Development of molecular hydrogen-bonding potentials (MHBPs) and their application to structure-permeation relations.

Hydrogen bonds are major forces of recognition in biochemistry and molecular pharmacology; they are an essential component of intermolecular interactions and determine to a significant extent the 3D-structure of bio-macromolecules. To explore three-dimensional H-bonding properties, a new tool called Molecular Hydrogen-Bonding Potentials (MHBPs) was created. The development of this tool is based on a stepwise procedure similar to the one used successfully to generate the Molecular Lipophilicity Potential (MLP). First, a H-bonding fragmental system was developed starting from published solvatochromic parameters. An atomic H-bonding donor fragmental value (alpha) is associated to each hydrogen atom in a polar moiety. Similarly, an atomic H-bonding acceptor fragmental value (beta) is associated to each polar atom. A distance function and an angle function were defined to take into account variations of the MHBPs in space. The fragmental system and the geometric functions were then combined to generate the MHBPs. These are calculated at each point of an adequate molecular surface or on a three-dimensional grid. The MHBPs were compared with GRID interactions energies and correlated with success to oral drug absorption data. Available examples demonstrate that the MHBPs are a promising computational tool in drug design. Their combination with CoMFA and VolSurf is being studied.

Lipophilicity behaviour of the Zwitterionic antihistamine cetirizine in phosphatidylcholine liposomes/water systems.

PURPOSE: The partitioning of cetirizine in a phosphatidylcholine liposomes/water system was compared with that of hydroxyzine and acrivastine to gain insight into the mechanisms of interaction of its various electrical species with membranes. METHODS: The lipophilicity profiles of the compounds were obtained from equilibrium dialysis and potentiometry, and compared with changes in NMR relaxation rates. RESULTS: The neutral form of hydroxyzine interacted mainly via hydrophobic interactions with the bilayer lipid core of the membrane, whereas for the cationic form both hydrophobic and electrostatic interactions were involved. Zwitterionic and anionic cetirizine were less lipophilic than its cation, which behaved like the corresponding species of hydroxyzine. Zwitterionic cetirizine interacted more by weak electrostatic interactions with the polar headgroups of phospholipids than by hydrophobic interactions with the membrane interior. The lipophilicity of its anion reflected the balance of repulsive electrostatic interactions between the carboxylate and phosphate groups and the hydrophobic interactions with the lipid core. CONCLUSION: The study confirms that various mechanisms influence the interaction of solutes with liposomes. Combining experimental techniques and using suitable reference compounds proves useful.

Evaluation and prediction of drug permeation.

A major challenge confronting the pharmaceutical scientist is to optimize the selective and efficient delivery of new active entities and drug candidates. Successful drug development requires not only optimization of specific and potent pharmacodynamic activity, but also efficient delivery to the target site. Following advances in rational drug design, combinatorial chemistry and high-throughput screening techniques, the number of newly discovered and promising active compounds has increased dramatically in recent years, often making delivery problems the rate-limiting step in drug research. To overcome these problems, a good knowledge of the pharmacokinetic barriers encountered by bioactive compounds is required. This review gives an overview of the properties of relevant physiological barriers and presents some important biological models for evaluation of drug permeation and transport. Physicochemical determinants in drug permeation and the relevance of quantitative and qualitative approaches to the prediction and evaluation of passive drug absorption are also discussed.

Molecular properties and pharmacokinetic behavior of cetirizine, a zwitterionic H1-receptor antagonist.

The ionization and lipophilicity behavior of the antihistamine (H1-receptor antagonist) cetirizine was investigated, showing the drug to exist almost exclusively as a zwitterion in the pH region 3.5-7.5. In this pH range, its octanol/water lipophilicity is constant and low compared to cationic antihistamines (log D = log PZ = 1.5), whereas its H-bonding capacity is relatively large (delta log PZ > or = 3.1). Conformational, electronic, and lipophilicity potential calculations revealed that zwitterionic cetirizine experiences partial intramolecular charge neutralization in folded conformers of lower polarity. Pharmacokinetic investigations have shown the drug to be highly bound to blood proteins, mainly serum albumin, and to have a low brain uptake, explaining its lack of sedative effects. As such, cetirizine does not differ from "second-generation" antihistamines. In contrast, its very low apparent volume of distribution in humans (0.4 L kg-1, smaller than that of exchangeable water) implies a low affinity for lean tissues such as the myocardium and is compatible with the absence of cardiotoxicity of the drug. The zwitterionic nature and modest lipophilicity of cetirizine may account for this pharmacokinetic behavior. The suggestion is offered that cetirizine and analogous zwitterions, whose physicochemical, pharmacokinetic, and pharmacodynamic properties differ from those of "first-" and "second-generation" drugs in this class, could be considered as "third-generation" antihistamines.

