DFMO inhibition of neuroblastoma tumorigenesis.

BACKGROUND: Most high-risk neuroblastoma patients who relapse succumb to disease despite the existing therapy. We recently reported increased event-free and overall survival in neuroblastoma patients receiving difluoromethylornithine (DFMO) during maintenance therapy. The effect of DFMO on cellular processes associated with neuroblastoma tumorigenesis needs further elucidation. Previous studies have shown cytotoxicity with IC50 values >5-15 mM, these doses are physiologically unattainable in patients, prompting further mechanistic studies at therapeutic doses. METHODS: We characterized the effect of DFMO on cell viability, cell cycle, apoptosis, neurosphere formation, and protein expression in vitro using five established neuroblastoma cell lines (BE2C, CHLA-90, SHSY5Y, SMS-KCNR, and NGP) at clinically relevant doses of 0, 50, 100, 500, 1000, and 2500 µM. Limiting Dilution studies of tumor formation in murine models were performed. Statistical analysis was done using GraphPad and the level of significance set at p = 0.05. RESULTS: There was not a significant loss of cell viability or gain of apoptotic activity in the in vitro assays (p > 0.05). DFMO treatment initiated G1 to S phase cell cycle arrest. There was a dose-dependent decrease in frequency and size of neurospheres and a dose-dependent increase in beta-galactosidase activity in all cell lines. Tumor formation was decreased in xenografts both with DFMO-pretreated cells and in mice treated with DFMO. CONCLUSION: DFMO treatment is cytostatic at physiologically relevant doses and inhibits tumor initiation and progression in mice. This study suggests that DFMO, inhibits neuroblastoma by targeting cellular processes integral to neuroblastoma tumorigenesis at clinically relevant doses.

Approval of DFMO for high-risk neuroblastoma patients demonstrates a step of success to target MYC pathway.

The "undruggable" MYC oncoproteins are deregulated in 70% human cancers. The approval of DFMO, an irreversible inhibitor of ornithine oxidase (ODC1) that is a direct transcriptional target of MYC, demonstrates that patients can benefit from targeting MYC activity via an indirect approach. However, the mechanism of action of DFMO needs further studies to understand how it works in post-immunotherapy neuroblastomas. Efforts to develop a more potent and safer drug to block MYC function will continue despite challenges.

Discordant Effects of Polyamine Depletion by DENSpm and DFMO on ß-cell Cytokine Stress and Diabetes Outcomes in Mice.

Type 1 diabetes (T1D) is an autoimmune disease leading to dysfunction and loss of insulin-secreting ß cells. In ß cells, polyamines have been implicated in causing cellular stress and dysfunction. An inhibitor of polyamine biosynthesis, difluoromethylornithine (DFMO), has been shown to delay T1D in mouse models and preserve ß-cell function in humans with recent-onset T1D. Another small molecule, N1,N11-diethylnorspermine (DENSpm), both inhibits polyamine biosynthesis and accelerates polyamine metabolism and is being tested for efficacy in cancer clinical trials. In this study, we show that DENSpm depletes intracellular polyamines as effectively as DFMO in mouse ß cells. RNA-sequencing analysis, however, suggests that the cellular responses to DENSpm and DFMO differ, with both showing effects on cellular proliferation but the latter showing additional effects on mRNA translation and protein-folding pathways. In the low-dose streptozotocin-induced mouse model of T1D, DENSpm, unlike DFMO, did not prevent or delay diabetes outcomes but did result in improvements in glucose tolerance and reductions in islet oxidative stress. In nonobese diabetic (NOD) mice, short-term DENSpm administration resulted in a slight reduction in insulitis and proinflammatory Th1 cells in the pancreatic lymph nodes. Longer term treatment resulted in a dose-dependent increase in mortality. Notwithstanding the efficacy of both DFMO and DENSpm in reducing potentially toxic polyamine levels in ß cells, our results highlight the discordant T1D outcomes that result from differing mechanisms of polyamine depletion and, more importantly, that toxic effects of DENSpm may limit its utility in T1D treatment.

Inhibition of polyamine biosynthesis preserves ß cell function in type 1 diabetes.

In preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing ß cell stress. However, the mechanism of DFMO action and its human tolerability remain unclear. In this study, we show that mice with ß cell ODC deletion are protected against toxin-induced diabetes, suggesting a cell-autonomous role of ODC during ß cell stress. In a randomized controlled trial (ClinicalTrials.gov: NCT02384889) involving 41 recent-onset T1D subjects (3:1 drug:placebo) over a 3-month treatment period with a 3-month follow-up, DFMO (125-1,000 mg/m2) is shown to meet its primary outcome of safety and tolerability. DFMO dose-dependently reduces urinary putrescine levels and, at higher doses, preserves C-peptide area under the curve without apparent immunomodulation. Transcriptomics and proteomics of DFMO-treated human islets exposed to cytokine stress reveal alterations in mRNA translation, nascent protein transport, and protein secretion. These findings suggest that DFMO may preserve ß cell function in T1D through islet cell-autonomous effects.

Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS) results from mutations in spermine synthase (SMS), which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonia, and seizures. Symptom management is the only treatment. Reduced SMS activity causes spermidine accumulation while spermine levels are reduced. The resulting exaggerated spermidine:spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity. Our studies aim to pharmacologically manipulate polyamine metabolism to correct this imbalance as a therapeutic strategy for SRS. Here we report the repurposing of 2-difluoromethylornithine (DFMO), an FDA-approved inhibitor of polyamine biosynthesis, in rebalancing spermidine:spermine ratios in SRS patient cells. Mechanistic in vitro studies demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of spermidine into spermine in hypomorphic SMS cells and induces uptake of exogenous spermine, altogether reducing the aberrant ratios. In a Drosophila SRS model characterized by reduced lifespan, DFMO improves longevity. As nearly all SRS patient mutations are hypomorphic, these studies form a strong foundation for translational studies with significant therapeutic potential.

Glioma Metabolic Feedback In Situ: A First-In-Human Pharmacodynamic Trial of Difluoromethylornithine + AMXT-1501 Through High-Molecular Weight Microdialysis.

BACKGROUND AND OBJECTIVES: No new drug has improved survival for glioblastoma since temozolomide in 2005, due in part to the relative inaccessibility of each patient's individualized tumor biology and its response to therapy. We have identified a conserved extracellular metabolic signature of enhancing high-grade gliomas enriched for guanidinoacetate (GAA). GAA is coproduced with ornithine, the precursor to protumorigenic polyamines through ornithine decarboxylase (ODC). AMXT-1501 is a polyamine transporter inhibitor that can overcome tumoral resistance to the ODC inhibitor, difluoromethylornithine (DFMO). We will use DFMO with or without AMXT-1501 to identify candidate pharmacodynamic biomarkers of polyamine depletion in patients with high-grade gliomas in situ . We aim to determine (1) how blocking polyamine production affects intratumoral extracellular guanidinoacetate abundance and (2) the impact of polyamine depletion on the global extracellular metabolome within live human gliomas in situ. METHODS: DFMO, with or without AMXT-1501, will be administered postoperatively in 15 patients after clinically indicated subtotal resection for high-grade glioma. High-molecular weight microdialysis catheters implanted into residual tumor and adjacent brain will be used for postoperative monitoring of extracellular GAA and polyamines throughout therapeutic intervention from postoperative day (POD) 1 to POD5. Catheters will be removed on POD5 before discharge. EXPECTED OUTCOMES: We anticipate that GAA will be elevated in tumor relative to adjacent brain although it will decrease within 24 hours of ODC inhibition with DFMO. If AMXT-1501 effectively increases the cytotoxic impact of ODC inhibition, we expect an increase in biomarkers of cytotoxicity including glutamate with DFMO + AMXT-1501 treatment when compared with DFMO alone. DISCUSSION: Limited mechanistic feedback from individual patients' gliomas hampers clinical translation of novel therapies. This pilot Phase 0 study will provide in situ feedback during DFMO + AMXT-1501 treatment to determine how high-grade gliomas respond to polyamine depletion.

Emerging compounds and therapeutic strategies to treat infections from Trypanosoma brucei: an overhaul of the last 5-years patents.

INTRODUCTION: Human African Trypanosomiasis is a neglected disease caused by infection from parasites belonging to the Trypanosoma brucei species. Only six drugs are currently available and employed depending on the stage of the infection: pentamidine, suramin, melarsoprol, eflornithine, nifurtimox, and fexinidazole. Joint research projects were launched in an attempt to find new therapeutic options for this severe and often lethal disease. AREAS COVERED: After a brief description of the recent literature on the parasite and the disease, we searched for patents dealing with the proposal of new antitrypanosomiasis agents and, following the PRISMA guidelines, we filtered the results to those published from 2018 onwards returning suitable entries, which represent the contemporary landscape of compounds/strategies against Trypanosoma brucei. In addition, some relevant publications from the overall scientific literature were also discussed. EXPERT OPINION: This review comprehensively covers and analyzes the most recent advances not only in the discovery of new inhibitors and their structure-activity relationships but also in the assessment of innovative biological targets opening new scenarios in the MedChem field. Finally, also new vaccines and formulations recently patented were described. However, natural and synthetic compounds were analyzed in terms of inhibitory activity and selective toxicity against human cells.

