Administration Of Difluoromethylornithine Research Articles

Berberine, a potential prebiotic to indirectly promote Akkermansia growth through stimulating gut mucin secretion

BackgroundAkkermansia spp. plays important roles in maintenance of host health. Increasing evidence reveals that berberine (BBR) may exert its pharmacological effects via, at least partially, promotion of Akkermansia spp. However, how BBR stimulates Akkermansia remains largely unknown. PurposeIn this study, we investigated the mechanism underlying the Akkermansia-promoting effect of BBR. Materials and methodsThe effect of BBR on Akkermansia was assessed in BBR-gavaged mice and direct incubation. The influence of BBR on intestinal mucin production was determined by alcian-blue staining and real-time PCR. The feces were analysis by gas chromatography–time-of-flight mass spectrometry (GC-TOF/MS) metabolomics. The role of polyamines in BBR-elicited mucin secretion and Akkermansia growth was evaluated by administration of difluoromethylornithine (DFMO) in mice. ResultsGavage of BBR dose-dependently and time-dependently increased the abundance of Akkermansia in mice. However, it did not stimulate Akkermansia growth in direct incubation, suggesting that BBR may promote Akkermansia in a host-dependent way. Oral administration of BBR significantly increased the transcription of mucin-producing genes and mucin secretion in colon. Untargeted metabolomics analysis showed that BBR increased polyamines production in feces which are known to stimulate goblet cell proliferation and differentiation, but treatment with eukaryotic polyamine synthase inhibitor DFMO did not abolish the stimulating effect of BBR on mucin secretion and Akkermansia growth, indicating that the gut bacteria-derived but not the host-derived polyamines may involve in the BBR-promoted Akkermansia growth. ConclusionsOur results reveal that BBR is a promising prebiotic for Akkermansia, and it promotes Akkermansia growth via stimulating mucin secretion in colon.

Anti-GD2 induced allodynia in rats can be reduced by pretreatment with DFMO.

Anti-GD2 therapy with dinutuximab is effective in improving the survival of high-risk neuroblastoma patients in remission and after relapse. However, allodynia is the major dose-limiting side effect, hindering its use for neuroblastoma patients at higher doses and for other GD2-expressing malignancies. As polyamines can enhance neuronal sensitization, including development of allodynia and other forms of pathological pain, we hypothesized that polyamine depletion might prove an effective strategy for relief of anti-GD2 induced allodynia. Sprague-Dawley rats were allowed to drink water containing various concentrations of difluoromethylornithine (DFMO) for several days prior to behavioral testing. Anti-GD2 (14G2a) was injected into the tail vein of lightly sedated animals and basal mechanical hindpaw withdrawal threshold assessed by von Frey filaments. Endpoint serum DFMO and polyamines, assessed 24h after 14G2a injection, were measured by HPLC and mass spectrometry. An i.v. injection of 14G2a causes increased paw sensitivity to light touch in this model, a response that closely mimics patient allodynia. Animals allowed to drink water containing 1% DFMO exhibited a significant reduction of 14G2a-induced pain sensitivity (allodynia). Increasing the dosage of the immunotherapeutic increased the magnitude (intensity and duration) of the pain behavior. Administration of DFMO attenuated the enhanced sensitivity. Consistent with the known actions of DFMO on ornithine decarboxylase (ODC), serum putrescene and spermidine levels were significantly reduced by DFMO, though the decrease in endpoint polyamine levels did not directly correlate with the behavioral changes. Our results demonstrate that DFMO is an effective agent for reducing anti-GD2 -induced allodynia. Using DFMO in conjunction with dinutuximab may allow for dose escalation in neuroblastoma patients. The reduction in pain may be sufficient to allow new patient populations to utilize this therapy given the more acceptable side effect profile. Thus, DFMO may be an important adjunct to anti-GD2 immunotherapy in addition to a role as a potential anti-cancer therapeutic.

