The Global Bioequivalence Harmonisation Initiative (GBHI): Report of the fifth international EUFEPS/AAPS conference.

The series of conferences of the Global Bioequivalence Harmonisation Initiative (GBHI) was started in 2015 by the European Federation for Pharmaceutical Sciences (EUFEPS). All GBHI meetings so far were co-organised together with the American Association of Pharmaceutical Scientists (AAPS). Beginning with the 3rd workshop US-FDA joined as co-sponsor - to support global harmonisation of regulatory recommendations for bioequivalence (BE) assessment. At the 5th GBHI conference, the following BE topics were intensively discussed, and the following main conclusions were drawn: (1) Statistical considerations for BE assessment in specific situations covering scaling approaches for highly variable drug (HVD) products, two-stage adaptive design and opportunities of modelling and simulation to support BE: even though special BE study concepts like adaptive designs are not often used in practise so far, a majority of the workshop participants were in favour of a more frequent application of such approaches. The regulatory conditions relevant in this context need further concretisation and harmonisation between the regions. Moreover, modelling and simulation were considered as a promising and evolving approach, also for BE development programmes. (2) Fed versus fasting conditions in BE trials: Findings that BE between generic products could be confirmed only after fasted administration but failed under fed conditions seem more an exception than the rule. Obviously, BCS class IV compounds are most problematic in this context. Differences in critical excipients such as surfactants or pH-modifiers may be relevant reasons for different sensitivity for interactions in fasted versus fed conditions. Consequently, such deviations in composition of generic preparations should be avoided. Moreover, confirmation of BE may be generally difficult comparing different dosage forms, such like capsules versus tablets, especially in fed state. (3) BE assessment of locally acting drug products applied topically to the skin: Appropriateness and potential benefit of in-vitro tests as alternatives to clinical efficacy studies have been comprehensively discussed. In addition to the already well-established in-vitro release and permeation tests, other techniques were suggested, e.g., Raman spectroscopy or dermal open flow microperfusion. Validation of those methods is challenging and, despite significant progress already achieved during previous years, more research is needed before they may be fully accepted for regulatory purposes. (4) BE evaluation of narrow therapeutic index (NTI) drugs: The discrepancies amongst regulatory agencies in necessity of tighter BE acceptance ranges, the recommendations for inclusion of peak and total drug exposure into BE assessment with more restrictive criteria and the importance of comparison of the product-related within-subject variability for NTI drugs were debated. Arguments in favour and against the different approaches were presented and discussed but need further consideration before harmonisation can be achieved. The highly interactive meeting and extensive exchange between regulators and scientists from industry and academia resulted in useful progress in open BE issues and supported the goal of science-driven harmonisation.

Phenotyping with sulfasalazine - time dependence and relation to NAT2 pharmacogenetics.

OBJECTIVE: N-acetyltransferase 2 (NAT2) genotype-phenotype relation with sulfasalazine as probe drug by means of detailed genotype analysis and kinetic data evaluation. BACKGROUND: Though phenotype analysis of sulfasalazine metabolism has been described before, genotype investigations in this regard are scarce. The influence of different single point mutations on the metabolism of the sulfasalazine metabolite sulfapyridine (SP) should give more insight into the functionality of different alleles especially with those still under discussion. METHODS: In two bioavailability studies performed under comparable conditions with 24 healthy subjects of both genders equally distributed, plasma levels of SP and acetylsulfapyridine (Ac-SP) were determined after oral intake of enteric coated formulations of sulfasalazine (500 mg and 1,000 mg, respectively). The resulting metabolic ratios were calculated. NAT2 genotype was analyzed in parallel for all subjects deducing haplotype set as well as putative functional phenotype as (homozygous or heterozygous) rapid acetylator (RA) or slow acetylator (SA) and correlated with the PK results. RESULTS AND DISCUSSION: RA genotype in the overall study population was seen with 45.5% (including 6.8% homozygous wildtype *4/*4) and SA genotype with 54.5%. Compared to RA genotype, apparent terminal elimination half-life of SP as well as of Ac-SP was prolonged in the SA genotype population, C(max) and AUC values of SP were higher whereas average C(max) value of Ac-SP was lower (with AUC only some tendency to lower values). In general, phenotype-genotype correlation was good with only few exceptions. Strongest functional effect on enzyme activity was noticed in slow acetylators carrying the 341T > C mutation, followed by 590G > A mutation whereas the influence of 857G > A was considerably less pronounced. Homozygous 803A > G mutation (lysine > arginine shift) did not reveal enzyme activity reduction.

