Protective role of arginase in a mouse model of colitis.

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement. There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.

Difluoromethylornithine-induced reversible hearing loss across a wide frequency range.

OBJECTIVES: Alpha-difluoromethylornithine (DFMO) is an antineoplastic agent that causes reversible hearing loss (HL) by an unknown mechanism. Previous neonatal gerbil studies have identified a dosing regimen of 1 g/kg per day of DFMO given for 3 weeks that results in reversible HL on click-evoked auditory brainstem response (ABR). The objectives of this study are 1) to measure HL and recovery at several frequencies in neonatal gerbils after DFMO therapy at two different dosing regimens and 2) to identify any effects of DFMO on cochlear histology. STUDY DESIGN: Prospective, nonrandomized, experimental design with placebo controls. METHODS: ABR to tone pips at 2, 4, 8, 16, and 32 kHz were recorded on 62 21-day-old Mongolian gerbils after daily subcutaneous injections of DFMO (group A at 1 g/kg, group B at 750 mg/kg) or saline from day 3 to day 20 after birth. Thirty-seven animals were retested after a 3-week drug-free recovery period. Twenty-seven animals were killed for analysis of cochlear histology. RESULTS: Animals that were administered DFMO demonstrated higher ABR thresholds across all five frequencies, with mean threshold differences of 21 to 29 dB in group A and 11 to 17 dB in group B as compared with controls. Higher thresholds were demonstrated at higher frequencies. Fewer side effects were noted at the lower dose. After a 3-week drug-free recovery period, auditory thresholds returned to pretreatment levels. No significant cochlear structural abnormalities were identified under light microscopy. CONCLUSION: An 18-day regimen of 750 mg/kg per day of DFMO given subcutaneously in neonatal gerbils causes minimal side effects with broad-frequency HL that, after 3 weeks of recovery, is fully reversible.

In situ detection of neoplastic transformation and chemopreventive effects in rat esophagus epithelium using angle-resolved low-coherence interferometry.

We present a quantitative study of the nuclear morphometry of epithelial cells in an animal model of esophageal carcinogenesis. Changes in the size and texture of cell nuclei as a result of neoplastic transformation and chemopreventive action are observed in situ using a new optical technique, angle-resolved low-coherence interferometry (a/LCI). The capabilities of a/LCI are demonstrated via quantitative in situ measurements of the nuclear morphometry of basal epithelial cells, approximately 50-100 microm beneath the tissue surface without the need for exogenous contrast agents or tissue fixation. The measurements quantify changes in nuclear size, characterized by average diameter, and nuclear texture, characterized by fractal dimension of the subcellular structures. Using this technique, we observed changes in the morphometry of rat esophageal epithelial cells in response to treatment with the carcinogen N-nitrosomethylbenzylamine. In addition, morphometric changes were observed in the esophagi of rats treated with N-nitrosomethylbenzylamine and two chemopreventive agents, difluoromethylornithine and perillyl alcohol. These agents induced either apoptosis in the basal epithelium (difluoromethylornithine) or both apoptosis and vacuolation of basal epithelial cells (perillyl alcohol). Vacuolation was associated with cellular toxicity. The light-scattering measurements were compared with histological images of the same tissues. The potential of a/LCI as a noninvasive means to investigate the development of epithelial neoplasia and for tracking the efficacy of chemopreventive agents appears high. This technique also may provide a new screening tool for intraepithelial neoplasia.

Inhibition of the development of metastatic squamous cell carcinoma in protein kinase C epsilon transgenic mice by alpha-difluoromethylornithine accompanied by marked hair follicle degeneration and hair loss.

The role of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated polyamine biosynthesis in the development of metastatic squamous cell carcinoma (mSCC) in protein kinase C epsilon (PKC epsilon) transgenic mice was determined. TPA treatment induced epidermal ornithine decarboxylase (ODC) activity and putrescine levels approximately 3-4-fold more in PKC epsilon transgenic mice than their wild-type littermates. Development of mSCC by the 7,12-dimethylbenz(a)anthracene (100 nmol)-TPA (5 nmol) protocol in PKC epsilon transgenic mice was completely prevented by administration of the suicide inhibitor of ODC alpha-difluoromethylornithine (DFMO, 0.5% w/v) in the drinking water during TPA promotion. However, DFMO treatment led to marked hair loss in PKC epsilon transgenic mice. DFMO treatment-associated hair loss in PKC epsilon transgenic mice was accompanied by a decrease in the number of intact hair follicles. These results indicate that TPA-induced ODC activity and the resultant accumulation of putrescine in PKC epsilon transgenic mice are linked to growth and maintenance of hair follicles, and the development of mSCC. Severe hair loss observed in PKC epsilon transgenic mice on DFMO during skin tumor promotion has not been reported before in the prevention of cancer in other animal models or in human cancer prevention trials.

