A survey of selected key areas of management of South African neurosurgical patients.

BACKGROUND: Previous surveys of neurosurgical patient management, including a South African (SA) survey conducted in 2001, confirmed the existence of significant dissimilarities in management on national and international levels. This survey aimed to determine current SA neurosurgical patient management and to compare this with international trends. METHOD: Questionnaires in multiple choice question (MCQ) and free text entry format covering key areas of neurosurgical practice were emailed to SA neurosurgeons following ethics approval. All responses were captured anonymously. RESULTS: The response rate was 53%. Demographically only 5.7% respondents were younger than 40 years, 59.3% obtained a local college fellowship, 14.8% an international fellowship, 40.7% a MMed and 16.6% obtained more than one postgraduate qualification. Public sector specialists predominantly practised intracranial surgery (69%) while private specialists practised mainly spinal surgery (58%). Years in specialist practice were negatively associated with endoscopic surgery (p = 0.014) and decompressive craniectomies (p = 0.008) but not with other more recently introduced techniques including pedicle screws, neuro-navigation and cell-saver techniques. Age per se had no influence on practice. In subarachnoid haemorrhage (SAH) disease, 88% routinely administered nimodipine, 8% restricting its use to managing vasospasm. Endovascular coiling, more recently introduced for intracranial aneurysm management, was preferred to surgical clipping (54%); 69% preferred total intravenous to inhalational anaesthesia with propofol primarily replacing thiopentone for brain protection. 27% still utilised the sitting position. Only one incident of a postoperative visual defect was recorded with prone positioning. CONCLUSION: With the exception of endoscopic and decompressive craniectomy surgery, overall management in key areas surveyed was in line with international trends.

Discovery of the imidazole-derived GPR40 agonist AM-3189.

As a follow-up to the GPR40 agonist AMG 837, which was evaluated in clinical trials for the treatment of type II diabetes, further optimization led to the discovery of AM-3189 (13k). AM-3189 is representative of a new class of compounds with minimal CNS penetration, superior pharmacokinetic properties and in vivo efficacy comparable to AMG 837.

Identification and pharmacological characterization of multiple allosteric binding sites on the free fatty acid 1 receptor.

Activation of FFA1 (GPR40), a member of G protein-coupling receptor family A, is mediated by medium- and long-chain fatty acids and leads to amplification of glucose-stimulated insulin secretion, suggesting a potential role for free fatty acid 1 (FFA1) as a target for type 2 diabetes. It was assumed previously that there is a single binding site for fatty acids and synthetic FFA1 agonists. However, using members of two chemical series of partial and full agonists that have been identified, radioligand binding interaction studies revealed that the full agonists do not bind to the same site as the partial agonists but exhibit positive heterotropic cooperativity. Analysis of functional data reveals positive functional cooperativity between the full agonists and partial agonists in various functional assays (in vitro and ex vivo) and also in vivo. Furthermore, the endogenous fatty acid docosahexaenoic acid (DHA) shows negative or neutral cooperativity with members of both series of agonists in binding assays but displays positive cooperativity in functional assays. Another synthetic agonist is allosteric with members of both agonist series, but apparently competitive with DHA. Therefore, there appear to be three allosterically linked binding sites on FFA1 with agonists specific for each of these sites. Activation of free fatty acid 1 receptor (FFAR1) by each of these agonists is differentially affected by mutations of two arginine residues, previously found to be important for FFAR1 binding and activation. These ligands with their high potencies and strong positive functional cooperativity with endogenous fatty acids, demonstrated in vitro and in vivo, have the potential to deliver therapeutic benefits.

AMG 837: a potent, orally bioavailable GPR40 agonist.

The discovery that certain long chain fatty acids potentiate glucose stimulated insulin secretion through the previously orphan receptor GPR40 sparked interest in GPR40 agonists as potential antidiabetic agents. Optimization of a series of ß-substituted phenylpropanoic acids led to the identification of (S)-3-(4-((4'-(trifluoromethyl)biphenyl-3-yl)methoxy)phenyl)hex-4-ynoic acid (AMG 837) as a potent GPR40 agonist with a superior pharmacokinetic profile and robust glucose-dependent stimulation of insulin secretion in rodents.

A scalable organoid model of human autosomal dominant polycystic kidney disease for disease mechanism and drug discovery.