Differences in bioavailability between 60 mg of nifedipine osmotic push-pull systems after fasted and fed administration.

OBJECTIVES: This study was designed to evaluate and compare the bioavailability of two osmotically active formulations of 60 mg nifedipine, Gen-Nifedipine extended release Test tablets (Genpharm ULC, Etobicoke, ON, Canada) and Adalat XL Reference tablets (Bayer Healthcare AG, Leverkusen, Germany) after single dose fasted and fed administration. MATERIALS AND METHODS: The study was performed following a 4-period crossover design with both investigational products obtained from marketed batches. The complete pharmacokinetic evaluation was carried out in 26 healthy male subjects with a median age of 29.5 years (range 18 - 44 years), mean weight of 79.7 kg (range 66.0 - 97.5 kg), and a mean body mass index (BMI) of 24.1 kg/m(2) (range 22.1 - 26.9 kg/m(2)). Tablets were administered with tap water either under fasting conditions or immediately following a high-fat, high-calorie breakfast. Blood samples were taken predose and at pre-defined time points until 48 h post dosing. Samples were protected from light during handling and frozen until analysis. A validated LC-MS/MS method was used for the quantification of nifedipine in plasma samples. All kinetic parameters were determined model-independently for each treatment directly from measured concentrations. Monitoring of subject safety was accomplished by routine monitoring of blood pressure, heart rate and probing for adverse events. RESULTS: In-vitro dissolution curves show later onset and considerably lower quantity of nifedipine release from Test compared to Reference tablets. Under fasting conditions total and maximum exposure, represented by geometric mean AUC(0-tlast)- and C(max)-values, respectively were 466.7 h*ng/ml (AUC(0-tlast)) and 21.9 ng/ml (C(max)) for Test and 507.8 h*ng/ml (AUC(0-tlast)) and 22.0 ng/ml (C(max)) for Reference tablets. However, the Test product exhibited a notably longer lag-time and less rapid onset of absorption than the Reference tablets. Moreover, the plateau phase is maintained for about 14 hours on Test but for almost 20 hours on Reference. Point estimates (PE) and associated 90% confidence intervals (CI) were determined as 91.8% and 79.9 - 105.5% for AUC(0-tlast), as well as 99.8% and 88.6 - 112.4% for C(max). Larger differences were found for AUC(0-9h) (PE: 54.8%; CI: 45.8 - 65.5%) determined as parameter for early exposure. Under fed conditions, although the mean plasma concentration time curves look similar in shape, concentrations of Test compared to Reference tablets are considerably lower at all time points until 36 hours after dosing. Again the lag time in onset of drug absorption is notably longer for the Test product. Both, total and maximum exposure, represented by geometric mean values for AUC(0-tlast) and C(max), were considerably lower (differences also statistically significant) after administration of Test with 481.8 h*ng/ml for AUC(0-tlast) and 25.3 ng/ml for C(max) in comparison to Reference tablets with 595.9 h*ng/ml for AUC(0-tlast) and 31.9 ng/ml for C(max). Test/Reference point estimates (PE) and associated 90% confidence intervals (CI) were determined as 80.7% and 73.7 - 88.5% for AUC(0-tlast), as well as 79.6% and 70.3 - 90.0% for C(max). Differences were also even more expressed for AUC(0-9h) (PE: 54.9%; CI: 47.4 - 63.5%) determined as parameter for early exposure. CONCLUSION: The results indicate that although both products are osmotic release systems they are not bioequivalent according to the accepted standards. This difference between both osmotic delivery systems might be substantiated by the fact that the core of the Test product is designed as a monolayer system (containing both, the active ingredient and the osmotic component) while Reference tablets consist of two separate layers. The observed pharmacokinetic differences may have an impact on blood pressure control in patients and thus, should be kept in mind when switching during treatment.