Polyamine blockade alters the neuroblastoma microenvironment to favor restriction of tumor growth

Abstract Despite improved treatments, neuroblastoma (NB) still accounts for ~15% of childhood cancer deaths. Amplification of MYCN results in NB with high-risk features and poor 5yr survival. Interestingly, MYCN-amplified NBs have elevated polyamine (PA) levels, as the gate-keeper enzyme in PA synthesis, ornithine decarboxylase (ODC1), is a direct Myc target. Inhibition of ODC1 activity by the FDA-approved medication difluoromethylornithine (DFMO) results in modest growth reduction of NB cell lines in vitro, whereas DFMO administration to mice that are predisposed to developing MYCN-driven NBs (TH-MYCN+/+ mice) leads to survival extensions that were more profound than that predicted by the tumor-intrinsic activity of the drug alone. These observations implied that DFMO might also exert tumor-extrinsic effects on the tumor microenvironment (TME). Indeed, DFMO treatment of TH-MYCN+/+ mice alters the NB TME in several ways, the most profound of which is an increase in NK cell frequency. We also find an increase in the percentage of NB cells expressing NKG2D ligands, and alterations in the expression of NK cell stimulatory and inhibitory receptors to favor activation. Collectively, these findings suggest that PA blockade induces distinct TME changes that allow for more efficient immune control of NB growth. We are now testing whether the activity of DFMO is dependent on NK cells, and are delineating how DFMO alters the transcriptional profiles of NBs and immune cells in the TME. We hope that these studies will complement the data from phase I/II clinical studies using DFMO in various therapeutic strategies for NB, and will allow for an increased understanding of how to more effectively combine PA blockade with other immunotherapies for this disease.

Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant

The acquired mutation (V617F) of Janus kinase 2 (JAK2) is observed in the majority of patients with myeloproliferative neoplasms (MPNs). In the screening of genes whose expression was induced by JAK2 (V617F), we found the significant induction of c-Myc mRNA expression mediated by STAT5 activation. Interestingly, GSK-3β was inactivated in transformed Ba/F3 cells by JAK2 (V617F), and this enhanced the protein expression of c-Myc. The enforced expression of c-Myc accelerated cell proliferation but failed to inhibit apoptotic cell death caused by growth factor deprivation; however, the inhibition of GSK-3β completely inhibited the apoptosis of cells expressing c-Myc. Strikingly, c-Myc T58A mutant exhibited higher proliferative activity in a growth-factor-independent manner; however, this mutant failed to induce apoptosis. In addition, knockdown of c-Myc significantly inhibited the proliferation of transformed cells by JAK2 (V617F), suggesting that c-Myc plays an important role in oncogenic activity of JAK2 (V617F). Furthermore, JAK2 (V617F) induced the expression of a target gene of c-Myc, ornithine decarboxylase (ODC), known as the rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO), prevented the proliferation of transformed cells by JAK2 (V617F). Importantly, administration of DFMO effectively delayed tumor formation in nude mice inoculated with transformed cells by JAK2 (V617F), resulting in prolonged survival; therefore, ODC expression through c-Myc is a critical step for JAK2 (V617F)-induced transformation and DFMO could be used as effective therapy for MPNs.

P067 JAK2 V617F mutation causes cytokine-independent growth and tumorigenesis – Roles of STAT5 and c-Myc

Introduction JAK2 is an essential signal transducer for various cytokine signaling including IL-3 or Epo. Mutation at V617F of JAK2 promotes cell growth and prevents apoptosis which causes a variety of myeloproliferative neoplasms (MPNs). How the JAK2 V617F mutation causes dysregulated proliferation, resistance to anti-tumor drugs and tumorigenesis in nude mice is to be investigated. Methods Ba/F3 cells harboring JAK2 V617F were established to study the constitutive activation of JAK2 and cytokine-independent growth. Tumorigenesis was tested by the inoculation of JAK2 V617F mutant in nude mice. DNA array was done to search genes expressed predominantly by JAK2 V617F and STAT5. Results (1) JAK2 V617F mutant caused aberrant activation of STAT5 and Akt when EpoR was expressed, which is critical for the JAK2 V617F-induced anti-apoptotic and oncogenic activities. (2) In search of genes induced by JAK2 V617F mutant, we observed significant induction of c-Myc. (3) While the phosphorylation at Thr58 by GSK-3b induced the proteasome-mediated degradation of c-Myc, GSK-3b was inactivated in JAK2 V617F mutant, which resulted in the enhancement of c-Myc expression. The enforced expression of c-Myc accelerated not only the cell proliferation but also apoptotic cell death; however, the inhibition of GSK-3b completely inhibited c-Myc-induced apoptosis. (4) JAK2 V617F mutant also induced expression of a target gene of c-Myc, ornithine decarboxylase (ODC) known as a rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO) prevented the proliferation of the JAK2 V617F mutant cells. Furthermore, administration of DFMO effectively delayed tumor formation in nude mice, resulting in a prolonged survival. Conclusion JAK2 V617F mutant caused aberrant activation of Akt and STAT5 pathways. ODC expression induced by c-Myc is critical for JAK2 V617F mutant-driven transformation and that targeted disruption of c-Myc-ODC axis may provide therapeutic utility for the treatment of MPNs. STAT5 and c-Myc have critical roles for JAK2 V617F mutant-induced tumorigenesis, which help clarify the mechanism how JAK2 V617F mutant causes MPNs.