Human pluripotent stem-cell-derived organoids are models for human development and disease. We report a modified human kidney organoid system that generates thousands of similar organoids, each consisting of 1-2 nephron-like structures. Single-cell transcriptomic profiling and immunofluorescence validation highlighted patterned nephron-like structures utilizing similar pathways, with distinct morphogenesis, to human nephrogenesis. To examine this platform for therapeutic screening, the polycystic kidney disease genes PKD1 and PKD2 were inactivated by gene editing. PKD1 and PKD2 mutant models exhibited efficient and reproducible cyst formation. Cystic outgrowths could be propagated for months to centimeter-sized cysts. To shed new light on cystogenesis, 247 protein kinase inhibitors (PKIs) were screened in a live imaging assay identifying compounds blocking cyst formation but not overall organoid growth. Scaling and further development of the organoid platform will enable a broader capability for kidney disease modeling and high-throughput drug screens.

Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors.

The citric acid cycle is central to the regulation of energy homeostasis and cell metabolism. Mutations in enzymes that catalyse steps in the citric acid cycle result in human diseases with various clinical presentations. The intermediates of the citric acid cycle are present at micromolar concentration in blood and are regulated by respiration, metabolism and renal reabsorption/extrusion. Here we show that GPR91 (ref. 3), a previously orphan G-protein-coupled receptor (GPCR), functions as a receptor for the citric acid cycle intermediate succinate. We also report that GPR99 (ref. 4), a close relative of GPR91, responds to alpha-ketoglutarate, another intermediate in the citric acid cycle. Thus by acting as ligands for GPCRs, succinate and alpha-ketoglutarate are found to have unexpected signalling functions beyond their traditional roles. Furthermore, we show that succinate increases blood pressure in animals. The succinate-induced hypertensive effect involves the renin-angiotensin system and is abolished in GPR91-deficient mice. Our results indicate a possible role for GPR91 in renovascular hypertension, a disease closely linked to atherosclerosis, diabetes and renal failure.

Structural basis for pharmacological modulation of the TRPC6 channel.

Transient receptor potential canonical (TRPC) proteins form nonselective cation channels that play physiological roles in a wide variety of cells. Despite growing evidence supporting the therapeutic potential of TRPC6 inhibition in treating pathological cardiac and renal conditions, mechanistic understanding of TRPC6 function and modulation remains obscure. Here we report cryo-EM structures of TRPC6 in both antagonist-bound and agonist-bound states. The structures reveal two novel recognition sites for the small-molecule modulators corroborated by mutagenesis data. The antagonist binds to a cytoplasm-facing pocket formed by S1-S4 and the TRP helix, whereas the agonist wedges at the subunit interface between S6 and the pore helix. Conformational changes upon ligand binding illuminate a mechanistic rationale for understanding TRPC6 modulation. Furthermore, structural and mutagenesis analyses suggest several disease-related mutations enhance channel activity by disrupting interfacial interactions. Our results provide principles of drug action that may facilitate future design of small molecules to ameliorate TRPC6-mediated diseases.

GPR40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo.

Long-chain fatty acids amplify insulin secretion from the pancreatic beta-cell. The G-protein-coupled receptor GPR40 is specifically expressed in beta-cells and is activated by fatty acids; however, its role in acute regulation of insulin secretion in vivo remains unclear. To this aim, we generated GPR40 knockout (KO) mice and examined glucose homeostasis, insulin secretion in response to glucose and Intralipid in vivo, and insulin secretion in vitro after short- and long-term exposure to fatty acids. Our results show that GPR40 KO mice have essentially normal glucose tolerance and insulin secretion in response to glucose. Insulin secretion in response to Intralipid was reduced by approximately 50%. In isolated islets, insulin secretion in response to glucose and other secretagogues was unaltered, but fatty acid potentiation of insulin release was markedly reduced. The Galpha(q/11) inhibitor YM-254890 dose-dependently reduced palmitate potentiation of glucose-induced insulin secretion. Islets from GPR40 KO mice were as sensitive to fatty acid inhibition of insulin secretion upon prolonged exposure as islets from wild-type animals. We conclude that GPR40 contributes approximately half of the full acute insulin secretory response to fatty acids in mice but does not play a role in the mechanisms by which fatty acids chronically impair insulin secretion.

A direct demonstration of closed-state inactivation of K+ channels at low pH.

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker alpha-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K(+) or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at -80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.

Discovery of AM-6226: A Potent and Orally Bioavailable GPR40 Full Agonist That Displays Efficacy in Nonhuman Primates.

GPR40 (FFA1) is a G-protein-coupled receptor, primarily expressed in pancreatic islets and enteroendocrine L-cells, and, when activated, elicits increased insulin secretion only in the presence of elevated glucose levels. We recently reported the discovery of AM-1638 (2), a full agonist of GPR40. Herein, we present further structure-activity relationships progressing from AM-1638 (2) to AM-6226 (14) that possesses a profile acceptable for dosing cynomolgus monkeys. The GPR40 full agonist AM-6226 (14) is the first molecule to display significant glucose lowering in cynomolgus monkeys providing additional evidence that GPR40 full agonists afford access to a powerful mechanism for maintaining glycemic control.