Adrenocorticotropic hormone may be transported directly from the pituitary to the brain.

Experiments were designed to test the hypothesis that pituitary hormones may be delivered directly to the brain. Concentrations of adrenocorticotropic hormone (ACTH) in the plasma were determined in blood samles obtained simultaneously from the carotid artery, the sagittal sinus, and the jugular vein of three awake sheep. Seizures were induced electrically to stimulate ACTH secretion, and at precise intervals thereafter several simultaneous comparisons were made in each animal. In many of the post-seizure comparisons, the ACTH plasma concentrations within the sagital sinus exceeded those within the carotid artery as well as those within the jugular vein, indicating that this hormone was released from the pituitary and carried directly through capillary beds of brain to the venous blood within the sagittal sinus. The experiment was repeated in one hypophysectomized sheep and, in this animal, ACTH concentration in the plasma was reduced, but that in the sagittal sinus still was elevated after the seizure, an indication that some ACTH (or ACTH-like material) was released from the brain itself.

Thyrotropin-releasing hormone selectively depresses glutamate excitation of cerebral cortical neurons.

The microiontophoretic application of thyrotropin-releasing hormone causes a selective reduction in neuronal excitation evoked by L-glutamate but not by acetylcholine in rat cerebral cortex. Thyrotropin-releasing hormone has no influence on the activity of acetylcholinesterase or on choline uptake and release from cerebral synaptosomes. This evidence for a selective interaction between a centrally acting peptide and an excitatory amino acid neurotransmitter may indicate a specific locus of thyrotropin-releasing hormone action at glutamate-activated receptor sites.

Endotoxin-stimulated opioid peptide secretion: two secretory pools and feedback control in vivo.

Small doses of endotoxin evoked a dramatic biphasic response of opioid peptide secretion into blood in sheep. The first phase began within minutes and coincided with a brief hypertensive response to endotoxin well before the appearance of fever or hypotension. The ratio of beta-endorphin to beta-lipotropin fell abruptly at the onset of the second phase of release, suggesting early depletion of a pool rich in beta-endorphin and subsequent emergence of a pool rich in unprocessed precursor. The concentration of cerebrospinal fluid opioids increased tenfold during the second phase. Naloxone administration augmented endotoxin-induced opioid secretion in both early and late phases, suggesting a short-loop feedback regulation of stress-induced endorphin secretion.

Significance of the biopharmaceutical properties of tramadol sustained-release formulations for chrono-pharmacologically optimized treatment of pain from various sources.

UNLABELLED: Tramadol is currently one of the most frequently used opioid analgesics in the world. OBJECTIVE: The objective of this study was to investigate the rate and extent of tramadol bioavailability following evening versus morning intake of an extended-release pellet system designed for once daily administration. Moreover, the suitability of the preparation for chrono-adjusted pharmacotherapy was to be investigated. METHODS: 18 male and female volunteers were enrolled in the study and treated with 200 mg tramadol extended-release capsules, which were to be taken in the fasted state between 7:30 and 8:00 a.m. or p.m., respectively. The parent compound and its O-desmethyl-metabolite were analyzed in plasma samples using a LC-MS/MS procedure. RESULTS: Maximum exposure of tramadol (geometric means of C(max)-values) was determined as 289.3 ng/ml after morning and 283.1 ng/ml after evening administration. Extent of tramadol exposure (geometric means of AUC(0-48)-values) was calculated as 4,802.5 ng x h/ml after morning and 4,767.0 ng x h/ml after evening administration. Also tmax-values were comparable after morning and evening administration (9.00 vs. 9.50 hours). Statistical analyses, based on conventional bioequivalence approach, revealed no evidence of any impact of the time-point of administration on the biopharmaceutical performance of the dosage form investigated here. CONCLUSIONS: Bioavailability of the extended-release tramadol capsules for once daily administration is not affected by the time-point of administration. Total and maximum exposure of the product was bioequivalent after intake in the morning and at night. Thus, the time-point of administration may be adjusted to the patient's needs without any significant change in the in-vivo performance.