Inhibition of placental ornithine decarboxylase by DL-alpha-difluoro-methyl ornithine causes fetal growth restriction in rat.

The roles of polyamines in intrauterine growth restriction (IUGR) is studied. The DL-alpha-difluoromethyl ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) which is a rate limiting enzyme of polyamine synthesis was administrated to pregnant rats so that we obtained rat fetuses with IUGR. The changes of maternal nutrition, damage of the placenta, and the direct effect of DFMO on the fetus were examined in this IUGR model. Administration of DFMO did not induced changes of maternal nutrition except for triglyceride and the fetal metabolic state. But the placental weight, ODC activity, and DNA in the placenta were decreased significantly. The ODC activity in the total placenta decreased to less than 10% of that of the control. Depression of ODC activity in the placenta may be the major cause of IUGR induced by DFMO administration, and polyamines play important roles to carry pregnancy.

2-difluoromethylornithine and dehydroepiandrosterone inhibit mammary tumor progression but not mammary or prostate tumor initiation in C3(1)/SV40 T/t-antigen transgenic mice.

Female transgenic mice that express SV40 T/t antigens under the regulatory control of the rat C3(1) gene spontaneously develop multifocal mammary lesions that predictably evolve into invasive, hormone-independent carcinomas, whereas male mice are prone to develop prostate cancer. Chemopreventive agents were administered to female C3(1)/SV40 large T-antigen mice from 7 to 19 weeks of age, during which time the mammary lesions developed and progressed to invasive carcinomas. No significant differences in the numbers of preinvasive mammary intraepithelial neoplasia lesions (histologically similar to human ductal carcinoma in situ) were observed after 2 or 8 weeks of treatment between mice receiving either vehicle alone, dehydroepiandrosterone (DHEA), or 2-difluoromethylornithine (DFMO). However, a dose-response reduction in invasive carcinoma growth was observed for both DFMO, an inhibitor of ornithine decarboxylase, and DHEA, the primary steroid precursor to both androgens and estrogens in primates. Despite unaltered expression of the transgene, tumor incidence was reduced approximately 20% by DFMO (8000 mg/kg) and 30% by DHEA (4000 mg/kg; P < 0.05). Tumor multiplicity was reduced by approximately 50% by both DFMO and DHEA (P < 0.05). DFMO had a dose-dependent effect on total tumor burden, which was reduced by 25% at low doses (4000 mg/kg) and 70% at high doses (8000 mg/kg). DHEA reduced tumor burden by 50% and 66% at low (2000 mg/kg) and high (4000 mg/kg) doses, respectively. Interestingly, despite its inhibitory effects on tumor development, DHEA caused a dose-dependent increase of serum estradiol levels that we have previously shown to increase mammary tumor formation in this model. No effect on the development of the prostate cancer precursor lesions (prostate intraepithelial neoplasia) was observed when mice were treated with DHEA, DFMO, tocopherol acetate, selenomethionine, or 9-cis-retinoic acid, although the effects on late-stage prostate cancer development were not determined. These results demonstrate that despite the expression of the highly transforming C3(1)/SV40 large T-antigen transgene, this transgenic model can be used to study the effects of chemopreventive agents on mammary cancer progression. The tumor-inhibitory effects of DHEA and DFMO on mammary cancer growth appear to occur after the development of preinvasive lesions, suggesting that these agents inhibit tumor progression but not initiation.

Delayed, reversible hearing loss caused by difluoromethylornithine (DFMO).

