D-2-Hydroxyglutarate in Glioma Biology.

Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in glioma, which result in the accumulation of an "oncometabolite", D-2-hydroxyglutarate (D-2-HG). Abnormally elevated D-2-HG levels result in a distinctive pattern in cancer biology, through competitively inhibiting α-ketoglutarate (α-KG)/Fe(II)-dependent dioxgenases (α-KGDDs). Recent studies have revealed that D-2-HG affects DNA/histone methylation, hypoxia signaling, DNA repair, and redox homeostasis, which impacts the oncogenesis of IDH-mutated cancers. In this review, we will discuss the current understanding of D-2-HG in cancer biology, as well as the emerging opportunities in therapeutics in IDH-mutated glioma.

Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies.

The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention.

Mutant IDH is sufficient to initiate enchondromatosis in mice.

Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

Preclinical evaluation of BAY 1075553, a novel (18)F-labelled inhibitor of prostate-specific membrane antigen for PET imaging of prostate cancer.

PURPOSE: Prostate-specific membrane antigen (PSMA) is a transmembrane protein overexpressed in prostate cancer and is therefore being explored as a biomarker for diagnosing and staging of the disease. Here we report preclinical data on BAY 1075553 (a 9:1 mixture of (2S,4S)- and (2R,4S)-2-[(18)F]fluoro-4-phosphonomethyl-pentanedioic acid), a novel (18)F-labelled small molecule inhibitor of PSMA enzymatic activity, which can be efficiently synthesized from a direct radiolabelling precursor. METHODS: The (18)F-radiolabelled stereoisomers of 2-[(18)F]fluoro-4-(phosphonomethyl)-pentanedioic acid were synthesized from their respective isomerically pure precursors dimethyl 2-{[bis(benzyloxy)phosphoryl]methyl}-4-(tosyloxy)pentanedioate. In vivo positron emission tomography (PET) imaging and biodistribution studies were conducted in mice bearing LNCaP, 22Rv1 and PC-3 tumours. Pharmacokinetic parameters and dosimetry estimates were calculated based on biodistribution studies in rodents. For non-clinical safety assessment (safety pharmacology, toxicology) to support a single-dose human microdose study, off-target effects in vitro, effects on vital organ functions (cardiovascular in dogs, nervous system in rats), mutagenicity screens and an extended single-dose study in rats were conducted with the non-radioactive racemic analogue of BAY 1075553. RESULTS: BAY 1075553 showed high tumour accumulation specific to PSMA-positive tumour-bearing mice and was superior to other stereoisomers tested. Fast clearance of BAY 1075553 resulted overall in low background signals in other organs except for high uptake into kidney and bladder which was mainly caused by renal elimination of BAY 1075553. A modest uptake into bone was observed which decreased over time indicating organ-specific uptake as opposed to defluorination of BAY 1075553 in vivo. Biodistribution studies found highest organ doses for kidneys and the urinary bladder wall resulting in a projected effective dose (ED) in humans of 0.0219 mSv/MBq. Non-clinical safety studies did not show off-target activity, effects on vital organs function or dose-dependent adverse effects. CONCLUSION: BAY 1075553 was identified as a promising PET tracer for PSMA-positive prostate tumours in preclinical studies. BAY 1075553 can be produced using a robust, direct radiosynthesis procedure. Pharmacokinetic, toxicology and safety pharmacology studies support the application of BAY 1075553 in a first-in-man microdose study with single i.v. administration.

N-acetylcysteine prevents spatial memory impairment induced by chronic early postnatal glutaric acid and lipopolysaccharide in rat pups.

BACKGROUND AND AIMS: Glutaric aciduria type I (GA-I) is characterized by accumulation of glutaric acid (GA) and neurological symptoms, such as cognitive impairment. Although this disease is related to oxidative stress and inflammation, it is not known whether these processes facilitate the memory impairment. Our objective was to investigate the performance of rat pups chronically injected with GA and lipopolysaccharide (LPS) in spatial memory test, antioxidant defenses, cytokines levels, Na+, K+-ATPase activity, and hippocampal volume. We also evaluated the effect of N-acetylcysteine (NAC) on theses markers. METHODS: Rat pups were injected with GA (5 umol g of body weight-1, subcutaneously; twice per day; from 5th to 28th day of life), and were supplemented with NAC (150 mg/kg/day; intragastric gavage; for the same period). LPS (2 mg/kg; E.coli 055 B5) or vehicle (saline 0.9%) was injected intraperitoneally, once per day, from 25th to 28th day of life. Oxidative stress and inflammatory biomarkers as well as hippocampal volume were assessed. RESULTS: GA caused spatial learning deficit in the Barnes maze and LPS potentiated this effect. GA and LPS increased TNF-α and IL-1ß levels. The co-administration of these compounds potentiated the increase of IL-1ß levels but not TNF-α levels in the hippocampus. GA and LPS increased TBARS (thiobarbituric acid-reactive substance) content, reduced antioxidant defenses and inhibited Na+, K+-ATPase activity. GA and LPS co-administration did not have additive effect on oxidative stress markers and Na+, K+ pump. The hippocampal volume did not change after GA or LPS administration. NAC protected against impairment of spatial learning and increase of cytokines levels. NAC Also protected against inhibition of Na+,K+-ATPase activity and oxidative markers. CONCLUSIONS: These results suggest that inflammatory and oxidative markers may underlie at least in part of the neuropathology of GA-I in this model. Thus, NAC could represent a possible adjuvant therapy in treatment of children with GA-I.