Characterization of the behavior of alginate-based microcapsules in vitro and in vivo.

OBJECTIVES: Functional formulations providing protection of nutritional products against gastric juice or a capable of delivering them to distinct areas of the gastrointestinal tract are increasingly utilized by the food industry. However, the application of functional excipients that are established in pharmaceutical applications is limited in case of food products, as they are typically not classified as Generally Recognized As Safe (GRAS). MATERIALS: Accordingly, we investigated whether two alginate-based microcapsule preparations (capsule diameter 1 - 2 mm) either based on alginate and maize starch (MS-type) or alginate and casein (OCF27-type) and both created from ingredients classified as food supplements provide functional properties with respect to regional gastrointestinal targeting. METHODS: For this purpose the in vitro disintegration and swelling of the microcapsules was tested in various media. Furthermore, individual microcapsules, magnetically labelled with 100 - 200 microg black iron oxide, were ingested by healthy volunteers under fasting and fed conditions. Gastrointestinal transit as well as the gastrointestinal disintegration behavior were determined by using Magnetic Marker Monitoring. RESULTS: The results of in vitro and in vivo investigations show that both types of microcapsules are resistant to gastric juice for approximately 10 hrs under fasting and fed conditions. However, the disintegration characteristics of the two types of microcapsules within the intestines are different. CONCLUSION: Whilst the MS-type of capsules disintegrated predominantly within the small intestine shortly after gastric emptying, the OCF27-type of capsules underwent a rather slow disintegration predominantly in the colon.

Lipoprotein apheresis is an optimal therapeutic option to reduce increased Lp(a) levels.

BACKGROUND: Lipoprotein(a) (Lp(a)) is a genetic risk factor for cardiovascular disease (CVD) and is associated with the induction and sustaining of atherosclerotic cardiovascular diseases (ASCVD). Since 2008 Lp(a) along with progressive CVD has been approved as an indication for regular lipoprotein apheresis (LA) in Germany. The German Lipoprotein Apheresis Registry (GLAR) has been initiated to provide statistical evidence for the assessment of extracorporeal procedures to treat dyslipidemia for both LDL-cholesterol (LDL-C) and Lp(a). The GLAR now allows prospective investigations over a 5-year period about annual incidence rates of cardiovascular events. Here Lp(a) patients (LDL-C < 100 mg/dl; Lp(a) > 60 mg/dl or >120 nmol/l) showed the same reduction of major coronary (83%) and non-coronary events (63%) as had been formerly shown in the Pro(a)LiFe study. However, Lp(a) is not only an apolipoprotein(a) (apo(a)) and LDL-C containing particle, which is covalently bound to a LDL-C core by a disulphide bridge. The composition of this particle, inter alia containing oxidized phospholipids, gives pro-atherosclerotic, pro-inflammatory, and pro-thrombotic properties, inducing atherosclerotic processes mainly in the arterial wall. However, recent investigations have shown that a reduction of inflammatory settings without LDL-C or Lp(a) reduction may reduce ASCVD events. Lipoprotein apheresis (LA) could not only reduce LDL-C and Lp(a) in parallel, but also different inflammatory and coagulation parameters. In summary lipoprotein apheresis is not only anti-atherosclerotic, but also anti-inflammatory and anti-thrombotic and therefore an ideal treatment option with respect to the shown reduction of major adverse coronary events (MACE) and major adverse non-coronary events (MANCE) by reducing Lp(a) levels.

[Importance of the formulation for a chronopharmacologically optimised way of pain therapy. Results of a comparative bioavailability study of tramadol extended-release capsules after single-dose evening versus morning administration]. / Bedeutung der Galenik in der "chrono- adjustierten" Schmerzbehandlung. Ergebnisse einervergleichenden Bioverfügbarkeitsuntersuchung mit einem Tramadol- Retardpräparat nach Einmalgabe am Abend und am Morgen.