Improvement of dermatological symptoms in patients with Bachmann-Bupp syndrome using difluoromethylornithine treatment.

Bachmann-Bupp syndrome (OMIM #619075) is a novel autosomal dominant disorder caused by variants in the c-terminus of the ornithine decarboxylase 1 gene, resulting in increased levels of ornithine decarboxylase. This case report includes two patients diagnosed with Bachmann-Bupp syndrome who were treated with difluoromethylornithine through compassionate use approval from the United States Food and Drug Administration. In both patients, treatment with difluoromethylornithine has resulted in improved dermatologic signs, including regrowth of eyebrow and scalp hair and cessation of recurrent follicular cyst development.

Ototoxicity of Long-Term α-Difluoromethylornithine for Skin Cancer Prevention.

OBJECTIVE: Evaluate the effects of α-difluoromethylornithine (DFMO) on hearing thresholds as part of a randomized, double-blind, placebo-controlled trial. METHODS: Subjects were randomized and assigned to the control (placebo) or experimental (DFMO) group. DFMO or placebo were administered orally (500 mg/m2 /day) for up to 5 years. RESULTS: Subjects taking DFMO had, on average, increased hearing thresholds from baseline across the frequency range compared to subjects in the control group. Statistical analysis revealed this was significant in the lower frequency range. CONCLUSIONS: This randomized controlled trial revealed the presence of increased hearing thresholds associated with long-term DFMO use. As a whole, DFMO may help prevent and treat certain types of cancers; however, it can result in some degree of hearing loss even when administered at low doses. This study further highlights the importance of closely monitoring hearing thresholds in subjects taking DFMO. Laryngoscope, 133:676-682, 2023.

Difluoromethylornithine (DFMO) Enhances the Cytotoxicity of PARP Inhibition in Ovarian Cancer Cells.

Ovarian cancer accounts for 3% of the total cancers in women, yet it is the fifth leading cause of cancer deaths among women. The BRCA1/2 germline and somatic mutations confer a deficiency of the homologous recombination (HR) repair pathway. Inhibitors of poly (ADP-ribose) polymerase (PARP), another important component of DNA damage repair, are somewhat effective in BRCA1/2 mutant tumors. However, ovarian cancers often reacquire functional BRCA and develop resistance to PARP inhibitors. Polyamines have been reported to facilitate the DNA damage repair functions of PARP. Given the elevated levels of polyamines in tumors, we hypothesized that treatment with the polyamine synthesis inhibitor, α-difluoromethylornithine (DFMO), may enhance ovarian tumor sensitivity to the PARP inhibitor, rucaparib. In HR-competent ovarian cancer cell lines with varying sensitivities to rucaparib, we show that co-treatment with DFMO increases the sensitivity of ovarian cancer cells to rucaparib. Immunofluorescence assays demonstrated that, in the presence of hydrogen peroxide-induced DNA damage, DFMO strongly inhibits PARylation, increases DNA damage accumulation, and reduces cell viability in both HR-competent and deficient cell lines. In vitro viability assays show that DFMO and rucaparib cotreatment significantly enhances the cytotoxicity of the chemotherapeutic agent, cisplatin. These results suggest that DFMO may be a useful adjunct chemotherapeutic to improve the anti-tumor efficacy of PARP inhibitors in treating ovarian cancer.

Polyamine Depletion Strategies in Cancer: Remodeling the Tumor Immune Microenvironment to Enhance Anti-Tumor Responses.

Polyamine biosynthesis is frequently dysregulated in cancers, and enhanced flux increases intracellular polyamines necessary for promoting cell growth, proliferation, and function. Polyamine depletion strategies demonstrate efficacy in reducing tumor growth and increasing survival in animal models of cancer; however, mechanistically, the cell-intrinsic and cell-extrinsic alterations within the tumor microenvironment underlying positive treatment outcomes are not well understood. Recently, investigators have demonstrated that co-targeting polyamine biosynthesis and transport alters the immune landscape. Although the polyamine synthesis-targeting drug 2-difluoromethylornithine (DFMO) is well tolerated in humans and is FDA-approved for African trypanosomiasis, its clinical benefit in treating established cancers has not yet been fully realized; however, combination therapies targeting compensatory mechanisms have shown tolerability and efficacy in animal models and are currently being tested in clinical trials. As demonstrated in pre-clinical models, polyamine blocking therapy (PBT) reduces immunosuppression in the tumor microenvironment and enhances the therapeutic efficacy of immune checkpoint blockade (ICB). Thus, DFMO may sensitize tumors to other therapeutics, including immunotherapies and chemotherapies.