OBJECTIVES/HYPOTHESIS: Difluoromethylornithine (DFMO) is an anticancer experimental drug that is ototoxic. The objectives of these three experiments were to: 1) determine a dose and dosing schedule of DFMO that produces significant hearing loss (HL) in newborn gerbils; 2) compare the HL level for control and newborn gerbils receiving daily subcutaneous injections of DFMO; and 3) to determine if DFMO-related HL is significantly reversible following discontinuation of DFMO treatment. STUDY DESIGN: Prospective, non-randomized experimental design with placebo controls. METHODS: Click-evoked auditory brainstem response (ABR) testing was performed for 21-day-old Mongolian gerbils following daily subcutaneous injections of DFMO or saline. Three experiments were carried out using different injection schedules and doses of DFMO. In experiment 3, animals were retested at 42 days of age following a 3-week recovery from DFMO. RESULTS: Animals administered an 18-day regimen of DFMO at 1 g/kg per day (from day 3 to day 20) had click thresholds of 25 to 65 dB nHL, whereas animals receiving daily injections of saline had thresholds of 5 to 20 dB nHL. Animals retested after 3 weeks of recovery from DFMO treatment had thresholds ranging from 5 to 20 dB nHL. Differences were statistically significant. CONCLUSIONS: DFMO causes mild to moderate HL in neonatal gerbils that recovers after discontinuation of the drug.

Bioavailability study of oral liquid and tablet forms of alpha-difluoromethylornithine.

The purpose of this study was to assess the bioavailability of two oral preparations of difluoromethylornithine (DFMO). The current preparation of DFMO is a liquid with a concentration of 0.2 gram/ml that must be drawn up into a syringe and dispensed into a small medicine glass. This form of DFMO causes wastage of the medication. The liquid form also makes compliance and blinding difficult. Recently, a new coated tablet preparation has become available from Ilex Oncology Services (San Antonio, TX). The coated tablets are 0.25 gram and are scored. The tablet form should increase compliance by making it much easier for the subject to take the medication. This report compares the bioavailability of both preparations with the goal of demonstrating equivalence of the preparations. Ten normal subjects entered the cross-over study in which the order in which they would receive the liquid or tablet preparation of DFMO was randomized. The study was designed with the objective of establishing the bioequivalence of a tablet preparation of DFMO at daily dose 0.5 gram/m2 and a liquid preparation of DFMO at the same daily dose. The mean area under the time-by-concentration curves (microM x hours) for the liquid and tablet preparations was 368.2 and 370.4, respectively. The peak concentrations for the liquid and tablet preparations were 47.3 and 48.2 microM, respectively. No statistically significant differences were seen in these parameters, in time to peak concentration, or in serum half-life.

Characterization of the ototoxicity of difluoromethylornithine and its enantiomers.

Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC), the essential enzyme in mammalian polyamine biosynthesis (Pasic et al., 1997, Arch. Otolaryngol. Head Neck Surg. 123[12], 1281-1286). This cancer chemotherapeutic agent has significant ototoxic potential. Because the DFMO enantiomers differ in their ability to block ODC, the present study was designed to compare the ototoxic potential of each enantiomer with the racemic form of this drug for the rat and guinea pig. Determining differential ototoxicity of the enantiomers is one preliminary step in determining the optimal form of DFMO to use in human cancer chemotherapy. Daily intubation with D,L-DFMO does not produce any auditory dysfunction in rats with doses between 200 mg/kg/day and 1. 2 g/kg/day for up to 8 weeks, despite the fact that doses of 800 and 1200 mg/kg/day depressed body weight gain. In contrast to the data observed in rats, substantial ototoxicity was observed when guinea pigs were injected ip with doses of D,L-DFMO between 500 mg/kg/day and 1 g/kg/day. D,L-DFMO produced loss of compound action potential sensitivity, but not of cochlear microphonic amplitude. This finding correlated with histological data revealing loss of both outer and inner hair cells in the cochlea with inner more affected than outer hair cells, particularly in the basal turn. Higher exposure doses (2-3 g/kg/day) resulted in significant general toxicity including impaired growth and some mortality. When the enantiomers were evaluated in the guinea pig, it was found that 1 g/kg/day D-DFMO did not produce any significant hearing impairment, whereas 1 g/kg/day of the L-enantiomer of DFMO generated a threshold shift that surpassed that of 1 g/kg/day of the D,L-DFMO treatment.

Minimal behavioral effects from developmental cerebellar stunting in young rats induced by postnatal treatment with alpha-difluoromethylornithine.