UNLABELLED: Objective of this study was to investigate the rate and extent of tramadol bioavailability following evening versus morning administration. METHODS: The study was performed following an open, randomised, cross-over study-design. 18 male and female volunteers were enrolled into the study and treated with 200 mg tramadol extended-release capsules (T-long), which were to be taken either in the morning or in the evening. RESULTS: Plasma concentration versus time profiles obtained after morning and evening administration were almost superimposable for both, tramadol and its active metabolite. Maximum exposure of tramadol and O-desmethyltramadol (geometric means of c(max)-values) as well as extent of exposure (geometric means of AUC(0-48)-values) were comparable after morning and eveningadministration. CONCLUSIONS: Time-point of administration does not have any relevant impact on the rate and extent of absorption in the investigated dosage form. Thus, time-point of administration may be adjusted to the patient's need in a chronopharmacologically optimised way for pain therapy.

Current insights into the German Lipoprotein Apheresis Registry (GLAR) - Almost 5 years on.

BACKGROUND: In recent years the Federal Joint Committee (G-BA), a paramount decision-making body of the German health care system required a reassessment of the approval of chronic lipoprotein apheresis therapy for regular reimbursement. Since 2005 an interdisciplinary German apheresis working group has been established by members of both German Societies of Nephrology. In 2009 the working group completed the indication for lipoprotein apheresis with respect to current cardiovascular guidelines and current scientific knowledge for the registry. In 2011 the German Lipoprotein Apheresis Registry (GLAR) was launched and data acquired over nearly 5 years can now be reported. METHODS AND RESULTS: All data were collected and analyzed during the time period 2012-2015. Over this time interval, 68 German apheresis centers collected retrospective and prospective observational data of 1.283 patients undergoing lipoprotein apheresis (LA) treatment of high LDL-cholesterol (LDL-C) levels and/or high lipoprotein(a) (Lp(a)) levels suffering from progressive cardiovascular disease (CVD). A total of 15,167 documented LA treatments were investigated. All patients treated by LA exhibited a median LDL-C reduction rate of 68.6%, and a median Lp(a) reduction rate of 70.4%. Analogue to the Pro(a)LiFe pattern, patient data were analyzed and compared with respect to the incidence rate of coronary events (MACE) 1 and 2 years before the start of LA treatment (y-2 and y-1) and prospectively one year on LA treatment (y+1). During the first year of LA treatment a MACE reduction of 97% was be observed. In the years considered, LA treatment side effects occurred at a low rate (ca. 5%) and mainly comprised puncture problems. CONCLUSIONS: For the first time data generated by the GLAR shows that LA lowers the incidence rate of cardiovascular events in patients with high LDL-C and/or high Lp(a) levels, progressive CVD and maximally tolerated lipid lowering medication. In addition LA treatments were found to be safe, exhibiting a low rate of side effects.

The German Lipoprotein Apheresis Registry (GLAR) - almost 5 years on.

BACKGROUND: Since 2005 an interdisciplinary German apheresis working group has been established by members of both German Societies of Nephrology and of Lipidologists and completed the data set for the registry according to the current guidelines and the German indication guideline for apheresis in 2009. In 2011 the German Lipoprotein Apheresis Registry (GLAR) was launched and data are available over nearly 5 years now. METHODS AND RESULTS: During the time period 2012-2016, 71 German apheresis centers collected retrospective and prospective observational data of 1435 patients undergoing lipoprotein apheresis (LA) treatment of high LDL-C levels and/or high Lp (a) levels suffering from cardiovascular disease (CVD) or progressive CVD. A total of 15,527 completely documented LA treatments were entered into the database. All patients treated by LA showed a median LDL-C reduction rate of 67.5%, and a median Lp (a) reduction rate of 71.1%. Analog to the Pro(a)LiFe pattern, patient data were analyzed to the incidence rate of coronary events (MACE) 1 and 2 years before the beginning of LA treatment (y-2 and y­1) and prospectively two years on LA treatment (y + 1 and y + 2). During two years of LA treatment a MACE reduction of 78% was observed. In the years considered, side effects of LA treatment were low (5.9%) and mainly comprised puncture problems. CONCLUSIONS: The data generated by the GLAR shows that LA lowers the incidence rate of cardiovascular events in patients with high LDL-C and/or high Lp (a) levels, progressive CVD, and maximally tolerated lipid lowering medication. In addition, LA treatments were found to be safe with a low rate of side effects.