Difluoromethylornithine inhibits crypt fission

Crypt fission is a physiologic mechanism of crypt reproduction. It increases in pathophysiologic situations where intestinal regeneration is required (e.g., radiation injury). Polyamine metabolism is important in the regulation of intestinal growth and recovery from injury in response to a variety of stimuli. Our aim was to determine whether inhibition of polyamine synthesis by difluoromethylornithine (DFMO) influenced crypt fission. Forty-eight rabbits underwent patch enteroplasty in the terminal ileum. One group served as a control group and the other took 2% DFMO orally. Animals (n = 6) from each group were killed at 7, 14, 21, and 28 days. Normal ileum adjacent to the enteroplasty was studied. Crypt dissection was performed 2 hours after vincristine was administered intravenously to determine crypt cell production rate, crypt depth, and proportion of bifurcating crypts (fission). DFMO administration decreased crypt fission (4 ± 2% vs. 11 ± 2% and 13 ± 1% vs. 34 ± 4% at 7 and 14 days) compared to control animals. There was a corresponding increase in crypt depth at 14 and 21 days. Crypt cell production rate was similar in both groups and did not change with time. Mucosal ornithine decarboxylase activity (11.9 ± 2.2 vs. 1.2 ± 0.3 specific activity at 21 days; P <0.05) and polyamine content (323 ± 32 vs. 17 ± 8 and 382 ± 89 vs. 160 ± 47 pmol/mg at 14 and 21 days, control vs. DFMO; P <0.05) were significantly lower in the DFMO group. The following conclusions were drawn: (1) DFMO administration inhibits crypt fission in stimulated intestinal epithelium; (2) this effect correlates temporally with reduced polyamine production; and (3) reduced crypt fission is another potential mechanism of inhibition of intestinal growth by altered polyamine metabolism.

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

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. 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). 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. 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.

Difluoromethylornithine inhibits crypt fission

Crypt fission is a physiologic mechanism of crypt reproduction. It increases in pathophysiologic situations where intestinal regeneration is required (e.g., radiation injury). Polyamine metabolism is important in the regulation of intestinal growth and recovery from injury in response to a variety of stimuli. Our aim was to determine whether inhibition of polyamine synthesis by difluoromethylornithine (DFMO) influenced crypt fission. Forty-eight rabbits underwent patch enteroplasty in the terminal ileum. One group served as a control group and the other took 2 % DFMO orally. Animals (n = 6) from each group were killed at 7, 14, 21, and 28 days. Normal ileum adjacent to the enteroplasty was studied. Crypt dissection was performed 2 hours after vincristine was administered intravenously to determine crypt cell production rate, crypt depth, and proportion of bifurcating crypts (fission). DFMO administration decreased crypt fission (4 ±2% vs. 11 ± 2% and 13 ±1% vs. 34 ±4% at 7 and 14 days) compared to control animals. There was a corresponding increase in crypt depth at 14 and 21 days. Crypt cell production rate was similar in both groups and did not change with time. Mucosal ornithine decarboxylase activity (11.9 ±2.2 vs. 1.2 ± 0.3 specific activity at 21 days; P <0.05)and polyamine content (323 ±32 vs. 17±8 and 382 ±89 vs. 160 ±47 pmol/mg at 14 and 21 days, control vs. DFMO; P <0.05) were significantly lower in the DFMO group. The following conclusions were drawn: (1) DFMO administration inhibits crypt fission in stimulated intestinal epithelium; (2) this effect correlates temporally with reduced polyamine production; and (3) reduced crypt fission is another potential mechanism of inhibition of intestinal growth by altered polyamine metabolism.

