescine and cadaverine levels than control cells, but there was no significant difference in cellular spermidine or spermine content or in production of 5'-methylthioadenosine, which is a measure of polyamine synthesis. Addition of putrescine to the control or resistant cells had no effect on their content of spermidine and spermine but addition of decarboxylated S-adenosylmethionine increased the content of spermidine and spermine. These results indicate that ornithine decarboxylase is not the rate-limiting step in polyamine synthesis in these L1210 cells. The growth of the alpha-difluoromethylornithine-resistant L1210 cells was inhibited when their ability to synthesize spermidine and spermine was blocked by the addition of the S-adenosylmethionine decarboxylase inhibitor, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine. Treatment with this compound produced a reduction of more than 85% in the production of 5'-methylthioadenosine and led to a large increase in the content of putrescine and a substantial decline in the content of spermidine and spermine. These results indicate the potential value of S-adenosylmethionine decarboxylase inhibitors as therapeutic agents in conditions where ODC inhibitors are ineffective.     

Decarboxylated-S-adenosylmethionine excretion: a biochemical marker of ornithine decarboxylase inhibition by alpha-difluoromethylornithine

In an attempt to define a biochemical marker of ornithine decarboxylase inhibition in humans, alpha-difluoromethylornithine hydrochloride (DFMO), an irreversible ornithine decarboxylase inhibitor, was infused i.v. in seven cancer patients over 10-day courses at doses of 10-90 g/day and 24-h urinary excretion of polyamines and decarboxylated-S-adenosylmethionine was determined before, during, and after treatment. DFMO produces marked increases in urinary decarboxylated-S-adenosylmethionine excretion, up to 84 times pretreatment values. This response appears to be time dependent, requiring several days to reach a maximum and lasting at least 4-5 days after stopping DFMO. In contrast, urinary excretion of the polyamines putrescine, cadaverine, spermidine, N1-monoacetylspermidine, N8-monoacetylspermidine, and spermine, were not consistently altered by DFMO. We conclude that urinary excretion of decarboxylated-S-adenosylmethionine represents a valid biochemical indicator of ornithine decarboxylase inhibition in humans, whereas urinary polyamines are of no value.     

Isolation and characterization of human breast adenocarcinoma cells made resistant to alpha-difluoromethylornithine

Human breast adenocarcinoma cells MCF-7 were selected for resistance to ornithine decarboxylase (ODC) inhibitor, alpha-difluoromethylornithine (DFMO). Stepwise increments of the concentration of DFMO resulted in selection of MCF-7 cells that were capable of growing in the presence of 1.0 mM DFMO. This capacity was associated with a 10-fold increase in ODC activity and marked enhancement in the synthesis rate of ODC protein as verified by a 2-hr [35S]methionine labeling of cellular proteins followed by immunoprecipitation and SDS-PAGE. The resistant cells had much higher concentration of putrescine, spermidine, and spermine than the control cells. A 25-fold increase in ED50 (effective dose causing 50% inhibition) for the antiproliferative action of DFMO in these resistant cells was observed. The susceptibility of wild-type and resistant cell lines to other inhibitors of the polyamine biosynthetic pathway and adriamycin is also reported.     

Estradiol control of ornithine decarboxylase mRNA,enzyme activity,and polyamine levels in MCF-7 breast cancer cells: therapeutic implications

Previous studies have shown that natural polyamines - putrescine, spermidine, and spermine - play a key role in the mechanism of action of estrogens in breast cancer. Ornithine decarboxylase (ODC) is the first enzyme of the polyamine biosynthetic pathway. To examine estrogenic regulation of polyamine biosynthesis in breast cancer, we measured ODC mRNA, ODC activity, and polyamine levels in G₁ synchronized MCF-7 cells. ODC mRNA and activity increased four-fold over that of cells in G₁ phase between 8 to 16 h after the addition of estradiol. Polyamine levels showed a sharp increase by 8 h after the addition of estradiol and decreased by 12 h. We further examined whether synthetic homologs of putrescine or spermidine could replace natural polyamines in supporting MCF-7 cell growth. Treatment of MCF-7 cells with 1 mM difluoromethylornithine (DFMO), an inhibitor of ODC, suppressed putrescine, spermidine, and spermine levels by 74, 78, and 10%, respectively, within 48 h. Cells treated with DFMO for 48 h were supplemented with either putrescine or its homologs or spermidine or its homologs. Diaminopropane, diaminobutane (putrescine), and diaminopentane were capable of fully or partially reversing the growth inhibitory effects of DFMO, whereas diaminoethane had no significant effect. Among a series of triamines, H₂N(CH₂)_nNH(CH₂)₃NH₂ (where n = 2 to 8; abbreviated as APn n = 4 for spermidine, or AP4), spermidine was most effective in reversing the effects of DFMO, whereas compounds with shorter or longer methylene bridging regions were less effective. AP8 was ineffective in reversing the growth inhibitory effects of DFMO. At 10 µM concentration, AP8 also inhibited DNA synthesis by 66%, as measured by [³H]-thymidine incorporation. These data show that MCF-7 cells have a strong requirement for polyamines for their growth and that estradiol stimulates the polyamine cascade by inducing the ODC mRNA level. Our results also suggest that polyamine homologs such as AP8 might be potentially useful in breast cancer therapy.     