Postnatal treatment with alpha-difluoromethylornithine (DFMO), a potent inhibitor of ornithine decarboxylase, reduces polyamine levels in rats. Because polyamines are critically involved in growth and development, body and/or brain weights are often decreased by DFMO treatment. Here, rats were injected subcutaneously with 0, 250, 500, or 750 mg/kg of DFMO on postnatal days (PNDs) 5-10. Behavioral assessments included righting reflex, negative geotaxis, forelimb hanging, open field activity, and rotarod performance. Additionally, day of eye opening was recorded and on PND 28, whole and regional brain weights were measured. Cerebellar/whole-brain ratio was decreased in a dose-dependent manner whereas frontal cortex/whole-brain ratio was increased. Eye opening was delayed to a similar extent in all treated groups whereas body weight was unaffected. alpha-difluoromethylornithine treatment had no significant effects on the assessed behaviors. These results indicate that 6 days of DFMO treatment can substantially impact cerebellar development, but this appears to have few effects on these early assessed behaviors. However, potential behavioral alterations may not be apparent until adulthood. Published by Elsevier Science Inc.

Chemopreventive efficacy of combined piroxicam and difluoromethylornithine treatment of Apc mutant Min mouse adenomas, and selective toxicity against Apc mutant embryos.

Genetic knockout or pharmacological inhibition of cyclooxygenase-2 decreases the number and size of adenomas in mouse models of familial adenomatous polyposis. Epidemiological and clinical studies in humans indicate that the entire class of nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit both COX-1 and COX-2 enzymes are promising colon cancer chemopreventive agents. We used the Apc mutant Min mouse model to test combinations of agents that might maximize preventive benefit with minimal toxicity because they act via different mechanisms. Min mice (n = 144) were exposed to low doses of the nonselective COX inhibitor piroxicam and the ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO), beginning at the time they were weaned and continuing throughout the duration of the experiment. Piroxicam at 12, 25, and 50 ppm in the diet caused dose-dependent decreases in the number of tumors in the middle and distal portions of the small intestine. This decrease in tumor multiplicity was associated with a striking decrease in the size of those tumors that did grow out. In contrast, none of the doses of piroxicam alone decreased tumor multiplicity in the proximal portion of the intestine (duodenum). Exposure to DFMO (0.5 or 1.0% in water) caused a dose-dependent decrease in tumor multiplicity in the middle and distal portions of the small intestine. However, this decreased multiplicity was not associated with a striking decrease in the size of the tumors. Combined treatment of mice with piroxicam plus DFMO was much more effective than either agent alone and resulted in a significant number of mice totally free of any intestinal adenomas (P < 0.001), in contrast to the 100% incidence and high multiplicity in control Min mice. In addition to this profound effectiveness in reducing tumor number, the few residual tumors in mice treated with the combined drugs were markedly smaller in size than tumors that arose from control Min mice. These experiments suggest that selective COX-2 inhibition combined with ODC inhibition is a very promising approach for colon cancer prevention. These COX-2 and ODC inhibitor drugs were not overtly toxic at the doses used when administered to mice after weaning. However, when treatment was begun in utero, the Mendelian expected progeny ratio of 1:1 that we routinely obtained in untreated control litters was no longer observed. Apc(min)/+ progeny of pregnant dams treated with piroxicam and/or DFMO were reduced in number and their ratio to Apc+/+ progeny was decreased to approximately 0.28:1. Thus, these agents are effective against adenomas that have homozygous mutation of the APC gene and also select against fetuses bearing a heterozygous mutation in the APC gene.

Difluoromethylornithine in combination with tamoxifen in female rats: 13-week oral toxicity study.