Relation of Ornithine Decarboxylase and Tyrosine Kinase Activity in the Jejunal Mucosa In Vivo

Our aim was to study the relationship between jejunal mucosal activity of ornithine decarboxylase and tyrosine kinase during proliferation in adolescent rats in vivo. Their relationship in the proliferating intestinal mucosa under in vivo conditions has not been reported before. From the results of in vitro studies, it was speculated that tyrosine kinase activity modulated ornithine decarboxylase activity during colonic mucosal proliferation (Majumdar AP. Am J Physiol 259:G626-G630, 1990). Jejunal mucosal hyperplasia was induced by Type 1 diabetes and suppressed in both control and diabetic rats by administration of difluoromethylornithine. Jejunal mucosal weight and enzyme activity were determined after 3, 6, and 10 days, and tyrosine-specific phosphorylated proteins after 10 days of induction of diabetes. Difluoromethylornithine suppressed jejunal mucosal proliferation and tyrosine kinase activity after the 6- and 10- day study periods. After the 3-day study period although jejunal mucosal growth was suppressed, tyrosine kinase activity was not. Activity of tyrosine kinase and ornithine decarboxylase were highly significantly correlated at all time periods in both control and diabetic rats. Tyrosine-specific phosphorylated proteins of 34, 54, 80, and 200 kDa proteins were observed in jejunal mucosa of both control and diabetic rats. In the difluoromethylornithine-treated rats, phosphorylation of the above proteins was negligible while the phosphorylation of a 14-kDa protein was prominent. We speculate that in vivo ornithine decarboxylase activity may be modulating tyrosine kinase activity and that phosphorylation of a 14-kDa protein was associated with suppressed mucosal growth in difluoromethylornithine-treated rats.

Precocious cessation of intestinal macromolecular transport and digestive enzymes development by prostaglandin E 2 in suckling rats

The effect of repeated oral administration of prostaglandin analogue (dmPGE 2) on intestinal macromolecular transport and digestive enzymes development were investigated in the suckling rats. By the administration of dmPGE 2 for 7 days, precocious induction of maltase activity, depression of amylase activity and enhancement of trypsin activity in the pancreas occurred. Absorption of bovine IgG was dose dependently depressed by drnPGE 2 treatments. The intestinal cessation was also observed in the adrenalectomized pups, but was not influenced by difluoromethyl ornithine administration. These results suggest that oral administration of PGE 2 induces precocious maturation of the small intestine and exocrine pancreas and that the intestinal cessation is not directly related to ornithine decarboxylase activity in the suckling rats.

Polyamines and Intestinal Epithelial Hyperplasia in Streptozotocin-Diabetic Rats

We measured specific activity of ornithine decarboxylase (ODC) and contents of putrescine and of the polyamines (spermidine and spermine) in isolated villus and crypt enterocytes from the jejunum of adolescent streptozotocin-diabetic and weight-matched control rats and diabetic and control rats treated with difluoromethyl ornithine (DFMO) 10 days after induction of diabetes. Consistent with previous observations by others of elevated ODC activity and contents of putrescine and of the polyamines in the intestinal epithelium undergoing hyperplasia, our studies showed elevated ODC activity and contents of putrescine and spermidine, but not of spermine, in the hyperplastic intestinal epithelium of diabetic rats. As in previous studies, suppression of ODC activity by DFMO prevented not only the jejunal epithelial hyperplasia in the diabetic rats, but also retarded jejunal epithelial growth in the control rats. DFMO administration lowered ODC activity by over 80% in both diabetic and control rat enterocytes and prevented the rise in enterocyte contents of putrescine and spermidine in the diabetic rat. The observation that, in both diabetic and control rats, treatment with DFMO lowered spermidine content in the crypt enterocytes but had no similar consistent effect on contents of putrescine or spermine suggested that spermidine could have been responsible for the intestinal epithelial hyperplasia in the diabetic rats and for the normal growth of the intestinal epithelium in control rats.