The use of a laryngeal mask airway after a prolonged suspension laryngoscopy to preserve a vocal cord fat graft.

A 62-yr-old man presented for a microdirect laryngoscopy and vocal cord fat grafting under jet ventilation. After a prolonged laryngoscopy, the patient developed hypercapnea and upper airway obstruction secondary to traumatic epiglottitis. The placement of a laryngeal mask airway provided ventilation and allowed for direct visualization of the patient's inflamed epiglottis without disruption of the patient's fat graft. Because of its placement above the cords and its effectiveness in providing adequate ventilation, we propose intermittent laryngeal mask airway ventilation as a bridge, in lieu of endotracheal intubation, in microdirect laryngoscopy cases in which ventilation during emergence may be difficult and the insertion of an endotracheal tube would disrupt the surgical procedure.

Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K+-free solutions at physiological pH.

Extracellular acidification and reduction of extracellular K(+) are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K(+)](o) at pH 7.4, it is very sensitive to [K(+)](o) at pH 6.4, and in the mutant, H463G, the removal of K(+)(o) virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K(+)(o) in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na(+) permeation through inactivated channels. Although the H463G currents were abolished in zero [K(+)](o), robust Na(+) tail currents through inactivated channels were observed. The appearance of H463G Na(+) currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could activate directly from closed-inactivated states. In wild-type channels, when intracellular K(+) was replaced by NMG(+) and the inward Na(+) current was recorded, addition of 1 mM K(+) prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction of [K(+)](o) inhibit Kv1.5 channels through a common mechanism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).

Synergistic inhibition of the maximum conductance of Kv1.5 channels by extracellular K+ reduction and acidification.

Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the functional classes of Kv channels, the Kv1 channels are the largest and the best studied and are known to play essential roles in excitable cell function, providing an essential counterpoint to the various inward currents that trigger excitability. The serum potassium concentration [K(+)(o)] is tightly regulated in mammals and disturbances can cause significant functional alterations in the electrical behavior of excitable tissues in the nervous system and the heart. At least some of these changes may be mediated by Kv channels that are regulated by changes in the extracellular K(+) concentration. As well as changes in serum [K(+)(o)], tissue acidification is a frequent pathological condition known to inhibit Shaker and Kv1 voltage-gated potassium channels. In recent studies, it has become recognized that the acidification-induced inhibition of some Kv1 channels is K(+)(o)-dependent, and the suggestion has been made that pH and K(+)(o) may regulate the channels via a common mechanism. Here we discuss P/C type inactivation as the common pathway by which some Kv channels become unavailable at acid pH and lowered K(+)(o). It is suggested that binding of protons to a regulatory site in the outer pore mouth of some Kv channels favors transitions to the inactivated state, whereas K(+) ions exert countereffects. We suggest that modulation of the number of excitable voltage-gated K(+) channels in the open vs inactivated states of the channels by physiological H(+) and K(+) concentrations represents an important pathway to control Kv channel function in health and disease.

Oliguria and tolazoline pharmacokinetics in the newborn.

Tolazoline treatment of neonates has been reported since 1965. Dosages increased from pulse doses of 1 to 2 mg/kg to continuous infusions of 10 mg/kg X h before neonatal plasma tolazoline concentrations were measured. We developed a microassay for tolazoline and determined neonatal distribution volume, 1.61 +/- 0.21 L/kg, and disposition rate constant (beta), 0.0027 +/- 0.005 min-1 (mean +/- SEM). Half-life (gamma) ranged from 1.47 to 41.25 (median = 4.43) hours and correlated inversely with urine output (x); y (h) = -0.46 + 7.63/x (mL/kg X h), r = .61, P less than .05. The highest plasma tolazoline concentration in a neonate was 20.3 mg/L. Lethal tolazoline concentrations in lambs ranged from 21.8 to 56.8 mg/L. Initial tolazoline concentrations during infusions and after 1- to 2-mg/kg pulse doses ranged from 0.35 to 2.3 mg/L and PaO2 increased greater than or equal to 15 mm Hg in 64% of 14 treatments. The average neonatal pharmacokinetics predict that each 1 mg of tolazoline HCl per kilogram pulse dose will increase the plasma concentration of tolazoline base by 0.5 mg/L. The plasma concentration should remain constant with infusion dose increments of 0.16 mg of tolazoline HCl per kilogram per hour for every 1.0-mg/kg loading dose. Tolazoline dose requirements for specific patients will vary, especially with renal dysfunction. Reduced tolazoline doses were used to treat two patients, concentrations remained constant, and PaO2 was maintained. Tolazoline doses derived from neonatal kinetics are less than current infusion doses and may avoid high concentrations.