Significant food interactions observed with a nifedipine modified-release formulation marketed in the European Union.

The objective of this study was to compare the rate and extent of nifedipine bioavailability after single dose administration of Adalat OROS 30 (Reference) and Nifedipine Sandoz retard 30 tablets (Test). Both modified release formulations are marketed in Member States of the European Union. Prior to the clinical study the in vitro dissolution characteristics were investigated. There was a significant pH dependency observed with the Test product but drug release with the Reference product was almost independent of the experimental conditions used. In the subsequent open, randomized, controlled, 4-way crossover study both pharmaceutical products were administered to 28 healthy male volunteers, either after fasting overnight or immediately after a high-fat American breakfast. Blood sampling was performed over 48 hours post-dose for the determination of pharmacokinetic profiles of nifedipine. Considerable differences were observed between the two formulations when administered to fasted subjects where maximum nifedipine plasma concentration (C(max)) were higher in the case of the Test formulation. Differences were even more pronounced after a high-fat American breakfast. Under these conditions a significant food interaction was detected in the case of Nifedipine Sandoz retard 30 with a three-fold increase in the mean C(max) when compared to values obtained in fasting subjects. In contrast, food intake had no clinically relevant effect on bioavailability of nifedipine (rate and extent) in the case of Adalat OROS 30. The pharmacokinetic findings in this study were reflected in the adverse event pattern which indicated a potential tolerability problem in the case of Nifedipine Sandoz retard 30. The results confirm the relationship between the in vitro dissolution profile results and the effects of the drug in vivo. Dose dumping after intake of a high-fat meal could be shown. Nifedipine Sandoz retard 30 is not bioequivalent to Adalat OROS 30 and produced highly variable and poorly predictable nifedipine plasma concentrations. The differences observed between the two products investigated may have direct therapeutic relevance when switching from one formulation to the other and, in particular, when administration conditions change i.e. administration in the fasting state and administration with a meal, since the pharmacological and therapeutic actions of nifedipine are closely associated with the concentration.

Felodipine quantification in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry.

A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C(18) analytical column (100 mm x 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL(-1) (r(2) > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL(-1)), 7.1% (0.6 ng mL(-1)) and 6.8% (7.5 ng mL(-1)). The between-run accuracy was +/- 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.

Connections of the hippocampal formation in humans: I. The mossy fiber pathway.

The hippocampal formation has been one of the most extensively studied cortical regions in rats, yet little is known about the anatomical connections of the hippocampus in primates, especially humans. With the use of an antibody against the calcium-binding protein, calbindin-D28K, in normal autopsy tissue and the neuronal tracers biocytin or biotinylated dextrans in in vitro slice preparations from tissue removed during surgery for intractable epilepsy, we examined the human hippocampal mossy fiber pathway. The injections of biocytin into the dentate granule cell layer labeled neurons in a Golgi-like manner, revealing the presence of basal dendrites on about 30% of the granule cells. The granule cell axons, the mossy fibers, initially formed a diffuse plexus of fibers in the polymorphic layer before organizing into fiber fascicles in the hilar pyramidal region. These fiber fascicles were much more prominent rostrally than caudally. Within the hilus and proximal portions of the extrahilar CA3 field, the mossy fibers ran through the pyramidal cell layer, and while near the transition to field CA2, the fibers turned superficially and crossed the pyramidal layer to run in the stratum lucidum. All of these features, seen following injections of tracer into hippocampal slices from the brains of epileptics, were confirmed by calbindin-staining of mossy fibers in normal brains. Biocytin-labeled mossy fiber axons revealed two characteristic types of enlargements: small varicosities and larger expansions. The expansions were found throughout the neuropil and were highly irregular, diaminobenzidine-dense profiles that had pleiomorphic modes of attachment to the parent axon. Electron microscopic images of these biocytin labeled expansions revealed that they were large synaptic boutons bearing asymmetric synapses. This study indicates that the human mossy fiber pathway shows some minor deviations from the rodent brain but little difference from monkeys. We argue that these changes mirror a phylogenetic growth of the CA3 pyramidal neurons (subfield CA3c) into the hilus rather than an evolutionary change of the mossy fiber pathway. This growth of subfield CA3c and the increase in mossy fibers running through the pyramidal layer (and a presumed accompanying increase in proximal basal dendritic contacts) may reflect a growing role of the projection from the dentate granule cells to subfield CA3c and from there to field CA1 in the primate hippocampus.