Alterations in bone marrow and blood mononuclear cell polyamine and methylglyoxal bis(guanylhydrazone) levels: phase I evaluation of alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) treatment of human hematological malignancies

Nine patients with hematological malignancies were treated with difluoromethylornithine and methylglyoxal bis(guanylhydrazone). The number of circulating blast cells decreased in all of the patients treated with DFMO and MGBG for longer than 1 wk. Morphological evidence of myeloid maturation was evident in four patients with leukemia and the circulating M Protein decreased in one patient with multiple myeloma. The polyamine content of the mononuclear cells in both the peripheral blood and bone marrow was transiently increased after the initial MGBG dose. During administration of DFMO decreases were achieved in the peripheral blood mononuclear cell putrescine levels in 7 patients, spermidine levels in 5 patients, and spermine levels in 4 patients. Alterations in bone marrow mononuclear cell polyamine levels were similar to those which occurred in the peripheral cells. An average of 9 days of DFMO treatment was required to lower mononuclear cell polyamine levels. Three of the 4 evaluable patients receiving multiple MGBG doses had an increased mononuclear cell content of MGBG after DFMO pretreatment. Enhancement of cellular MGBG levels was not directly correlated to the degree of cellular polyamine depletion.     

in vivo effects of 4-amidinoindan-l-one 2′-amidinohydrazone (CGP 48664A) and α-difluoromethylornithine (DFMO) on L1210 growth,cell-cycle phase distribution and polyamine contents

We studied the in vivo effects of 4-amidinoindan-l-one 2′-amidinohydrazone (CGP 48664A), α-difluoromethylornithine (DFMO) and a combination of CGP 48664A-DFMO on tumor growth, cell-cycle phase distribution and polyamine contents. DBA-2 mice were inoculated i.p. with 10⁵ L1210 cells on day 0, treated i.p. on days 1–4 and killed on day 5. As compared to controls, CGP 48664A, DFMO and the CGP 48664A-DFMO combination reduced L1210 cell numbers by 33, 43 and 85%, respectively. CGP 48664A did not affect cell-cycle phase distribution. DFMO and the CGP 48664A-DFMO combination caused a moderate and a heavy accumulation in G₀/G₁- and G₂/M-phases, respectively. Compared with controls, the CGP 48664A-DFMO combination reduced putrescine, spermidine and total polyamines, but did not affect spermine. Compared with CGP 48664A, the CGP 48664A-DFMO combination caused lower putrescine and total polyamines, higher spermine, but no change in spermidine. Compared with DFMO, the CGP 48664A-DFMO combination caused higher putrescine and spermidine, lower spermine, but no change in total polyamine levels. We conclude that CGP 48664A potentiates the cystostatic effect of DFMO in vivo. The resulting growth inhibition is accompanied by an accumulation in G₀/G₁- and G₂/M-phases and a reduction of putrescine and spermidine. The data suggest that perturbed polyamine composition rather than reduced spermidine or total polyamine pool size causes a profound growth inhibition. © 1995 Wiley-Liss, Inc.     

