The paradox-breaking panRAF plus SRC family kinase inhibitor, CCT3833, is effective in mutant KRAS-driven cancers.

BACKGROUND: KRAS is mutated in ∼90% of pancreatic ductal adenocarcinomas, ∼35% of colorectal cancers and ∼20% of non-small-cell lung cancers. There has been recent progress in targeting G12CKRAS specifically, but therapeutic options for other mutant forms of KRAS are limited, largely because the complexity of downstream signaling and feedback mechanisms mean that targeting individual pathway components is ineffective. DESIGN: The protein kinases RAF and SRC are validated therapeutic targets in KRAS-mutant pancreatic ductal adenocarcinomas, colorectal cancers and non-small-cell lung cancers and we show that both must be inhibited to block growth of these cancers. We describe CCT3833, a new drug that inhibits both RAF and SRC, which may be effective in KRAS-mutant cancers. RESULTS: We show that CCT3833 inhibits RAF and SRC in KRAS-mutant tumors in vitro and in vivo, and that it inhibits tumor growth at well-tolerated doses in mice. CCT3833 has been evaluated in a phase I clinical trial (NCT02437227) and we report here that it significantly prolongs progression-free survival of a patient with a G12VKRAS spindle cell sarcoma who did not respond to a multikinase inhibitor and therefore had limited treatment options. CONCLUSIONS: New drug CCT3833 elicits significant preclinical therapeutic efficacy in KRAS-mutant colorectal, lung and pancreatic tumor xenografts, demonstrating a treatment option for several areas of unmet clinical need. Based on these preclinical data and the phase I clinical unconfirmed response in a patient with KRAS-mutant spindle cell sarcoma, CCT3833 requires further evaluation in patients with other KRAS-mutant cancers.

Development of vigabatrin-induced lesions in the rat brain studied by magnetic resonance imaging, histology, and immunocytochemistry.

Vigabatrin, the gamma-aminobutyric acid transaminase (GABA-T)-inhibiting anticonvulsant drug, was given orally at a dose of 275 mg/kg/day to rats (n = 6) in their feed for a period of 12 weeks, during which T2-weighted magnetic resonance images (MRIs) and diffusion-weighted MRIs (DWIs) were collected at weeks 1, 3, 6, 9, and 12. Half the rats (n = 3; and half their age-matched littermate controls; n = 3) were then killed for histopathological confirmation of the observed VGB-induced cerebellar and cortical white-matter lesions. VGB was removed from the diet and additional MRIs of the remaining rats taken at weeks 14, 17, 20, and 24, at which time they (n = 3), along with remaining controls (n = 3), were also killed for histopathology. The T2-weighted MRIs acquired were used to compute T2 relaxation time maps. Statistically significant VGB-induced T2 increases were observed in the frontal and occipital cortices and in the cerebellar white matter (CWM). The cerebellar lesions were more clearly discerned by eye in the DWIs than by T2-contrast alone. During the recovery period the VGB-treatment group CWM-T2 and CWM-DWI hyperintensity greatly decreased as the reversible lesion disappeared. As expected, histological and immunocytochemical examinations demonstrated the presence of intra-myelinic edema, microvacuolation, and reactive astrocytosis in the CWM and cortex after 12 weeks VGB-treatment. In the remaining animals microvacuolation of the white matter had not completely resolved during the 12-week recovery phase. The data show that quantitative MRI T2-relaxometry can be used to detect VGB-induced CNS pathology, and also suggest that DWI is particularly sensitive to the cerebellar lesion. The reversible neurotoxicity of global GABA-elevation in experimental animals is discussed.

Mechanism of action of chromogranin A on catecholamine release: molecular modeling of the catestatin region reveals a beta-strand/loop/beta-strand structure secured by hydrophobic interactions and predictive of activity.

