D,L-alpha-difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC) that is being investigated to treat cancers such as pediatric neuroblastoma. A novel and sensitive LC-MS/MS assay was developed and validated to quantify DFMO concentrations in support of pre-clinical pharmacokinetic studies in mice. The study was performed using a Shimadzu triple quad LC-MS/MS equipped with an Atlantis HILIC Silica 3 µm 2.1 × 100 mm column, and an isocratic mobile phase (75:25 acetonitrile and 0.2 % formic acid) at a flow rate of 0.5 mL/min. Multiple Reaction Monitoring (MRM) was used to identify the precursor ion (183 m/z) with quantification of daughter ions at transitions of 183 > 120.10, 183 > 166.10, and 183 > 80.05. Plasma standards and quality control samples (20 µL) were processed using protein precipitation with cold acetonitrile. The lower limit of detection (LLOQ) was 5 ng/mL. Assay performance was determined from multiple runs (n = 10) with standards ranging from 250-50,000 ng/mL and three levels of quality control (500, 4000, and 40,000 ng/mL). Standard curves were linear with r2 values between 0.9960 and 0.9999. Quality control samples were stable and exhibited maximum inter-day % bias of ≤3 % and CV% of ≤0.7 %. The assay was successfully applied to an in vivo study to determine the pharmacokinetics of DFMO in athymic nu/nu mice.

Polyaminopathies are a relatively new family of rare genetic syndromes recently described in the literature. These syndromes are involved in the biosynthesis of polyamines, which include putrescine, spermidine, and spermine. Polyamines are aliphatic molecular that are found in most life forms, including humans, and are essential for embryogenesis, organogenesis, and tumorigenesis. The five known polyaminopathies that have been described to date include Snyder-Robinson Syndrome (SRS), Bachmann-Bupp Syndrome (BABS), Faundes-Banka Syndrome (FABAS), as well as neurodevelopmental disorders associated with variants in DHPS and DOHH. These syndromes share many overlapping clinical phenotypes, including developmental delay, hypotonia, and intellectual disability. Here we describe details for identifying and obtaining high-quality biological samples from patients with polyaminopathies. This includes special considerations for the informed consent process and the collection and shipment of biological samples for patients with rare diseases, many of whom live in countries around the world. We also detail the technical protocols for the collection, processing, storage, and tracking of biological samples for downstream research analysis specific to research in polyaminopathies, as well as biobanking for future use.

Sulfasalazine is a sepiapterin reductase and System x − c inhibitor with anti-neuroblastoma activity. The objective was to develop cellular redox glutathione stimuli-responsive Sulfasalazine-loaded albumin-chitosan hybrid nanocarriers with a target profile of < 100 nm, polydispersity index (PDI) of < 0.3, charge of 3 to 10 mV, and drug load of > 8

We here describe the design, synthesis, and biological activity of novel ornithine decarboxylase (ODC) inhibitors that show significantly higher potency in vitro than α-difluoromethylornithine (DFMO), a U.S. Food and Drug Administration (FDA) approved drug. We report two X-ray structures of ODC complexed with new ODC inhibitors, computational docking, molecular dynamics, and binding free energy calculations to validate the experimental models. The X-ray structures reveal that covalent adducts with pyridoxal phosphate (PLP) are formed in the active site of the human ODC enzyme, as verified by their preparation and enzymatic testing. Finally, we verified that the cellular activity of endogenous ODC was inhibited, and polyamine levels were reduced. Given that ODC is a clinically validated target, combined with the fact that DFMO is currently the only ODC inhibitor in clinical use for several indications, the further development of more potent ODC inhibitors with superior activity and physical properties is warranted.

Polyamines are aliphatic molecules that include putrescine, spermidine, and spermine. Polyamines are present in most living organisms including humans. These positively charged molecules play important roles in cell physiology and pathology by contributing to embryonic cell development, regulation of cell division and, if overproduced, the stimulation of cancer cell proliferation and tumorigenesis. We recently discovered Bachmann-Bupp Syndrome (BABS); a rare neurodevelopmental disorder linked to de novo mutations in the ornithine decarboxylase 1 (ODC1) gene. ODC1 gene mutations that are linked to BABS always produce C-terminally truncated versions of the enzyme ornithine decarboxylase (ODC). These shortened ODC proteins remain enzymatically active and are not cleared by the proteasome, therefore leading to ODC protein accumulation in cells. ODC is a key enzyme of polyamine biosynthesis by converting ornithine to putrescine, and if accumulated, can lead to high putrescine levels in human cells including red blood cells (RBCs) and primary dermal fibroblasts. Here we describe how to quantitatively measure ODC enzymatic activity and the polyamines by a radiolabeled 14C-ornithine assay and by reverse phase (RP)-HPLC, respectively. While these methods have been developed decades ago, many publications provide incomplete protocols with omission of experimental details, which inadvertently can lead to mistakes, inconclusive results, and failed experiments. There is a growing number of laboratories that have become interested in exploring polyamines (in part due to metabolomics analyses in human health-related studies). The detailed protocols of this chapter provide step-by-step guidance detailing how to measure ODC activity and polyamines in human RBCs.