Blood-to-brain transfer of various oxicams: effects of plasma binding on their brain delivery.

PURPOSE: The objective of this work was to assess the influence of binding to plasma proteins and to serum on the brain extraction of four antiinflammatory oxicams. METHODS: The brain extraction of isoxicam, tenoxicam, meloxicam and piroxicam was investigated in rats using the carotid injection technique. Blood protein binding parameters were determined by equilibrium dialysis using human serum, human serum albumin (HSA) and alpha-l-acid glycoprotein (AAG) solutions at various concentrations. RESULTS: All oxicams had low values of brain extraction, between 19% and 39% when dissolved in serum, i.e. under physiological conditions. Brain efflux rate constants calculated from the wash-out curves were the same in the absence or presence of serum. Brain efflux was inversely related to the polarity of the oxicams, such that the higher their H-bonding capacity, the lower their brain efflux. The free dialyzable drug fraction was inversely related to protein concentration. However, rat brain extraction was always higher than expected from in vitro measurements of the dialyzable fraction. CONCLUSIONS: Except for piroxicam whose brain extraction was partially decreased in the presence of proteins, the serum unbound and initially bound fractions of oxicams both seem available for transfer into the brain. Modest affinities for AAG rule out any related effect. More surprising is the apparent lack of effect on brain transfer of the high-affinity binding to HSA and serum. The enhanced brain uptake of meloxicam in the presence of AAG could be a result of interactions between this globular protein and the endothelial wall.

Spurious elevation of plasma immunoreactive adrenocorticotropic hormone in cyclic Cushing's syndrome.

We studied a 40-year-old woman with cyclic Cushing's syndrome who demonstrated abnormal high-dose dexamethasone suppression and metyrapone stimulation tests. These results, associated with persistent elevations of plasma adrenocorticotropic hormone (ACTH) levels, suggested ectopic secretion of ACTH. Surprisingly, an adrenal adenoma with atrophy of the contralateral adrenal gland was found at exploratory laparotomy. Subsequent ACTH determinations that extract ACTH from the plasma before assay suggested that the apparent increase in ACTH concentration in our routine assay was due to the presence of an interfering substance(s). We conclude that the diagnosis of Cushing's syndrome continues to depend on a battery of adrenal function tests and radiographic procedures and recommend that measurements of ACTH be performed only after extraction of ACTH from specimens.

Peripheral resistance to thyroid hormone in a family: heterogeneity of clinical presentation.

We describe a family with thyroid hormone resistance. Juvenile Graves disease was diagnosed in the propositus, an 8-month-old boy. He was initially given propylthiouracil, and at 22 months of age underwent subtotal thyroidectomy. A diagnosis of hyperthyroidism was made in a younger sister at 3 months of age. Because of the unusual occurrence of juvenile Graves disease in two siblings, we evaluated the parents. The mother was euthyroid on physical examination and by thyroid hormone measurements. The father, although clinically euthyroid, had markedly elevated thyroid hormone values. In the three affected members, serum thyrotropin concentrations and results of thyrotropin-releasing hormone infusion tests were inappropriate for the elevated serum thyroid hormone levels. The father was given increasing doses of triiodothyronine. Complete suppression of TRH-induced TSH release did not occur until a daily dose of 300 micrograms triiodothyronine was administered. Furthermore, this large dose of T3 did not produce clinical evidence of hyperthyroidism or result in changes in his systolic ejection time intervals. This family therefore had the unusual feature of clinical heterogeneity. The two children had mainly pituitary resistance to thyroid hormone and were hyperthyroid; the euthyroid father, on the other hand, had generalized tissue resistance to thyroid hormone.

Ketotic hypoglycaemia.

Ketotic hypoglycaemia is the most common form of childhood hypoglycaemia. This disorder classically manifests itself between the ages of 18 months and 5 years, and generally remits spontaneously before 8 or 9 years of age. A presumptive diagnosis is made by documenting a low blood sugar in association with ketonuria, ketonaemia and typical symptoms of hypoglycaemia. The definitive diagnosis is established by demonstrating an inability to tolerate a provocative ketogenic diet, or a fast. Susceptible or affected children develop severe hypoglycaemia and ketosis on this diet within 24 hours. Plasma alanine concentrations on either a normal or ketogenic diet were significantly lower in ketotic hypoglycaemic children compared with normal children. In contrast to adults, even normal children develop hypoglycaemia and ketonaemia when calorically deprived for relatively short periods of time (32 to 36 hrs).

Constant intravenous insulin infusion during labor and delivery in diabetes mellitus.