A novel fragment of chromogranin A, known as 'catestatin' (bovine chromogranin A344-364), inhibits catecholamine release from chromaffin cells and noradrenergic neurons by acting as a non-competitive nicotinic cholinergic antagonist, and may therefore constitute an endogenous autocrine feedback regulator of sympathoadrenal activity. To characterize how this activity depends on the peptide's structure, we searched for common 3-dimensional motifs for this primary structure or its homologs. Catestatin's primary structure bore significant (29-35.5% identity, general alignment score 44-57) sequence homology to fragment sequences within three homologs of known 3-dimensional structures, based on solved X-ray crystals: 8FAB, IPKM, and 2IG2. Each of these sequences exists in nature as a beta-strand/loop/beta-strand structure, stabilized by hydrophobic interactions between the beta-strands. The catestatin structure was stable during molecular dynamics simulations. The catestatin loop contains three Arg residues, whose electropositive side chains form the terminus of the structure, and give rise to substantial uncompensated charge asymmetry in the molecule. A hydrophobic moment plot revealed that catestatin is the only segment of chromogranin A predicted to contain amphiphilic beta-strand. Circular dichroism in the far ultraviolet showed substantial (63%) beta-sheet structure, especially in a hydrophobic environment. Alanine-substitution mutants of catestatin established a crucial role for the three central arginine residues in the loop (Arg351, Arg353, and Arg358), though not for two arginine residues in the strand region toward the amino-terminus. [125I]Catestatin bound to Torpedo membranes at a site other than the nicotinic agonist binding site. When the catestatin structure was 'docked' with the extracellular domain of the Torpedo nicotinic cholinergic receptor, it interacted principally with the beta and delta subunits, in a relatively hydrophobic region of the cation pore extracellular orifice, and the complex of ligand and receptor largely occluded the cation pore, providing a structural basis for the non-competitive nicotinic cholinergic antagonist properties of the peptide. We conclude that a homology model of catestatin correctly predicts actual features of the peptide, both physical and biological. The model suggests particular spatial and charge features of the peptide which may serve as starting points in the development of non-peptide mimetics of this endogenous nicotinic cholinergic antagonist.

Metabolic precursors and compartmentation of cerebral GABA in vigabatrin-treated rats.

The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by 13C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2(-13)C2]acetate (15 mumol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2(-13)C2]glucose (15 mumol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by 13C NMR. In vigabatrin-treated animals [13C]glutamine, a common intermediate for [13C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2(-13)C2]glucose or [1,2(-13)C2]acetate. However, [13C]GABA accumulation was sevenfold higher during [1,2(-13)C2]glucose infusions or twofold higher during [1,2(-13)C2]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near-equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [13C]GABA accumulation occurs initially in the neuronal compartment.

Characteristic metabolic profiles revealed by 1H NMR spectroscopy for three types of human brain and nervous system tumours.

Cell culture techniques, high-resolution in vitro 1H NMR spectroscopy, and chromatographic analyses were used to compare the properties of three types of human brain and nervous system tumours. Cell lines were immunocytochemically characterized at all stages in culture with specific antibodies. Intracellular metabolites present in cell extracts were analysed by 1H NMR spectroscopy and by high performance liquid chromatography (HPLC). The spectra from meningiomas, neuroblastomas, and glioblastomas displayed, in addition to similarities-including the presence of signals from leucine, isoleucine, valine, threonine, lactate, acetate, glutamate, choline-containing compounds and glycine-certain distinguishing metabolic features. Spectra from meningiomas featured relatively high signals from alanine. Intense signals from creatine were present in neuroblastoma spectra, while in spectra from glioblastoma they were not detectable. We found statistically significant differences by 1H NMR spectroscopy in the amounts of alanine, glutamate, creatine, phosphorylcholine and threonine among the types of tumours examined. HPLC determinations confirmed that there were also other metabolites specific to a type of tumour, such as taurine, gamma-aminobutyric acid, and serine. We suggest that these findings have potential relevance for the development of non-invasive diagnosis of tumour lineage by 1H NMR spectroscopy in vivo.

Cell type-specific fingerprinting of meningioma and meningeal cells by proton nuclear magnetic resonance spectroscopy.