Rare diseases impact approximately 1 in 10 people worldwide, and yet, less than 5% of all rare diseases currently have an approved treatment option available. This is due to many challenges unique to rare diseases, including small, diverse patient populations, the cost of drug development that is not proportionate to the number of patients who could potentially benefit from treatment, and difficulty with clinical trial design to validate new therapeutics. As a result, drug repurposing has become an increasingly promising option for finding treatment options for rare diseases. First described in 2018, Bachmann-Bupp Syndrome (BABS) is a rare neurodevelopmental disorder that is caused by gain-of-function variants in the ornithine decarboxylase (ODC1) gene and is characterized by developmental delay, hypotonia, and alopecia. Through collaboration and the use of a unique drug repurposing strategy, the first patient identified with BABS was treated with the repurposed drug eflornithine, also known as α-difluoromethylornithine (DFMO), in just 16 months. Currently, five additional patients with BABS are being treated with DFMO. This model of drug repurposing of an FDA-approved drug for use in another indication can serve as an example of what is possible in the scope of other rare diseases, specifically in other polyaminopathies.

BACKGROUND/AIM:Allicin is a small-molecule natural product found in garlic (Allium sativum). We previously showed that allicin inhibits ornithine decarboxylase (ODC) in vitro and induces apoptotic cell death in pediatric neuroblastoma (NB) cancer cell cultures. However, its potency as an anticancer agent in vivo has not been sufficiently explored. MATERIALS AND METHODS:In this study, we used cell proliferation assays, immunoblotting techniques, and light microscopy to study NB tumor cell cultures and human primary neonatal skin fibroblast control cells as well as a MYCN-amplified NB patient-derived xenograft (PDX) mouse tumor model to study the efficacy of allicin in vivo. RESULTS:Allicin strongly inhibits NB tumor cell proliferation in a dose-dependent manner while non-cancerous human primary neonatal skin fibroblast control cells were largely unaffected. Importantly, two intra-tumoral injections of allicin over a two-week trial period significantly reduced the NB tumor burden in mice compared to controls (N=4-9 mice/group). Excised tumor tissues revealed that allicin treatment increased the cyclin-dependent kinase inhibitor p27Kip1 protein levels, suggesting that in vivo, allicin increases p27Kip1-mediated G1/S cell cycle arrest. CONCLUSION:Our findings warrant further preclinical development of allicin as a potential anticancer agent, especially for those types of cancers that are treatable by intra-tumoral injections, including neuroblastoma, glioblastoma, and medulloblastoma.

ABSTRACT Polyaminopathies are a recently described family of rare genetic neurodevelopmental disorders. Polyaminopathies disrupt the biosynthesis of the primary polyamines: putrescine, spermidine, and spermine. Snyder–Robinson syndrome results from hemizygous loss‐of‐function variants in the spermine synthase ( SMS ) gene, resulting in decreased or complete loss of spermine synthase enzyme activity. Bachmann–Bupp syndrome results from heterozygous gain‐of‐function variants in the ornithine decarboxylase 1 ( ODC1 ) gene, resulting in increased ornithine decarboxylase enzyme activity. Faundes–Banka syndrome results from heterozygous loss‐of‐function variants in the eukaryotic translation initiation factor 5A ( EIF5A ) gene, impairing eIF5A protein function. DHPS (deoxyhypusine synthase) deficiency is an autosomal recessive disease and results from bi‐allelic hypomorphic variants in the deoxyhypusine synthase ( DHPS ) gene, which results in reduced deoxyhypusine synthase enzyme activity. Finally, DOHH (deoxyhypusine hydroxylase) disorder is an autosomal recessive disorder caused by bi‐allelic loss‐of‐function variants in the deoxyhypusine hydroxylase ( DOHH ) gene, which causes decreased deoxyhypusine hydroxylase enzyme activity. Snyder–Robinson syndrome was first described in 1969, while the other four syndromes have only been identified in the past 7 years. A comprehensive phenotypic and genotypic description of these five syndromes is needed. We review the clinical and genetic features of these five polyaminopathies to create an inclusive clinical resource. A systematic keyword search strategy was used to identify all published cases in PubMed, Web of Science, and Scopus databases. The five known syndromes associated with the polyamine pathway share many similar clinical phenotypes, and yet patients with each syndrome present with distinctive syndromic features. This review will serve as a valuable resource for clinicians diagnosing and caring for patients with these rare polyaminopathies.