Connections of the hippocampal formation in humans: II. The endfolial fiber pathway.

We investigated the anatomical connections of the pyramidal neurons located within the hilar region of the dentate gyrus of the human hippocampus, neurons which do not have a rodent equivalent. The myeloarchitectural patterns of the human hippocampus indicated the presence of a distinct fiber pathway, the endfolial fiber pathway, in the stratum oriens of the hilus and field CA3. By using the fluorescent lipophilic dye DiI in formalin-fixed human hippocampal tissue, we demonstrated that this is a continuous fiber pathway between the deep hilar region and CA2. This fiber pathway did not enter the fimbria or alveus along the entire distance of the traced pathway and ran exclusively in the stratum oriens of the hilus and CA3. Tracing studies with biocytin in in vitro human hippocampal slices indicated that the hilar and CA3 pyramidal neurons contributed to this pathway. Out distally in field CA3, the long transverse fibers became short and choppy, suggesting that they were beginning to move out of the plane of the tissue slice. Numerous fibers from this pathway were seen crossing the pyramidal layer. Based on comparative studies, we propose that the endfolial fiber system is a component of the hilar Schaffer collateral system in humans. The presence of a significant Schaffer collateral system from the pyramidal neurons in the hilar region would indicate that these neurons are anatomically related to the CA3 pyramidal neurons. Therefore, we suggest the inclusion of the human hilar pyramidal neurons within Lorente de No's field CA3 and, in particular, within subfield CA3c.

Body composition, resting metabolic rate, and energy requirements of short- and normal-stature, low-income Guatemalan children.

We examined body composition using bioelectrical impedance analysis and isotope dilution (18O and 2H), resting metabolic rate (RMR) by indirect calorimetry, and total energy expenditure (TEE) by doubly labeled water in 15 short-stature (height-for-age < or = -1.5 SD) and 15 normal-stature (height-for-age > -1.5 SD) Guatemalan children aged 4-6 y. Although, in absolute terms significant group differences were found in fat-free mass (FFM), fat mass, and total body water (TBW), there were no significant differences in fat mass and TBW after adjustment for FFM. RMR of the short-stature children (3791 +/- 376 kJ/d) was not significantly different from that of normal-stature children (4038 +/- 531 kJ/d), and the regression between RMR and FFM was also not significantly different between groups. TEE was not significantly different in short-stature (4753 +/- 761 kJ/d) compared with normal-stature children (5304 +/- 1020 kJ/d); the regression between TEE and FFM was not significantly different between the two groups. There were no significant group differences in RMR and TEE after adjustment for FFM. FFM was the strongest predictor of TEE, but could only explain 29% of the variance. We conclude that 1) the lower TBW and fat mass in the short-stature group is proportional to their lower FFM, 2) there is no significant difference in either RMR or TEE between short- and normal-stature children, and 3) TEE is highly variable among these children and cannot be explained by differences in body size alone.

Neurophysiologic correlates of implicit face memory in intracranial visual evoked potentials.

Visual evoked potentials were recorded in the amygdala, hippocampus, mid- and inferotemporal cortex, orbitofrontal cortex, and lateral frontal cortex of seven epileptic patients while they were engaged in a difficult task requiring the discrimination between repeated and nonrepeated faces. The explicit recognition of previously seen faces was at chance levels, as measured by the accuracy of push-button responses. Nevertheless, all subjects showed clear-cut differential evoked responses to repeated versus nonrepeated faces, indicating implicit encoding of the distinction between the two types of stimuli. Differential responses were more frequent in neocortical recording sites (especially in the mid- and inferotemporal leads) than in limbic recording sites such as the amygdala and hippocampus. The authors conclude that implicit encoding processes are modulated by neocortical visual association areas of the temporal lobes.