Inhibitory action of alpha-difluoromethylornithine on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced rat urinary bladder carcinogenesis

We previously have shown that urine components capable of stimulating ornithine decarboxylase activity of urothelium can enhance rat urinary bladder carcinogenesis, and that alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, suppresses carcinogen-initiated rat urinary bladder carcinogenesis. The present investigation was conducted to determine whether DFMO's suppressive effect is stage specific during carcinogenesis and whether the suppressive effect lasts with its continued use. Following initiation with 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine in drinking water for 6 wk, male Fischer 344 rats initially weighing 125 to 150 g were randomly divided into two groups, the first receiving 0.2% DFMO in drinking water ad libitum and the second receiving tap water only. Groups of animals were killed at regular intervals until the completion of the experiment at 75 wk. The effect of DFMO was evaluated by monitoring the incidence of tumors, the mean number of tumors per rat, the mean volume of individual tumors, and the mean total tumor volume per rat. The results showed that continuous treatment with DFMO significantly reduced tumor formation until 60 wk (P less than 0.017). The effect was only of borderline significance (0.017 less than P less than 0.035) at 75 wk. Discontinuation of DFMO treatment at 40 wk resulted in the loss of protective effect in all comparisons except for the borderline effect on the tumor number and total tumor volume per rat. DFMO had no significant effect on the incidence or development of preneoplastic early lesions. Mucosal polyamine (spermidine and spermine) levels were reduced and correlated well with the reduction in tumor growth, suggesting that the reduction in tumor growth rate by DFMO may be due to its ability to reduce polyamine levels in urothelium. There were no side effects attributable to DFMO treatment. DFMO may be a useful chemopreventive agent to retard the recurrence of human superficial bladder cancer.     

Phase I study of alpha-difluoromethylornithine and methyl-GAG

α-Difluoromethylornithine (DFMO) is a potent inhibitor of the syntheiss of putrescine (pu) and spermidine (sd) in some benign and malignant tissues. Intracellular deprivation of pu and sd has been shown to induce an enhanced uptake of polyamine-analogs such as methyl-GAG (MGBG). The purpose of this study was to investigate the tolerance and the toxicity of the combination of DFMO and MGBG. Thirty-six patients received 4 × 2 g of DFMO/day orally and every 2 weeks 250–500 mg/m² of MGBG as a 2-hr infusion, starting on day 14. Besides the well known acute and late side-effects of methyl-GAG, dose-limiting toxicity consisted also of thrombocytopenia, leucopenia, dyspnea, hemolysis and jaundice. The maximal toleranted dose of MGBG for one course was 350 mg/m² and for repeated courses 250 mg/m², due to cumulative toxicity.Furthermore, after 8 weeks of continuous administration of DFMO 70% of the patients had a severe hearing loss, which was reversible after a treatment delay of 4–6 weeks. Since the hearing loss prohibited the continuous use of DFMO, two diferent schedules of intermittent DFMO-administration together with two different infusion periods of MGBG have been investigated in 15 patients. In none of these patients did hearing loss occur. The schedule of continuous administration of 4 × 2 g of DFMO/day orally for 21 days and 250 mg/m² of MGBG as a 24-hr infusion on days 7, 14 and 21, repeated on day 42, was tolerated best. In 28 evaluable patients two partial remissions were seen. Pretreatment with DFMO significantly enhanced the toxicity of MGBG and the combination of both drugs produced side-effects not seen with eithe drug alone.     

Synergistic inhibition of polyamine synthesis and growth by difluoromethylornithine plus methylthioadenosine in methylthioadenosine phosphorylase-deficient murine lymphoma cells

The antiproliferative effects of the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) are limited by the inability of the compound to deplete completely cellular polyamine pools. 5'-Deoxy-5'-methylthioadenosine (MeSAdo), the purine end product of the polyamine biosynthetic pathway, is an inhibitor of spermine and spermidine synthesis. Furthermore, a substantial number of human tumors are deficient in MeSAdo phosphorylase, and cannot degrade MeSAdo. It therefore seemed possible that DFMO and MeSAdo could interact synergistically to inhibit polyamine synthesis in MeSAdo phosphorylase-deficient malignant cells. To test this hypothesis, we have analyzed the effects of DFMO, in combination with MeSAdo, on polyamine synthesis and growth in a MeSAdo phosphorylase-deficient murine lymphoma cell line (R1.1-H), and a MeSAdo resistant mutant (R1.1-H3). Cultivation of the R1.1-H3 cells in medium containing 250 microM DFMO and 500 microM MeSAdo caused profound depletion of putrescine, spermidine, and spermine, and the accumulation of both decarboxylated S-adenosylmethionine and its acetylated derivative to levels that exceeded by nearly 3-fold the total cellular content of S-adenosylmethionine. Similarly, DFMO sensitized the lymphoma cells to the growth inhibitory effects of MeSAdo. Supplementation of the medium with putrescine, spermidine, or spermine partially protected R1.1-H3 cells from the DFMO-MeSAdo drug combination. It is conceivable that MeSAdo, or related nucleosides, may potentiate the cytostatic effects of DFMO toward MeSAdo phosphorylase-deficient tumors.     