Population Pharmacodynamic Modeling of Eflornithine-Based Treatments Against Late-Stage Gambiense Human African Trypanosomiasis and Efficacy Predictions of L-eflornithine-Based Therapy.

Eflornithine is a recommended treatment against late-stage gambiense human African trypanosomiasis, a neglected tropical disease. Standard dosing of eflornithine consists of repeated intravenous infusions of a racemic mixture of L- and D-eflornithine. Data from three clinical studies, (i) eflornithine intravenous monotherapy, (ii) nifurtimox-eflornithine combination therapy, and (iii) eflornithine oral monotherapy, were pooled and analyzed using a time-to-event pharmacodynamic modeling approach, supported by in vitro activity data of the individual enantiomers. Our aim was to assess (i) the efficacy of the eflornithine regimens in a time-to-event analysis and (ii) the feasibility of an L-eflornithine-based therapy integrating clinical and preclinical data. A pharmacodynamic time-to-event model was used to estimate the total dose of eflornithine, associated with 50% reduction in baseline hazard, when administered as monotherapy or in the nifurtimox-eflornithine combination therapy. The estimated total doses were 159, 60 and 291 g for intravenous eflornithine monotherapy, nifurtimox-eflornithine combination therapy and oral eflornithine monotherapy, respectively. Simulations suggested that L-eflornithine achieves a higher predicted median survival, compared to when racemate is administered, as treatment against late-stage gambiense human African trypanosomiasis. Our findings showed that oral L-eflornithine-based monotherapy would not result in adequate efficacy, even at high dose, and warrants further investigations to assess the potential of oral L-eflornithine-based treatment in combination with other treatments such as nifurtimox. An all-oral eflornithine-based regimen would provide easier access to treatment and reduce burden on patients and healthcare systems in gambiense human African trypanosomiasis endemic areas. Graphical abstract.

Polycystic ovarian syndrome-current pharmacotherapy and clinical implications.

Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women is characterized by polycystic ovaries, chronic anovulation and hyperandrogenism. The treatment in PCOS is mainly symptomatic and involves lifestyle interventions and medications such as Metformin, Oral contraceptives and Antiandrogens. However, the management of PCOS is challenging and current interventions are not able to deal with outcomes of this syndrome. This review encompasses latest pharmacotherapeutic and non-pharmacotherapeutic interventions currently in use to tackle various symptomatic contentions in PCOS. Our focus has been mainly on novel therapeutic modalities for treatment/management of PCOS, like use of newer insulin sensitizers viz., Inositols, Glucagon-like peptide-1(GLP-1) agonists, Dipeptidyl pepdidase-4 (DPP-4) inhibitors, and sodium-glucose transport protein 2 (SGLT2) inhibitors. Also, evidence suggesting the use of vitamin D, statins, and Letrozole as emerging therapies in PCOS have been summarized in this review. Additionally, novel cosmetic techniques like electrolysis, laser and use of topically applied eflornithine to tackle the most distressing feature of facial hirsutism associated with PCOS, non-pharmacological therapy like acupuncture and the role of herbal medicine in PCOS management have also been discussed.

Expanding the phenotype: Four new cases and hope for treatment in Bachmann-Bupp syndrome.

Bachmann-Bupp syndrome (BABS) is a rare syndrome caused by gain-of-function variants in the C-terminus of ornithine decarboxylase (ODC coded by the ODC1 gene). BABS is characterized by developmental delay, macrocephaly, macrosomia, and an unusual pattern of non-congenital alopecia. Recent diagnosis of four more BABS patients provides further characterization of the phenotype of this syndrome including late-onset seizures in the oldest reported patient at 23 years of age, representing the first report for this phenotype in BABS. Neuroimaging abnormalities continue to be an inconsistent feature of the syndrome. This may be related to the yet unknown impact of ODC/polyamine dysregulation on the developing brain in this syndrome. Variants continue to cluster, providing support to a universal biochemical mechanism related to elevated ODC protein, enzyme activity, and abnormalities in polyamine levels. Recommendations for medical management can now be suggested as well as the potential for targeted molecular or metabolic testing when encountering this unique phenotype. The natural history of this syndrome will evolve with difluoromethylornithine (DFMO) therapy and raise new questions for further study and understanding.

Repurposing eflornithine to treat a patient with a rare ODC1 gain-of-function variant disease.