Acute Difluoromethylornithine Treatment Increases Skin Flap Survival in Rats

Difluoromethylornithine (DFMO) pretreatment for 7 days improved survival of rat abdominal skin flaps in previous studies. The purpose of this study was to determine if acute administration of DFMO enhances survival. Each rat had a 7 x 7-cm abdominal skin flap raised on a single epigastric neurovascular pedicle. Within 1 minute of pedicle ligation, the rats were given 0, 1, or 4 gm/kg of body weight of DFMO intraperitoneally. Putrescine was administered to additional rats alone or with DFMO. After 48 hours, the percentage of flap survival was estimated using fluorescein injection and planimetry to quantify the perfused and unperfused areas. Flap survival increased from 71 +/- 3% in controls to 83 +/- 2% and 92 +/- 3% in rats treated with 1 and 4 gm/kg of DFMO, respectively (p less than 0.005). Putrescine reversed the protective effect of DFMO, suggesting a specific polyamine-related mechanism. This study indicates that there may be both short- and long-term polyamine pools through which DFMO acts. In summary, DFMO may prove to be important in preventing cell death following acute ischemia.

Differential stimulation of S-adenosylmethionine decarboxylase by difluoromethylornithine in the rat colon and small intestine

The effects of chronic inhibition of ornithine decarboxylase (ODC) by the specific inhibitor difluoromethylornithine (DFMO) in the rat colon and small intestine on mucosal contents of polyamines, decarboxylated S-adenosylmethionine (decarboxylated AdoMet) and S-adenosylmethionine decarboxylase (AdoMet decarboxylase) activity were studied. Administration of 1% DFMO in the drinking water for 10 or 15 weeks resulted in inhibition of ODC and decreases in intracellular putrescine and spermidine contents in both proximal and distal segments of small intestine and colon. At both time points DFMO administration resulted in a dramatic stimulation of AdoMet decarboxylase activity and a rise in decarboxylated AdoMet content in the proximal and distal small-intestinal segments compared with controls, which was not seen in either colonic segment of DFMO-treated animals. This differential stimulation of AdoMet decarboxylase by DFMO in the small intestine and colon could not be entirely explained on the basis of differences in polyamine contents, which are known to regulate this enzyme activity. Kinetic and inhibition studies of AdoMet decarboxylase in control small and large intestine revealed that: (1) there was no difference in Vmax. values between the tissues; (2) the Km for AdoMet was higher in the small intestine than in the colon; and (3) the Ki for product inhibition by decarboxylated AdoMet was higher in the small intestine than in the colon. These results suggest that the differential stimulation of AdoMet decarboxylase by DFMO in the small intestine and colon may be due to different isoenzymes and could play a significant role in the regulation of polyamine contents throughout the gut.

Effects of Timing of Administration and Dose of Difluoromethylornithine on Rat Colonic Carcinogenesis

During azoxymethane (AOM)-induced colonic carcinogenesis in rats, biphasic induction of ornithine decarboxylase (ODC) activity occurs in the colonic mucosa. The relative contributions of these two phases of ODC induction to the carcinogenesis process were examined by studying the effects of the specific ODC inhibitor, difluoromethylornithine (DFMO), administered during either the initial phase of ODC increase or during both phases continuously. The effects of 1% and 0.25% DFMO administered continuously were also compared. Continuous oral administration of DFMO at 1% (approximately 8 mg/g body wt/wk) and 0.25% (approximately 2 mg/g body wt/wk) produced 93% inhibition of ODC induction by AOM in the right and left colons throughout the study. Despite suppression of ODC activity to near-normal levels, colon tumor incidence at 26 weeks in the right colon was not affected by either initial-phase or continuous administration of DFMO. In contrast, tumor incidence in the left colon was reduced from 35% to 5% by DFMO given continuously at doses of both 1% and 0.25% over the entire 26 weeks (P less than .05). No significant reduction in left colon tumor incidence resulted from the short initial 11-week course of DFMO although the tumor incidence was reduced (15% vs. 35%). Results suggest that the second ("post-initiation") phase of ODC induction may be of particular importance in carcinogenesis.