Combination chemotherapy involving alpha-difluoromethylornithine and 1-beta-D-arabinofuranosylcytosine in murine L1210 leukemia

Administration of 2% aqueous alpha-difluoromethylornithine (DFMO) as the sole drinking-fluid to C57BL X DBA/2 F1 (hereafter called BD2F1) mice for 5 days resulted in significant reduction of labeling index, percentage of cells in S phase, and intracellular putrescine and spermidine concentrations of bone marrow cells. DFMO treatment also resulted in moderate leukopenia. The decreases in body weight, peripheral leukocyte and erythrocyte counts, and subsequent recovery following administration of 1-beta-D-arabinofuranosylcytosine (ara-C), an S-phase-specific cytotoxic agent, to animals receiving DFMO was comparable to those observed in animals which received ara-C alone. In animals inoculated i.p. with L1210 cells, DFMO treatment produced a marginal increase in survival time and significant reductions in putrescine and spermidine concentrations of the tumor cells. However, in contrast to normal bone marrow cells, depletion of polyamines in tumor cells resulted in the progressive accumulation of these cells in S phase; by Day 5 following DFMO treatment, greater than 70% of the tumor cells were found to be in S phase compared to only 55% for cells from untreated animals. A significant synergistic increase in survival time with a combination of DFMO and ara-C was observed when the ara-C therapy was initiated at the time of maximum accumulation of tumor cells in S phase. The implications of these findings for the management of neoplasia are discussed.     

Effect of Ornithine in Parenteral Nutrition Regimens on Difluoromethylornithine-Induced Platelet Suppression and Changes in Tumor Polyamine Content

We have demonstrated that DFMO-induced thrombocytopenia can be ameliorated with concomitant ornithine (Orn) in chow-fed rats; a reversal in DFMO-associated tumor polyamine reduction and antitumor activity, however, was also evident. To determine the effect of Orn in total parenteral nutrition (TPN) regimens on DFMO-induced thrombocytopenia and changes in tumor polyamine concentrations, Ward-colon-tumor-bearing (WCT) rats were given TPN with arginine (ENA) or with ornithine substituted for arginine (ENO) alone or with DFMO (1.5 g/day) added directly to the infusate. After 4 days, the peripheral blood platelet counts for ENA (917 ± 151 × 10³/mm³) or ENO (908 ±67x 10³/mm³) were equivalent to those of chow-fed rats (901 ±42 × 10³/mm³). ENA/DFMO rats had significant thrombocytopenia (607 ± 185 × 10³/mm³), which was completely ameliorated for ENO/DFMO rats (939 ± 111 × 10³/mm³). Peripheral white blood count, hematocrit, and other hematological parameters were not affected. Tumor putrescine content for ENA rats (46.9 ±8.7 nmol/g) was equal to that for chow-fed rats (44.8 ± 6.2 nmol/g) and ENO rats (53.6± 8.3 nmol/g). The reduction in tumor putrescine content for ENO/DFMO rats (19.6 ±6.9 nmol/g) was equivalent to that of ENA/DFMO rats (14.7 ± 3.0 nmol/g). Tumor spermidine was reduced only for the ENA/DFMO rats while spermine was slightly elevated. Tumor spermine content for ENO/DFMO rats (57.2 ±12.0 nmol/g) was equal to that for ENO rats (65.6 ±8.7 nmol/g) but was significantly (p = 0.004)reduced when compared with rats receiving ENA/DFMO (89.4 ± 20.4 nmol/g). The results of this study show that TPN with Orn substituted for arginine can be used with a chemotherapeutic dose of DFMO to ameliorate the thrombocytopenia. The DFMO-induced reduction in tumor putrescine content, however, was not affected when Orn was substituted for arginine in a parenteral nutrition regimen. These results suggest that the antitumor activity of DFMO would not be adversely affected by coadministering DFMO with a TPN regimen with Orn substituted for arginine.