We compared the properties of six human meningiomas with normal rat meningeal cells using cell culture techniques, high resolution in vitro 1H-NMR (nuclear magnetic resonance) spectroscopy, and chromatographic analysis. Cell cultures were immunocytochemically characterized at all stages with specific antibodies. Quantitative and qualitative metabolite assessments in cell extracts were obtained from 1H-NMR spectra and chromatographic analysis. Human meningioma cells expressed a characteristic spectrum of metabolites including free amino acids, compounds related to membrane phospholipid metabolism, energy metabolites, and other intermediary products. These spectral characteristics, although different in some respects, were strikingly similar to the ones of rat meningeal cells. Particularly, several metabolites that allow discrimination between meningeal cells and other cell types of the central nervous system were preserved in meningiomas. These similarities suggest that the regulation of intracellular levels of such metabolites is so intrinsic to the identity of cell type as to be conserved across species and through transformation. Additionally, human meningioma cultures expressed some spectroscopic characteristics that enabled them to be clearly distinguished from primary rat meningeal cultures. Thus, human meningiomas may be both specifically recognizable by 1H-NMR spectroscopy and also distinguishable from normal rat meningeal tissue. Our results raise the eventual possibility of using NMR in the noninvasive diagnosis of brain tumors in vivo.

Experimental encephalomyelitis modulates inositol and taurine in the spinal cord of Biozzi mice.

In this high resolution magnetic resonance spectroscopic study of experimental allergic encephalomyelitis (EAE) and Semliki Forest virus (SFV) infection of the Biozzi AB/H mouse, marked increases in the initially low levels of N-trimethyl compounds in the spinal cord were observed during probable demyelinating episodes. There was also a pronounced and reproducible modulation of the levels of taurine and myo-inositol during acute and again during chronic relapsing EAE. The ratio of N-acetyl-aspartate to creatine in the spinal cord of mice infected with the mutant M9 strain of SFV decreased to approximately 70% of that seen in normal mice.

Vigabatrin-induced lesions in the rat brain demonstrated by quantitative magnetic resonance imaging.

Rats treated with 250 mg/kg/day vigabatrin showed lesions detected by magnetic resonance imaging (MRI) in the cerebellar white matter in vivo. No lesions were seen in any control animal. As well as these visually apparent lesions, quantitative T2 relaxation time measurements showed a 12 ms increase in cerebellar white matter from 66 +/- 4 ms (SD, n = 5) to 78 +/- 2 ms (SD, n = 7). This region, as expected from previous studies, showed microvacuolation on post-mortem pathology. Additionally, significant increases in T2 relaxation times of 4-9 ms were found in the cerebral cortex, thalamus and hippocampus. Microvacuolation was not detected by post-mortem histopathology in the cerebral cortex or hippocampus, however, immunohistochemical staining for glial fibrillary acidic protein and for macrophages (ED1) showed reactive astrocytes (gliosis) and in more severe cases, microglial proliferation in these regions; such changes were also seen in association with the microvacuoles. No T2 increase was found in the cerebellar grey matter or olfactory bulbs. MRI techniques, including T2 relaxometry, are therefore sensitive for detecting vigabatrin-induced changes, including reactive astrocytosis, microglial proliferation and vacuolation in the rat brain. These results suggest that quantitative MRI should be a useful method for evaluating whether vigabatrin has neuropathological effects when given to patients.

Nuclear magnetic resonance detection of increased cortical GABA in vigabatrin-treated rats in vivo.

1H Nuclear magnetic resonance ([1H]NMR) spectroscopy was used to detect elevation of gamma-aminobutyric acid (GABA) in rat brain after administration of the antiepileptic drug vigabatrin (VGB). Rats were treated for 3 weeks with VGB added to their drinking water to deliver a dose of 250 mg/kg body weight per day. NMR spectroscopy was performed noninvasively in vivo, and a GABA concentration of 6.0 +/- 2.3 mmol/kg wet weight (mean +/- SD, n = 5) was measured. GABA could not be detected in control animals in vivo. Postmortem GABA levels of 1.3 +/- 0.5 and 4.5 +/- 1.0 mmol/kg (mean +/- SD, n = 5) were measured in perchloric acid extracts of frozen brain from control and treated animals, respectively. Noninvasive measurement of increased cerebral GABA should allow detailed studies of the pharmacology of GABA-increasing drugs in vivo. With future developments, these measurements may be feasible in human subjects.

Determining 15N to 14N ratios in biofluids by single-pulse 1H nuclear magnetic resonance.

In the 1H NMR spectra of acidified biofluids from our studies of nitrogen metabolism the 1:1 doublet signal (1J-1H-15N = 73.2 Hz) from the protons of 15N-enriched ammonia is clearly resolved from the 1:1:1 triplet signal (1J-1H-14N = 52.3 Hz) from (naturally abundant) 14N ammonia. The five spectral lines, infinitely broad at neutral pH, produce a "pseudo-pentet" at 7.1 ppm from which the fractional 15N-enrichment is easily calculated. 1H NMR spectra of plasma and urine from rats infused with 15N-enriched ammonium acetate illustrate the technique. The greater sensitivity of 1H NMR over 15N and 14N NMR spectroscopy suggests 15N-ammonia determinations by this method will compare favorably with other methods for determining nitrogen labeling.