PURPOSE: Cancer chemoprevention is the use of pharmacologic or natural agents to inhibit the development of cancer. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in the biosynthesis of polyamines. DFMO has demonstrated chemopreventive efficacy in animal models of tumorigenesis. Tamoxifen (TAM) is currently used for treatment of estrogen receptor-positive breast carcinoma and has demonstrated efficacy in chemoprevention of breast cancer in women at high risk for the disease. The administration of tamoxifen with DFMO is being considered for development by the National Cancer Institute as a potential drug regimen for the chemoprevention of breast carcinoma. METHODS: The toxicity of DFMO in combination with TAM was evaluated in female rats following 13 weeks of daily administration by gavage. Dose groups were vehicle control, DFMO (1000 mg/kg per day), low TAM (0.25 mg/kg per day), high TAM (2.5 mg/kg per day), low combination (1000 + 0.25) and high combination (1000 + 2.5). RESULTS: No mortalities occurred in the study. Clinical signs of toxicity were limited to dermal lesions consisting of scab formation and abrasions produced by DFMO. Administration of either DFMO or TAM resulted in decreased body weight gains, with coadministration having an additive effect. Serum albumin, total protein, cholesterol and triglyceride levels were decreased in all drug-treated dose groups, although histologic evidence of liver lesions were not seen. TAM resulted in increased numbers of red blood cells, whereas DFMO produced a slightly anemic response. DFMO produced lesions in the small intestine consisting of necrosis of crypt epithelium and crypt microabscess, which were enhanced by TAM coadministration. Administration of TAM resulted in histologic changes in the ovaries, fallopian tube, vagina, cervix and uterus, indicating that inhibition of ovulation and reproductive cycle arrest in the proestrus stage had occurred. Coadministration with DFMO did not affect the changes to the reproductive system induced by TAM. CONCLUSIONS: Coadministration of DFMO with tamoxifen did not result in toxicity unique to the combination drug regimen, but rather toxicity resulted from administration of each drug. Under the conditions of the study, the overall toxicity produced by dual administration of DFMO with tamoxifen was additive with respect to the toxicity associated with each agent alone.

Developmental toxicity studies of 2-(difluoromethyl)-dl-ornithine (DFMO) in rats and rabbits.

DFMO, an irreversible inhibitor of ornithine decarboxylase (ODC), is under development as a chemopreventive drug against cancers with pronounced proliferative phases. In support of human clinical trials, preclinical developmental toxicity studies were conducted in pregnant rats and rabbits. Rats were treated during GD 6-17, and fetuses were obtained by C-section on GD 20. Rabbits were treated during GD 7-20, and fetuses were obtained by C-section on GD 29. The dose range-finding study in rats (5/group at 0, 50, 125, 300, 800, or 1000 mg/kg/day) revealed maternal toxicity at doses > or = 800 mg/kg/day (decreased body weights and food consumption) and developmental toxicity at doses > or = 300 mg/kg/day (increased early resorptions and reduced fetal body weights). In the main study, rats (25/group) received 0, 30, 80, or 200 mg/kg/day. Developmental toxicity in the absence of maternal toxicity was observed at 200 mg/kg/day as significantly decreased fetal weights and increased incidence of litters with skeletal variations of 14th rudimentary rib, 14th full rib, and/or 27th presacral vertebrae. There were no treatment-related fetal skeletal malformations or external or visceral anomalies at any dose level. The dose range-finding study in rabbits (5/group at 0, 30, 60, 120, 240, or 500 mg/kg/day) revealed developmental toxicity at doses > or = 60 mg/kg/day (increased resorptions and reduced fetal body weights) in the absence of maternal toxicity. In the main study, rabbits (20/group) received 0, 15, 45, or 135 mg/kg/day. Developmental toxicity in the absence of maternal toxicity was observed at 135 mg/kg/day as nonsignificantly increased early resorptions, decreased implantation sites, decreased viable fetuses, and reduced fetal weights. There were no external, visceral, or skeletal anomalies at any dose level. Thus, in the main developmental toxicity studies, DFMO produced developmental but not maternal toxicity at 200 and 135 mg/kg/day in rats and rabbits, respectively. Accordingly, in rats, the maternal no-observable-effect level (NOEL) was 200 mg/kg/day and the fetal NOEL was 80 mg/kg/day; while in rabbits the maternal NOEL was 135 mg/kg/day and the fetal NOEL was 45 mg/kg/day. These fetal NOELs are several-fold higher than the dose level currently used in Phase II and III clinical trials (approximately 13 mg/kg).

Development of difluoromethylornithine (DFMO) as a chemoprevention agent.