We treated 30 diabetic women (31 pregnancies) during the peripartum period with a continuous insulin infusion. A mean infusion rate of 1.0 micron/h maintained the mean plasma glucose concentration below 100 mg/dl in 84% of the patients; the plasma glucose concentration was below 100 mg/dl within an hour of delivery in 71% of the women. Mild hypoglycemia developed during the infusion in three women and after delivery in another patient. Only two infants of the diabetic mothers developed transient and asymptomatic hypoglycemia. We conclude that continuous insulin infusion is a practical, safe, and effective method for treating diabetic mothers during the peripartum period and suggest that this technique may decrease the frequency and severity of neonatal hypoglycemia.

Differences in circulating gluconeogenic substrates during short-term fasting in men, women, and children.

To determine whether the differences in fasting glucose responses in men, women and children could be related to differences in glucoregulatory hormone secretion or availability of circulating gluconeogenic substrates, 20 adults (10 men and 10 women) were fasted for 86 hr and 15 children (6.1 +/- 0.8 yr, mean +/- SE) were fasted for 30 hr. Circulating concentrations of glucose, ketone bodies, potential gluconeogenic substrates and glucoregulatory hormones were determined at frequent intervals. In the postprandial state (1-14 hr fasting), substrate and hormone concentrations were similar in all groups with the exception of plasma glutamine concentrations which were higher in men (640 +/- 20 microM) than in women of children (490 +/- 20 microM and 480 +/- 50 microM, respectively, p less than 0.01 for both). Following 30 hr fasting children had the lowest glucose (53 +/- 3 mg/dl) and alanine (167 +/- 17 microM) concentrations and men had the highest (72 +/- 3 mg/dl and 279 +/- 16 microM, respectively) whereas those of women were intermediate (64 +/- 3 mg/dl and 226 +/- 19 microM. respectively). Following 30 hr of fasting betahydroxybutyrate concentrations were highest in children and lowest in men (the children, 3.7 +/- 0.4 mM; women, 1.7 +/- 0.2 mM and men 0.9 +/- 0.2 mM, p less than 0.02 between all groups). An inverse relationship (p less than 0.001 was observed between beta-hydroxybutyrate and glucose concentrations throughout the fast for each group (r greater than 0.92). Although plasma cortisol concentrations were higher in women and children when compared to those of the men, no differences in growth hormone, glucagon, or insulin concentrations were observed among the three groups studied. Differences in plasma substrate responses to fasting among the groups studied may reflect differences in glucose requirements among men, women and children, as well as the relative availability of both gluconeogenic substrate and ketone bodies.

Elevation of the free thyroxine index in psychiatric patients.

We calculated a free triiodothyronine (FT3) index on 124 patients who exhibited symptoms and signs of hyperthyroidism and elevations of the free thyroxine (FT4) index on initial screening. A thyrotropin-releasing hormone (TRH) test served as the final arbiter of thyroid function if the clinical presentation was not characteristic of hyperthyroidism or if the FT3 index was not elevated. Forty-one of the 124 patients had normal TRH tests and were thus classified as euthyroid. Of these patients with the so-called euthyroid sick syndrome, 23 had psychiatric disorders. In a separate study, we measured a FT4 index on 100 unselected admissions to the psychiatric ward. Of six patients with elevated FT4 values, only one had hyperthyroidism. We conclude that false positive FT4 index elevations occur commonly in psychiatric patients. The mechanism(s) for the FT4 index elevations remain obscure. Despite some limitations, the TRH test is a valuable diagnostic adjunct for diagnosing hyperthyroidism in the mentally ill patient when other tests and serial observations are inconclusive.

Thyroxine toxicosis. A common variant of hyperthyroidism.

To determine the frequency of thyroxine (T4) toxicosis, we calculated a free triiodothyronine index (FT3I) on 124 patients who displayed elevations of the free T4 index (FT4I) on initial screening. If the clinical presentation was not characteristic of hyperthyroidism or if the FT3I was not elevated, a thyrotropin-releasing hormone (TRH) test was performed. Of 83 hyperthryoid patients, 70 displayed elevations of both the FT4 and FT3 indices. Thirteen patients, however, had elevations of the FT4I alone. This frequency of T4 toxicosis is higher than generally appreciated. Forty-one patients, most of whom were ill with nonthyroidal diseases, had normal TRH test results and were classified euthyroid. We conclude that FT4I elevations owing to T4 toxicosis and nonthyroidal illnesses are common and that the laboratory differentiation between the syndromes may be difficult.

Adaptations of alpha2- and beta-cells of rat and mouse pancreatic islets to starvation, to refeeding after starvation, and to obesity.