Gastric mucosal ornithine decarboxylase: localization and stimulation by gastrin.

Gastrin injection and refeeding fasted rats are effective trophic stimuli for the oxyntic gland mucosa of the stomach. Neither stimulus increases detectable ornithine decarboxylase (ODC) activity in the tissue. Difluoromethylornithine (DFMO), a potent inhibitor of ODC, blocks the mucosal growth response, indicating that ODC activity is necessary for growth. Elevated levels of spermidine and spermine are detectable in the mucosa after gastrin administration. Using a highly specific, polyclonal antiserum to ODC, we determined that the enzyme is present in oxyntic gland mucosa confined to a narrow band of cells at the base of the gastric pits and openings of the glands. In antral mucosa, ODC is present throughout the lower 20% of the mucosa, which consists of the necks and pyloric glands. Using antiserum dilution techniques, we show that gastrin administration increases immunoreactive ODC in the oxyntic gland area but not in the antral mucosa, where it has no trophic effect. Elevated cellular content of ODC is apparent within 2 h after injection of gastrin, peaks at 4 h, and declines to basal levels by 12 h. Gastrin-stimulated increase in ODC is confined to the narrow band of cells in which low levels of the enzyme protein were detected in control animals. The decarboxylating activity detectable in oxyntic gland mucosal extracts is not inhibited by administration of DFMO or cycloheximide, each of which inhibits ODC activity in other tissues. Addition of unlabeled lysine to the decarboxylation assay reaction of oxyntic gland mucosa extract inhibits the decarboxylation of radiolabeled ornithine substrate. Thus it is likely that the stomach possesses nonspecific decarboxylase activity, which accounts for most of the measured activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyamines increase in human peripheral blood and bone marrow mononuclear cells following administration of methylglyoxal bis(guanylhydrazone).

Eight patients who had refractory leukemia and 1 patient with refractory multiple myeloma were treated with the polyamine biosynthesis inhibitors methylgloxal bis(guanylhydrazone) (MGBG) and difluoromethylornithine (DFMO). After the first dose of MGBG there was an increase in polyamine content in the mononuclear cells of both the peripheral blood and the bone marrow despite the administration of DFMO in all patients with leukemia. Putrescine levels increased in the mononuclear cells of all patients, cellular spermidine levels increased in 4 and cellular spermine levels increased in 5 patients. The cellular polyamine levels remained elevated above the pretreatment levels for up to 1 week in some patients. Subsequent treatment with MGBG, administered after 1-2 weeks of DFMO treatment, also promoted increases in mononuclear cell polyamine concentrations. Since enhanced tumor cell uptake of MGBG after DFMO priming is hypothesized to be dependent on a decrease in cellular polyamine levels, the increase in cellular polyamines after MGBG has important implications for the scheduling of this drug combination. From these observations, withholding MGBG until DFMO treatment has produced a decrease in tumor cell polyamine concentrations would be the schedule most likely to enhance the uptake of MGBG.

Sustained delivery of difluoromethylornithine (DFMO) by means of implantable ceramic devices.

A total of 25 Sprague-Dawley albino male rats were assigned to five groups, each group consisting of five rats. Polylactic acid-impregnated ALCAP capsules filled with 40 mg DFMO were implanted subcutaneously (sc) or intraperitoneally (ip) in group I and II rats, respectively. Rats in group III were implanted with empty ALCAP capsules (ALCAP control). Group IV rats were administered orally 3% DFMO in drinking water. Rats in group V served as control. Blood samples were collected every week for six weeks via the tail artery. The concentration of DFMO was determined by a modified ninhydrin hydrindantin method. Data obtained in this investigation showed that the levels of DFMO in the serum of rats in groups I and II were 41.87 +/- 6.8 and 237.62 +/- 13.3 micrograms/mL, respectively. Body, spleen, liver, prostate, seminal vesicles, and testicular weights of the controls and DFMO treated rats were not significantly different (p less than 0.05) over the entire duration of this study. The diarrhea often noted in rats treated orally with DFMO was not observed in rats implanted with ALCAP capsules filled with DFMO. The results of this study suggest that (1) polymer-impregnated ALCAP ceramic capsules can be used to deliver DFMO in a sustained manner for long durations of time, (2) side effects associated with oral administration of DFMO can be avoided by using DFMO-filled ALCAP ceramic implants, and (3) a ceramic system can be designed to deliver DFMO and drugs such as DFMO in a sustained manner over long periods of time in humans.