Supplementary Figure 1 from Inhibition of S-adenosylmethionine decarboxylase by inhibitor SAM486A connects polyamine metabolism with p53-Mdm2-Akt/protein kinase B regulation and apoptosis in neuroblastoma

Supplementary Figure 2 from Inhibition of <i>S</i>-adenosylmethionine decarboxylase by inhibitor SAM486A connects polyamine metabolism with p53-Mdm2-Akt/protein kinase B regulation and apoptosis in neuroblastoma

Supplementary Tables S1-S2 from Expression of Prenylated Rab Acceptor 1 Domain Family, Member 2 (PRAF2) in Neuroblastoma: Correlation with Clinical Features, Cellular Localization, and Cerulenin-Mediated Apoptosis Regulation

Supplementary Figure 1 from Inhibition of <i>S</i>-adenosylmethionine decarboxylase by inhibitor SAM486A connects polyamine metabolism with p53-Mdm2-Akt/protein kinase B regulation and apoptosis in neuroblastoma

Supplementary Table 1 from Inhibition of <i>S</i>-adenosylmethionine decarboxylase by inhibitor SAM486A connects polyamine metabolism with p53-Mdm2-Akt/protein kinase B regulation and apoptosis in neuroblastoma

Supplementary Figure 1 from Inhibition of <i>S</i>-adenosylmethionine decarboxylase by inhibitor SAM486A connects polyamine metabolism with p53-Mdm2-Akt/protein kinase B regulation and apoptosis in neuroblastoma

Supplementary Figure 2 from Inhibition of <i>S</i>-adenosylmethionine decarboxylase by inhibitor SAM486A connects polyamine metabolism with p53-Mdm2-Akt/protein kinase B regulation and apoptosis in neuroblastoma

ABSTRACT The eukaryotic translation initiation factor 5A1 (eIF5A1) and 5A2 (eIF5A2) are important proteins in a variety of physiological and pathophysiological processes and their function has been linked to neurodevelopmental disorders, cancer, and viral infections. Here, we report two new genome-edited mouse models, generated using a CRISPR-Cas9 approach, in which the amino acid residue lysine 50 is replaced with arginine 50 (K50R) in eIF5A1 or in the closely related eIF5A2 protein. This mutation prevents the spermidine-dependent post-translational formation of hypusine, a unique lysine derivative that is necessary for activation of eIF5A1 and eIF5A2. Mouse brain lysates from homozygous eif5a2-K50R mutant mice (eif5a2K50R/K50R) confirmed the absence of hypusine formation of eIF5A2, and metabolomic analysis of primary mouse dermal fibroblasts revealed significant alterations in the metabolite landscape compared to controls including increased levels of tryptophan, kyrunenine, pyridoxine, nicotinamide adenine dinucleotide, riboflavin, flavin adenine dinucleotide, pantothenate, and coenzyme A. Further supported by new publicly available bioinformatics data, these new mouse models represent excellent in vivo models to study hypusine-dependent biological processes, hypusination-related disorders caused by eIF5A1 and eIF5A2 gene aberrations or mRNA expression dysregulation, as well as several major human cancer types and potential therapies.

Bachmann-Bupp syndrome (BABS) is a neurodevelopmental disorder characterized by developmental delay, hypotonia, and varying forms of non-congenital alopecia. The condition is caused by 3'-end mutations of the ornithine decarboxylase 1 (ODC1) gene, which produce carboxy (C)-terminally truncated variants of ODC, a pyridoxal 5'-phosphate-dependent enzyme. C-terminal truncation of ODC prevents its ubiquitin-independent proteasomal degradation and leads to cellular accumulation of ODC enzyme that remains catalytically active. ODC is the first rate-limiting enzyme that converts ornithine to putrescine in the polyamine pathway. Polyamines (putrescine, spermidine, spermine) are aliphatic molecules found in all forms of life and are important during embryogenesis, organogenesis, and tumorigenesis. BABS is an ultra-rare condition with few reported cases, but it serves as a convincing example for drug repurposing therapy. α-Difluoromethylornithine (DFMO, also known as eflornithine) is an ODC inhibitor with a strong safety profile in pediatric use for neuroblastoma and other cancers as well as West African sleeping sickness (trypanosomiasis). Patients with BABS have been treated with DFMO and have shown improvement in hair growth, muscle tone, and development.

Recent identification of four additional polyaminopathies, including Bachmann–Bupp syndrome, have benefited from previous research on Snyder–Robinson syndrome in order to advance from research to treatment more quickly. As a result of the discovery of these conditions, the potential for treatment within this pathway, and for other possible unidentified polyaminopathies, the International Center for Polyamine Disorders (ICPD) was created to help promote understanding of these conditions, research opportunities, and appropriate care for families. This case study provides insights from two new patients diagnosed with Bachmann–Bupp syndrome, further expanding our understanding of this ultra-rare condition, as well as a general discussion about other known polyaminopathies. This work also presents considerations for collaborative research efforts across these conditions, along with others that are likely to be identified in time, and outlines the role that the ICPD hopes to fill as more patients with these polyaminopathies continue to be identified and diagnosed.