D,L-alpha-difluoromethylornithine (DFMO) was synthesized over 20 years ago. It was hoped that this enzyme-activated, irreversible inhibitor of ornithine decarboxylase, the first enzyme in polyamine synthesis, would be effective as a chemotherapy for hyperproliferative diseases, including cancer and/or infectious processes. DFMO was generally found to exert cytostatic effects on mammalian cells and tissues, and its effectiveness as a therapeutic agent has been modest. DFMO was also found to cause treatment-limiting (but reversible) ototoxicity at high doses. This side effect, along with its minimal therapeutic activity, contributed to the loss of interest by many clinicians in further developing DFMO as a cancer therapeutic agent. However, DFMO was subsequently shown to inhibit carcinogen-induced cancer development in a number of rodent models, and interest in developing this compound as a preventive agent has increased. The rationale for the inhibition of ornithine decarboxylase as a cancer chemopreventive agent has been strengthened in recent years because this enzyme has been shown to be transactivated by the c-myc oncogene in certain cell/tissue types and to cooperate with the ras oncogene in malignant transformation of epithelial tissues. Recent clinical cancer chemoprevention trials, using dose de-escalation designs, indicate that DFMO can be given over long periods of time at low doses that suppress polyamine contents in gastrointestinal and other epithelial tissues but cause no detectable hearing loss or other side effects. Current clinical chemoprevention trials are investigating the efficacy of DFMO to suppress surrogate end point biomarkers (e.g., colon polyp recurrence) of carcinogenesis in patient populations at elevated risk for the development of specific epithelial cancers, including colon, esophageal, breast, cutaneous, and prostate malignancies.

Thirteen-week oral toxicity study of difluoromethylornithine in combination with tamoxifen citrate in female dogs.

PURPOSE: Cancer chemoprevention is the use of pharmacologic or natural agents to inhibit the development of cancer. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in the biosynthesis of polyamines. DFMO has demonstrated chemopreventive efficacy in animal models of tumorigenesis. Tamoxifen (TAM), a nonsteroidal antiestrogen, is approved for use in the treatment of estrogen receptor-positive breast carcinoma and has demonstrated efficacy in chemoprevention of breast cancer in women at high risk for the disease. The administration of TAM with DFMO is being considered for development by the National Cancer Institute as a potential drug regimen for the chemoprevention of breast carcinoma. METHODS: The toxicity of DFMO in combination with TAM was evaluated in female Beagle dogs following 13 weeks of daily oral administration by capsule. Dose levels in milligrams per kilogram body weight per day were: 0 (vehicle control), 100 DFMO, 0.1 TAM, 1.0 TAM, 0.1 TAM + 100 DFMO and 1.0 TAM + 100 DFMO. RESULTS: No mortalities occurred. Diarrhea was produced by TAM and vaginal discharge, due to reproductive tract lesions, was produced by both DFMO and TAM, either alone or in combination. DFMO decreased reticulocyte counts and TAM increased counts of mature neutrophils. DFMO alone resulted in lesions to the intestines and ovaries, and cornified epithelium of vagina and cervix. TAM produced cornified epithelium of vagina and cervix, and numerous lesions in the ovaries, fallopian tube, uterus, cervix and vagina which were likely due to an estrogen agonist effect. Coadministration of DFMO increased the incidence and/or severity of these reproductive tract lesions. Each compound alone produced ovarian atrophy, and antral follicles and corpora lutea were completely absent in the 1.0 TAM + 100 DFMO group. CONCLUSIONS: Coadministration of DFMO and TAM resulted in additive toxicity involving the female reproductive system.

Potentiation by 2-deoxy-D-glucose tetraacetate of the cytostatic action of alpha-difluoromethylornithine in tumoral insulin-producing cells.

Insulin-producing tumoral cells of the RINm5F line were cultured for 8 to 96 h in the absence or presence of 2-deoxy-D-glucose tetraacetate (0.01 mM to 1.0 mM) and/or á-difluoromethylornithine (also 0.01 mM to 1.0 mM). The ester of the glucose analog potentiated the inhibitory action of the ornithine decarboxylase inhibitor. For instance, when both agents were tested in combination at a concentration of 0.01 mM each, the generation of formazan from 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium was decreased, within 2 days, to 23.2+/- 6.1% of its paired control value, whilst neither the ester of 2-deoxy-D-glucose nor the ornithine analog exerted any significant effect upon cell growth when used separately at the same low concentration and over the same length of culture. These findings suggest that 2-deoxy-D-glucose tetraacetate could be used to sensitize tumoral cells to the cytostatic action of established chemotherapeutic agents in the treatment of neoplastic diseases.

Inhibition of breast cancer in nude mouse model by anti-angiogenesis.