Haematological cell proliferation and differentiation responses to perturbations of polyamine biosynthesis.

Combined administration of methylglyoxal-bis-guanylhydrazone (MGBG) (25 mg/kg) with difluoromethylornithine (DFMO), or MGBG alone at a higher dose (50 mg/kg), to mice resulted in a decreased white cell count (WBC) in the peripheral blood while DFMO or MGBG alone at a lower dose (25 mg/kg) had no effect. As expected, DFMO alone increased the number of colony forming units spleen (CFU-s), colony forming units diffusion chamber granulocyte (CFU-dg) and colony forming units culture (CFU-c) in the bone marrow. MGBG treatment led to an increase in CFU-dg alone. Combined treatment seemingly had no effect on marrow stem cells. Total tibial and differential counts were not affected by any of the treatments. Cell proliferation in diffusion chamber cultures, as judged by CFU-dg colony formation, was impaired by MGBG alone or in combination with DFMO, at dose levels which had no effect or increased the precursor cell number in the bone marrow. This effect was partially reversed with either putrescine or spermidine. Determination of intracellular polyamine concentrations, demonstrated decreased putrescine and spermidine levels after DFMO administration. As expected, MGBG treatment resulted in decreased spermidine and spermine levels, concomitant with an increase in putrescine. In mice which received both agents, rather than only MGBG, after 3 days higher intracellular polyamine concentrations were observed. After 11 days, however, there was no significant difference between the two groups.

Regulation of Epidermal Proliferation in Mouse Epidermis by Combination of Difluoromethyl Ornithine (DFMO) and Methylglyoxal Bis(guanylhydrazone) (MGBG)

Topical methylglyoxal bis(guanylhydrazone) (MGBG) and alpha-difluoromethylornithine (DFMO) individually have been shown to produce partial clinical improvement in psoriasis. In an effort to further enhance therapeutic activity, studies were designed to optimize percutaneous penetraton of DFMO in vitro and to determine the effects of the combination of DFMO and MGBG on DNA synthesis and polyamine levels in hairless mouse skin. MGBG was shown to be more effective than DFMO in inhibiting DNA synthesis in vitro and in vivo. Maximum in vitro percutaneous penetration of DFMO (5%) was obtained in Vehicle N containing 10% Azone (297 micrograms/h/cm2). Topical administration of the combination of 5% DFMO and 0.1% MGBG in this vehicle produced a greater inhibition of DNA synthesis and depletion of polyamine levels than either drug individually. The simultaneous topical administration of DFMO and MGBG, allowing the use of lower concentrations of the more toxic MGBG, may be useful for therapy of psoriasis and other cutaneous disorders associated with abnormalities in polyamine metabolism.

Experimental Chemotherapy in a Transplantable Renal Adenocarcinoma

Difluoromethylornithine (DFMO) and methylglyoxal bis (guanylhydrazone) (MGBG), inhibitors of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AMDC), respectively, were tested for antitumor activity in the BALB/C mouse renal adenocarcinoma model, wherein ODC and AMDC activity are elevated compared to the normal kidney. Additionally, an indirect effector of AMDC synthesis, arabinofuranosyladenine and an inhibitor of AMDC synthesis, cycloleucin (CL), were tested in this model. Simultaneous administration of both DFMO and MGBG affected the growth of renal adenocarcinoma less than did administration of DFMO or MGBG alone. Combinations of inhibitors of polyamine synthesis demonstrated a high toxicity with low therapeutic activity. The most effective tumor suppression was observed with CL alone but associated toxicity was severe. Suramin, an ODC stimulator, may have enhanced tumor growth.