Experimental allergic encephalomyelitis raises betaine levels in the spinal cord of strain 13 guinea-pigs.

Chronic relapsing experimental allergic encephalomyelitis, an animal model of multiple sclerosis, was induced in Strain 13 guinea-pigs by subcutaneous injection of spinal cord homogenate and Freund's incomplete adjuvant supplemented with Mycobacterium tuberculosis. High resolution 1H NMR spectra of CNS tissue extracts indicated that the levels of choline metabolites, particularly betaine, were elevated in the spinal cord tissue, the principal site of lesion formation in this guinea-pig strain. The spectra also show that N-acetylated compounds are slightly depleted in the disease. The results are discussed in relation to the biochemical interpretation of NMR spectra obtained in vivo from patients with multiple sclerosis.

Studies on the disposition and metabolism of hydrazine in rats in vivo.

1. Rats were given various doses of hydrazine orally and their plasma and liver hydrazine levels were determined (at various times up to 270 min after dosing) by gas chromatography/mass spectrometry. 2. The increase in the peak plasma level and in the area under the plasma concentration-time curve (AUC) were not directly proportional to the dose. 3. The ratio of plasma to liver hydrazine varied with dose suggesting saturation of an uptake mechanism might be occurring. 4. In a separate experiment hydrazine was still detectable in the plasma and liver 24 h after dosing with hydrazine i.p. 5. Rats were given the same doses of hydrazine and urine was collected for 24 h after dosing and assayed for hydrazine and acetylhydrazine. The proportion of hydrazine and acetylhydrazine excreted declined with dose. 6. Liver samples were taken for histopathological examination 96 h after dosing. Only after the highest dose (81 mg kg-1) was there evidence of fatty liver, 96 h after a single dose, and a reduction in both liver and body weight.

19F NMR studies of enflurane elimination and metabolism.

The elimination and metabolism of enflurane, a fluorinated ether anaesthetic, was studied by 19F NMR in vivo in both rat liver and brain as well as human body fluids. In the liver of thiobarbitone-anaesthetized rats the half-life for enflurane following exposure to 0.15% (v/v) for 30 min was 76 min but this could be decreased to 39 min by pretreatment of the animals with isoniazid (0.1% in the drinking water for 7 days), an agent known to enhance enflurane metabolism. In these animals the major organic metabolite difluoromethoxy difluoroacetate (DFMDFA) was also detected by 19F NMR in vivo. This metabolite was detected along with fluoride ion in rat and human urine and plasma by high resolution 19F NMR. Human urine also contained signals from a probable DFMDFA conjugate and unexpectedly from trifluoroacetate.

A biochemical and NMR spectroscopic study of hydrazine in the isolated rat hepatocyte.

Using isolated rat hepatocytes the biochemical effects of hydrazine have been investigated using both conventional assay techniques and high resolution proton NMR. High resolution proton NMR revealed that hydrazine caused a significant increase in alanine and lactate levels in the incubation buffer, whereas levels of beta-hydroxybutyrate were decreased. NMR also detected metabolites of hydrazine notably acetylhydrazine and a cyclised hydrazone formed with alpha-ketoglutarate. Changes were detected in NADH and NADPH, ATP, succinate dehydrogenase (SDH) and total non-protein sulphydryl groups (TNPSH). However, the changes in pyridine nucleotides occurred at higher concentrations than those affecting succinate dehydrogenase and ATP. Similarly, the depletion of TNPSH occurred at a higher concentration and with a different time course to that seen with ATP depletion and inhibition of succinate dehydrogenase.

Determination of hydrazine in biofluids by capillary gas chromatography with nitrogen-sensitive or mass spectrometric detection.