The mechanism by which DL-alpha-difluoromethylornithine (DFMO) inhibits angiogenesis is generally thought to involve the inhibition of the rate-limiting enzyme, ornithine decarboxylase (ODC), leading to polyamine depletion in cells and the ultimate cytostatic effect on proliferating endothelial cells. Another mechanism for inhibiting tumor growth involves pentosan polysulfate (PPS) which binds heparin-binding growth factors, known to be crucial for tumor angiogenesis. To quantitate the combined anti-angiogenic effect of DFMO and PPS on tumors, blood vessels were stained using monoclonal antibodies against the platelet endothelial cell adhesion molecule (PECAM). When compared to untreated mice, DFMO/PPS-treated mice exhibited significantly lower (6-fold) blood vessel counts. Furthermore, mice receiving the combination drug treatment had prolonged life compared to untreated tumor-bearing mice, but less than normal tumor-free mice. The prolonged life span of drug treated tumor-bearing mice also correlated with reduced tumor burden in these mice. The use of single drug treatment results in rapid tumor growth and eventual death of tumor-bearing mice. We have demonstrated that there was significant difference in survival time which correlated to less tumor burden in treated groups of mice as compared to the controls. Inhibition of tumor angiogenesis by the drug combinations suggest that these compounds are anti-angiogenic agents for potential use in clinical trial.

In vitro cytotoxic effect of difluoromethylomithine increased nonspecifically by peptide coupling.

Difluoromethylomithine (DFMO)-peptide conjugates were synthesized as prodrugs to improve the cytotoxic efficacy of DFMO. All conjugates inhibited cell growth in different cell lines more effectively than DFMO itself. The best cytotoxic effect was achieved in all cell lines by DFMO-Glu-His-Phe-Arg-Trp-Gly-OMe, where the carrier peptide is a melanotropin hormone fragment. Although this conjugate is capable of displacing labeled melanotropin from its receptor, its cytotoxic effect on the receptor-positive human melanoma cell line has not been proven to be receptor-mediated. The differences in the cytotoxicities of the congeners seem to be influenced, at least in part, by the nature of the carrier molecule.

Chronic toxicity studies of the potential cancer preventive 2-(difluoromethyl)-dl-ornithine.

The synthetic compound 2-(difluoromethyl)-dl-ornithine irreversibly inhibits ornithine decarboxylase and reduces the intracellular levels of the polyamine cell cycle factors putrescine and spermidine. The drug has shown chemopreventive efficacy in numerous laboratory epithelial cancer models and is a prototype for antiproliferative agents. Chronic toxicity studies in rats and dogs were performed to characterize the toxicities of the compound at high dosages and to support its further development in clinical trials as a potential chemopreventive agent. Chronic administration (52 weeks) by gavage to Charles River CD rats at dosages of 400, 800, and 1600 mg/kg produced weight loss, increased platelets, alopecia and skin abrasions, dermatitis, liver necrosis, and gastric inflammation. The no-effect dose in this study was considered 400 mg/kg. Chronic administration by capsule to dogs at dosages of 50, 100, and 200 mg/kg produced conjunctivitis, hyperkeratosis and alopecia, and cystic intestinal crypts. A no-effect dose was not determined in this study. The toxicities demonstrated in these studies may be minimized at lower dosages and support the further development of this compound in chemopreventive clinical investigations.

Zinc is required for the expression of ornithine decarboxylase in a difluoromethylornithine-resistant cell line.

Dilution of quiescent L1210-DFMOr (difluoromethylornithine-resistant) cells in fresh medium containing serum led to the induction of ornithine decarboxylase (ODC) and to the expression of its mRNA, as determined by a sensitive solution-hybridization-RNase-protection assay. Addition of the chelating agent diethylenetriaminepentaacetic acid (DTPA) at seeding time caused an inhibition of the induction of ODC activity by up to 90%, and only Zn2+ of the bivalent metal ions tested was effective in reversing this effect. The inhibition of the induction of ODC activity was accompanied by a marked decrease, prevented by Zn2+ supplementation, of the accumulation of immunoreactive ODC protein and ODC mRNA. DTPA treatment also caused a slight acceleration of ODC turnover. These results indicate that a restricted Zn2+ availability in L1210-DFMOr cells impairs ODC induction remarkably, mainly by affecting the expression of the messenger.