Phase I, pharmacokinetic and biological correlative study of OSI-7904L, a novel liposomal thymidylate synthase inhibitor, and cisplatin in patients with solid tumors.

PURPOSE: To evaluate the safety and describe the pharmacokinetic profile of OSI-7904L, a novel liposomal thymidylate synthase inhibitor, in combination with cisplatin (CDDP) in adults with advanced solid tumors. EXPERIMENTAL DESIGN: CDDP was administered as a 2-h intravenous infusion followed by OSI-7904L intravenously over 30 min, both given every 3 weeks. Doses of each drug were escalated in separate cohorts of patients. Five dose levels of CDDP/OSI-7904L were explored: 60/6, 60/9, 60/12, 60/7.5, and 75/7.5 mg/m2. Pharmacokinetic samples, baseline plasma homocysteine, and genotype polymorphisms were evaluated. RESULTS: Twenty-seven patients were treated with 101 total courses of CDDP/OSI-7904L. Dose-limiting toxicity was observed in 2 patients in the CDDP/OSI-7904L 60/12 mg/m2 cohort. One patient experienced rash, stomatitis, dehydration, renal failure, hyperbilirubinemia, and fatal neutropenic sepsis, whereas the other patient experienced grade 3 nausea, vomiting, and ileus. Therefore, the CDDP/OSI-7904L 60/9 mg/m2 cohort was expanded, with 2 of 6 patients reporting significant fatigue. Other toxicities were mild or moderate. Intermediate dose levels of 60/7.5 and 75/7.5 mg/m2 were evaluated, and the latter was identified as the recommended dose for phase II studies. No major pharmacokinetic interactions between CDDP and OSI-7904L were observed. Three patients had partial responses (gastric adenocarcinoma and heavily pretreated breast cancer). There was no significant relationship between baseline homocysteine and toxicity. CONCLUSIONS: The recommended doses for CDDP and OSI-7904L administered once every 3 weeks are 75 and 7.5 mg/m2, respectively. Pharmacokinetic interaction between the agents was not apparent. Preliminary clinical activity was observed in breast and gastric cancer.

Multicentre phase II pharmacokinetic and pharmacodynamic study of OSI-7904L in previously untreated patients with advanced gastric or gastroesophageal junction adenocarcinoma.

A two-stage Simon design was used to evaluate the response rate of OSI-7904L, a liposome encapsulated thymidylate synthase inhibitor, in advanced gastric and/or gastroesophageal adenocarcinoma (A-G/GEJA), administered intravenously at 12 mg m(-2) over 30 min every 21 days. Fifty patients were treated. Median age was 64 years (range 35-82), 62% were male and 89% had ECOG PS of 0/1. A total of 252 cycles were administered; median of 4 per patient (range 1-21). Twelve patients required dose reductions, mainly for skin toxicity. Investigator assessed response rate was 17.4% (95% CI 7.8-31.4) with one complete and seven partial responses in 46 evaluable patients. Twenty-one patients (42%) had stable disease. Median time to progression and survival were 12.4 and 36.9 weeks, respectively. NCI CTCAE Grade 3/4 neutropenia (14%) and thrombocytopenia (4%) were uncommon. The main G3/4 nonhaematological toxicities were skin-related 22%, stomatitis 14%, fatigue/lethargy 10%, and diarrhea 8%. Pharmacokinetic data showed high interpatient variability. Patients with higher AUC were more likely to experience G3/4 toxicity during cycle 1 while baseline homocysteine did not predict toxicity. Response did not correlate with AUC. Elevations in 2'-dU were observed indicating target inhibition. Analysis of TS genotype, TS protein and expression did not reveal any correlation with outcome. OSI-7904L has activity in A-G/GEJA similar to other active agents and an acceptable safety profile.

The central nervous system effects, pharmacokinetics and safety of the NAALADase-inhibitor GPI 5693.