Plasma and liver levels of hydrazine were determined at 10, 30, 90 and 270 min in rats given 0.09, 0.27, 0.84 and 2.53 mmol of hydrazine per kg body weight orally by capillary gas chromatography-mass spectrometry of its pentafluorobenzaldehyde adduct (DFBA, m/z 388) using selected ion monitoring with 15N2-labelled hydrazine as the internal standard (adduct, m/z 390). The mean half-life for hydrazine in the plasma was approximately 2 h but varied with dose. Urinary excretion (0-24 h) of hydrazine and its metabolite acetylhydrazine were determined employing nitrogen-phosphorus detection of the adducts utilising a novel internal standard, pentafluorophenylhydrazine, the adduct of which structurally resembles DFBA. The fraction of the original dose excreted as hydrazine (and acetylhydrazine) declined with increasing dose.

Identification of novel hydrazine metabolites by 15N-NMR.

15N-NMR has been used to study the metabolism of hydrazine in rats in vivo. Single doses of [15N2]hydrazine (2.0 mmol/kg: 98.6% g atom) were administered to rats and urine collected for 24 hr over ice. A number of metabolites were detected by 15N-NMR analysis of lyophilized urine. Ammonia was detected as a singlet at 0 ppm and unchanged [15N2]hydrazine was present in the urine detectable as a singlet at 32 ppm. Peaks were observed at 107 and 110 ppm which were identified as being due to the hydrazido nitrogen of acetylhydrazine and diacetylhydrazine, respectively. A resonance at 85 ppm was ascribed to carbazic acid, resulting from reaction of hydrazine with carbon dioxide. A singlet detected at 316 ppm was thought to be due to the hydrazono nitrogen of the pyruvate hydrazone. The resonance at 56 ppm was assigned to 15N-enriched urea, this together with the presence of ammonia indicates that the N-N bond of hydrazine is cleaved in vivo, possibly by N-oxidation, and the resultant ammonia is incorporated into urea. A doublet centred at 150 ppm and a singlet at 294 ppm were assigned to a metabolite which results from cyclization of the 2-oxoglutarate hydrazone. Therefore 15N-NMR spectroscopic analysis of urine has yielded significant new information on the metabolism of hydrazine.

Effects of glutathione depletion, chelation and diuresis on iron nitrilotriacetate-induced lipid peroxidation in rats and mice.

1. Rats and mice dosed with iron nitrilotriacetate (FeNTA) i.p. (2-12 mg Fe/kg) showed evidence of lipid peroxidation as indicated by increased exhalation of ethane and increased malondialdehyde formation in liver and kidney. 2. Buthionine sulphoximine (BSO) administered i.p. to rats and mice decreased the total glutathione (GSH) content of liver and kidney. When the rodents were pretreated i.p. with BSO prior to injection of FeNTA the increases in ethane exhalation, and in liver and kidney malondialdehyde production, were greater than with FeNTA alone, and the total GSH of liver and kidney were decreased. 3. Diuresis produced by i.p. administration of furosemide to mice substantially decreased the ethane exhalation resulting from FeNTA administration, had a lowering effect on kidney MDA, but had no significant effect on liver MDA production. 4. Similarly, desferrioxamine beta-mesylate administered i.p. to mice markedly decreased the ethane exhalation and kidney MDA production resulting from FeNTA administration.

NMR studies of drug metabolism and disposition.

The ways in which NMR is being used in vivo to study drug metabolism and disposition are reviewed. We also assess the role of this technique as a non-invasive method for monitoring the fate of drugs in human and animals, and for providing information about pharmacology and toxicity.

Course of ATP depletion in hydrazine hepatotoxicity.

The effect of hydrazine on ATP levels has been investigated in rats in vivo and in hepatocytes in vitro. Hydrazine was found to cause a dose-dependent depletion of hepatic ATP in vivo 3 h after dosing. In isolated hepatocytes in vitro hydrazine also caused a concentration-dependent depletion of ATP which preceded cytotoxicity as indicated by loss of cell viability. The ATP depletion in isolated hepatocytes was also significant at a concentration of hydrazine which was not cytotoxic. Attempts to determine hepatic ATP depletion in vivo over time using topical 31P NMR were confounded by the effects of the thiopentobarbitone used to anaesthetise the animals. This was found to ameliorate the effects of hydrazine on ATP depletion but potentiate the lethality of hydrazine. Consequently, although ATP depletion was detected in some hydrazine-treated animals, this was only observed in animals which subsequently died. The results indicate that ATP depletion may underlie the hepatotoxicity of hydrazine.