Background: Polyamine levels are intricately controlled by biosynthetic, catabolic enzymes and antizymes. The complexity suggests that minute alterations in levels lead to profound abnormalities. We described the therapeutic course for a rare syndrome diagnosed by whole exome sequencing caused by gain-of-function variants in the C-terminus of ornithine decarboxylase (ODC), characterized by neurological deficits and alopecia. Methods: N-acetylputrescine levels with other metabolites were measured using ultra-performance liquid chromatography paired with mass spectrometry and Z-scores established against a reference cohort of 866 children. Results: From previous studies and metabolic profiles, eflornithine was identified as potentially beneficial with therapy initiated on FDA approval. Eflornithine normalized polyamine levels without disrupting other pathways. She demonstrated remarkable improvement in both neurological symptoms and cortical architecture. She gained fine motor skills with the capacity to feed herself and sit with support. Conclusions: This work highlights the strategy of repurposing drugs to treat a rare disease. Funding: No external funding was received for this work.

Inhibition of Polyamine Biosynthesis Using Difluoromethylornithine Acts as a Potent Immune Modulator and Displays Therapeutic Synergy With PD-1-blockade.

Polyamines are known to play a significant role in cancer progression and treatment using difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, has shown some clinical promise. It is interesting to note that, while DFMO is directly cytostatic in vitro, recent work has suggested that it achieves its antitumor efficacy in vivo by enhancing adaptive antitumor immune responses. On the basis of these data, we hypothesized that DFMO might act as an immune sensitizer to increase tumor responsiveness to checkpoint blockade. To test this hypothesis, we treated tumors with DFMO, in either the presence or absence of additional PD-1 blockade, and subsequently analyzed their immunological and therapeutic responses. Our data demonstrates that treatment with DFMO significantly enhances both the viability and activation status of intratumoral CD8+ T cells, most likely through an indirect mechanism. When combined with PD-1 blockade, this increased viability resulted in unique proinflammatory cytokine profiles and transcriptomes within the tumor microenvironment and improved therapeutic outcomes. Taken together, these data suggest that DFMO might represent a potential immunomodulatory agent that can enhance current PD-1-based checkpoint therapies.

Capryloyl glycine and soy isoflavonoids in hypertrichosis: An experimental and placebo-controlled clinical study.

BACKGROUND: The management of acquired hypertrichosis (HT) is based on the search of the causes and subsequent specific treatment. However, simultaneous hair removal is important. No single method for hair removal is appropriate for all patients and skin areas. Treatment options are actually limited and clinical results are often unsatisfactory. Ornithine decarboxylase 1 (ODC1), an enzyme present in hair follicles, is considered as a potential target to inhibit hair growth. Only eflornithine hydrochloride, an inhibitor of ODC1, showed to be partially effective in the management of acquired HT. AIMS: The aim of our study was to evaluate the potential inhibition of ODC1 activity by a cream containing 4% capryloyl glycine, an ODC1 inhibitor, and 1% glycine soy-fermented extract (soy isoflavonoids). Furthermore, we present the results of a placebo-controlled clinical study that evaluated the efficacy and tolerability of this cream. METHODS: The ODC1 activity was detected by measuring absorbance at 340 nm. In the presence of ODC1 inhibitors, absorbance decreases as a function of inhibition. Difluoromethylornithine (DFMO) was provided as an inhibitor control. ODC1 activity inhibition was expressed as percentage of control (untreated sample). All data were presented as mean ± standard deviation of three independent experiments. To determine if statistically significant differences between treatments were present, a t test analysis was performed. The differences between groups were considered significant at p < 0.05. Twelve Caucasian female adult patients, with HT located on the forearms, were enrolled. The study cream (product A) was applied twice/day for four months on the right forearm. A placebo cream (product B) was applied twice/day for four months on the left forearm. Clinical efficacy was evaluated by means of macrophotography. RESULTS: The cream significantly inhibited ODC1 activity (35.1 ± 0.5% inhibition, equivalent to a 64.9 ± 0.5% ODC1 activity). DFMO completely abolished the enzymatic activity (100 ± 5% inhibition, equivalent to 0 ± 5% ODC1 activity). All patients were considered evaluable. In 11 out of 12 patients (91.7%), who were treated with product A, an improvement was observed. No improvement was observed in patients treated with product B. The global assessment showed good efficacy in 7 patients (58.3%) and moderate efficacy in 5 patients (41.7%) treated with the product A. No efficacy was detected in patients treated with product B. CONCLUSIONS: The study cream showed to be effective in Caucasian, adult, female patients with hypertrichosis located on the forearms.