AIM: The aim was to assess the central nervous system (CNS) effects, pharmacokinetics and safety of GPI 5693, an inhibitor of a novel CNS-drug target, NAALADase which is being evaluated for the treatment of neuropathic pain. METHODS: This was a double-blind, placebo-controlled, exploratory study in healthy subjects receiving oral GPI 5693 single ascending doses of 100, 300, 750, 1125 mg with a placebo treatment randomly interspersed. An open-label, parallel extension examined the effects of food and sex on the pharmacokinetics of 750, 1125 and 1500 mg doses. Blood samples were collected for pharmacokinetic and biochemical/haematological safety analysis, vital signs, ECG and adverse event checks were performed regularly up to 48 h postdose. Postdose CNS effects were assessed using eye movements, adaptive tracking, electroencephalography (EEG), body sway and Visual Analogue Scales (VAS). RESULTS: CNS effects were mainly observed after the 1125 mg dose, showing a significant decrease of adaptive tracking performance, VAS alertness and VAS mood, and an increase of EEG occipital alpha and theta power. Gastro-intestinal (GI) adverse effects were frequent at higher doses. No clinically significant changes in vital signs or ECG were noted during any of the treatments. The therapeutically relevant concentration range (950-11 100 ng ml(-1)) as determined from animal experiments was already reached after the 300 mg dose. C(max) after the 300 mg and 750 mg dose was 2868 and 9266 ng ml(-1) with a t(1/2) of 2.54 and 4.78 h, respectively. Concomitant food intake (with the 750 mg and 1125 mg doses) reduced C(max) by approximately 66% and AUC by approximately 40%. With concomitant food intake, the dose-normalized C(max) also decreased significantly by -5.6 (CI: -2.6 to -8.7) ng ml(-1) mg(-1). The pharmacokinetic variability was largest after the 300 mg and 750 mg dose, resulting in a SD of approximately 50% of the C(max). CONCLUSION: NAALADase inhibition with GPI 5693 was safe and tolerable in healthy subjects. Plasma concentrations that were effective in the reversal of hyperalgesia in the chronic constrictive injury animal model of neuropathic pain were obtained at doses of 300, 750 and 1125 mg in the fasted state. Comcomitant food intake reduced C(max) and AUC. CNS effects and GI AEs increased in incidence over placebo only at the 1125 mg dose.

NMDA receptor activation and respiratory chain complex V inhibition contribute to neurodegeneration in d-2-hydroxyglutaric aciduria.

The inherited neurometabolic disease d-2-hydroxyglutaric aciduria is complicated by progressive neurodegeneration of vulnerable brain regions during infancy and early childhood, frequently presenting with hypotonia, epilepsy and psychomotor retardation. Here, we report that the pathogenetic role of the endogenously accumulating metabolite d-2-hydroxyglutarate (D-2), which is structurally similar to the excitatory amino acid glutamate, is mediated by at least three mechanisms. (i) D-2-induced excitotoxic cell damage in primary neuronal cultures from chick and rat involved N-methyl-d-aspartate (NMDA) receptor activation. Indeed, D-2 activated recombinant NMDA receptors (NR1/NR2A, NR1/NR2B) but not recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptors in HEK293 cells. (ii) Fluorescence microscopy using fura-2 as a calcium indicator and the oxidant-sensitive dye dihydrorhodamine-123 revealed that D-2 disturbed intracellular calcium homeostasis and elicited the generation of reactive oxygen species. (iii) D-2 reduced complex V (ATP synthase) activity of the mitochondrial respiratory chain, reflecting an impaired energy metabolism due to inhibition of ATP synthesis but without affecting the electron-transferring complexes I-IV. Thus, D-2 stimulates neurodegeneration by mechanisms well-known for glutamate, NMDA or mitochondrial toxins. In conclusion, excitotoxicity contributes to the neuropathology of d-2-hydroxyglutaric aciduria, highlighting new neuroprotective strategies.

Inactivation of rhinovirus on human fingers by virucidal activity of glutaric acid.

Hand lotion containing 2% glutaric acid was significantly more effective than placebo in inactivating rhinovirus serotype 2 on the fingers of human volunteers. It may be possible to develop safe, cosmetically acceptable hand lotions which have durable virucidal activity and can interrupt the hand-to-hand transmission of rhinovirus colds.

Effects of 3-hydroxy-3-methylglutaric acid on plasma and low-density lipoprotein cholesterol levels in familial hypercholesterolemia.

Different doses of 3-hydroxy-3-methyl-glutaric acid (HMG) on plasma lipids were studied in a double-blind trial in 36 patients with familial hypercholesterolemia (type IIa or hyper-beta-lipoproteinemia). The patients were randomly assigned to five groups, and each group received one of the following treatments: placebo, or HMG 750 mg, 1500 mg, 2250 mg, or 3000 mg per day. As compared to placebo, the mean plasma cholesterol levels during the eight-week treatment period were 11 and 13 per cent lower in the 2250-mg and 3000-mg HMG-treated groups (P less than 0.034 and less than 0.021, respectively). At the same dosage levels LDL cholesterol was decreased by 8 per cent. HMG had no significant effect on plasma triglycerides as compared to placebo. Discontinuation of the medication did not result in a rebound of plasma cholesterol. There were no clinical or biologic adverse effects due to the administration of HMG, and all patients maintained excellent compliance to the medication. Because of its lack of toxicity, HMG may be useful as an adjunct to diet in the treatment of familial hypercholesterolemia.