Disposition of dietary caffeine in milk, saliva, and plasma of lactating women.

Caffeine is present in many dietary substances; its appearance from these sources in human milk has not previously been studied in detail. Fifteen lactating women ingested a known amount of a caffeinated beverage (36 to 335 mg). Simultaneous milk and saliva samples were collected at intervals for the subsequent 12 hours and assayed for caffeine content. Eleven of 15 mothers excreted measurable caffeine in milk. Caffeine was detected by 15 minutes in saliva and milk; peak levels in milk (2.09 to 7.17 micrograms/mL) and saliva (1.24 to 9.22 micrograms/mL) were achieved within 1 hour. Elimination half-lives were 1.3 to 13.5 hours (mean 4.0 +/- 3.7 [SD] hours) for saliva and 1.5 to 14.5 hours (mean 6.1 +/- 4.4 [SD] hours) for milk. Assuming each infant would ingest 90 mL of milk every three hours for 24 hours after maternal ingestion of caffeine, it is possible to estimate potential exposure of the nursing infant to caffeine. The amount of caffeine available for infant absorption ranged from 0.01 to 1.64 mg or 0.06% to 1.5% of the maternal dose. Caffeine was not present in the infants' urine collected for five hours after the first nursing period. The maternal ingestion of a single cup of caffeinated beverage does not appear to present significant doses of caffeine to the nursing infant.

Relationship between representation of hindpaw and hindpaw barrel subfield (HBS) in layer IV of rat somatosensory cortex.

We describe the organization of the hindpaw barrel subfield (HBS) in layer IV of rat somatosensory cortex (SI) and relate this organization to the representation of the hindpaw. The ovoid-shaped, HBS is oriented anterior to posterior and comprises barrels and barrel-like structures, the most prominent of which consist of at least five anteriorly-located elongated barrel bands. Posterior to these elongated bands is a cluster of four barrels. Two additional barrels are found, one lateral, the other medial. The lateral border is formed by a nearly continuous band that overlaps portions of the anterior elongated bands and posterior barrels. The HBS shows considerable variability in size and shape; nevertheless, the overall pattern reflects a common plan of organization. Electrophysiological mapping confirmed that hindpaw representation is somatotopically organized. The glabrous toes are represented anteriorly, the pads posteriorly, and the dorsal hairy skin of the toes and hindpaw laterally. By aligning physiological and morphological (HBS) maps according to lesion sites, our data suggest that the elongated anteriorly-located barrel bands represent the hindpaw toes, the four toe pads are represented immediately posterior followed by barrels representing the plantar pads. The representations of dorsal hairy skin of toe and dorsal hindpaw form the lateral border; the heel and ankle are represented most posterior. We interpret our findings as support that individual barrels in the HBS are associated with discrete regions of the hindpaw; however, the precise relationship of structure and function reported between the vibrissae and posteromedial barrel subfield (PMBSF) and between the forepaw and the forepaw barrel subfield (FBS) were not observed.

The surprisingly beneficial effect of soft donors on the performance of early transition metal olefin polymerisation catalysts.

Group 4 metal complexes containing phenoxy-amide ligands bearing soft pendant donors are shown to give more highly active ethylene polymerisation catalysts than counterparts containing hard donors or systems without a pendant donor.

Improving the Pharmacokinetics of GPR40/FFA1 Full Agonists.

We recently reported the discovery of a potent GPR40 full agonist AM-1638 (1). Herein, we describe our efforts in improving the drug-like properties of the full agonists through the systematic introduction of polar groups in the C-, D-, and A-rings. This led to the discovery of new GPR40 full agonists with significantly improved pharmacokinetic propeties. Compound 8 and 20 also showed potent in vivo efficacy in oral glucose tolerance tests in mice in addition to the improvement in properties.

Optimization of GPR40 Agonists for Type 2 Diabetes.

GPR40 (FFA1 and FFAR1) has gained significant interest as a target for the treatment of type 2 diabetes. TAK-875 (1), a GPR40 agonist, lowered hemoglobin A1c (HbA1c) and lowered both postprandial and fasting blood glucose levels in type 2 diabetic patients in phase II clinical trials. We optimized phenylpropanoic acid derivatives as GPR40 agonists and identified AMG 837 (2) as a clinical candidate. Here we report our efforts in searching for structurally distinct back-ups for AMG 837. These efforts led to the identification of more polar GPR40 agonists, such as AM-4668 (10), that have improved potency, excellent pharmacokinetic properties across species, and minimum central nervous system (CNS) penetration.