The effects of starvation and refeeding and of obesity on pancreatic alpha2- and beta-cell responses to glucose or tolbutamide were studied with the isolated rat or mouse pancreas perfused with an amino acid mixture in the presence and absence of glucose. It was observed that the physiological adaptation to a regimen of fasting and realimentation and to obesity differed greatly in the two types of endocrine cells. Whereas beta-cells of rats showed a dramatic reduction of glucose- and tolbutamide-stimulated insulin release during starvation that was reversed by refeeding, alpha2-cells preserved their response to stimulators and inhibitors during this experimental manipulation. Amino acid stimulation of glucagon release occurred equally well with the pancreas from fed and starved rats and was suppressed efficiently by glucose and tolbutamide in both nutritional states. Surprisingly, the rate of onset of glucose suppression of alpha2-cells was significantly higher in the fasted than in the fed state. This glucose hypersensitivity was apparent 2 d after after food deprivation and had disappeared again on the 2nd d of refeeding. In the pancreas from animals starved for 3 d, glucose and tolbutamide suppression of alpha2-cells took place in the absence of demonstrable changes of insulin release. In the isolated perfused pancreas taken from the hyperphagic obese hyperglycemic mouse (C57 Black/6J; ob/ob), the observed rate of insulin secretion as a result of a combined stimulus of amino acids and glucose and of glucagon release stimulated by amino acids was about four times higher than achieved by the pancreas of lean controls. However, glucose was unable to suppress the alpha2-cells in the pancreas of obese animals, in spite of the hypersection of the beta-cells, again in contrast to the alpha2-cells of controls that were readily inhibited by glucose. These data imply that the acute suppression of alpha2-cells by glucose is largely independent of a concomitant surge of extracellular insulin levels and that the adaptation of the islet organ to starvation leads to decreased glucose sensitivity of beta-cells, which contrasts with an improved glucose responsiveness of alpha2-cells. However, hyperphagia, which is assumed to be the primary abnormality in the ob/ob mouse, leads to overproduction of insulin and glucagon by the pancreas while greatly reducing the alpha2-cell sensitivity to glucose. An attempt is made to incorporate these data on starvation, refeeding, and obesity, as well as previous results with experimental diabetes, in a comprehensive picture describing a regulative principle underlying the glucose responsivness of alpha2-cells.

Defective activation of the pyruvate dehydrogenase complex in subacute necrotizing encephalomyelopathy (Leigh disease).

Autopsy examination confirmed the diagnosis of subacute necrotizing encephalomyelopathy (SNE) in a 7-month-old male infant who underwent several metabolic studies before death. Intermittent lactic acidemia and fumaric aciduria, an extreme hyperglycemic response to an intravenous bolus of alanine, and an elevated total body flux rate of glucose (58.4 mumoles . kg-1 . min-1) suggested a disturbance in the oxidative decarboxylation of pyruvate. Enzymological studies of postmortem samples revealed low nonactivated pyruvate dehydrogenase activity in liver (19.4%) and brain (53.8%). The lowest brain pyruvate dehydrogenase activities were noted in the midbrain and pontine regions. Supramaximal activation of the hepatic pyruvate dehydrogenase complex (135% of control values) occurred in vitro. Spontaneous reactivation following in vitro inactivation of the complex with adenosine triphosphate was significantly less (p less than 0.02) in the patient's samples compared to controls. The biochemical defect was not apparent in fibroblasts. These enzymological observations point to an in vivo defect in the activation mechanism of the pyruvate dehydrogenase complex as the biochemical disturbance in SNE. The findings suggest that dichloroacetate may be beneficial in treating SNE.

Starvation diabetes in the rat: onset, recovery, and specificity of reduced responsiveness of pancreatic beta-cells.

A 24-h starvation markedly diminished the stimulant action of 8 mM glucose on insulin secretion from isolated perifused rat islets of Langerhans. The response to a supramaximal glucose stimulus (27.5 mM) remained normal, but prolonged fasting (48 or more) also reduced its efficacy. Refeeding of 24-h fasted animals resulted in complete restoration of glucose sensitivity within 24 h. The responses to glyceraldehyde (2 mM) and alpha-ketoisocaproate (8 mM) at concentrations which elicit approximately half-maximal stimulation were unaltered by a 24-h fast, while that to a half-maximally effective dose of mannose (15 mM) was decreased. Theophylline (5 mM) could not normalize the reduced secretory response to glucose seen in this state. The islets' ability to metabolize glucose, using various in vitro pretreatment protocols and different incubation times, was not affected by a 24-h fast. Mannose and glyceraldehyde metabolism were also unaltered. Prolonged fasting (48 h) reduced glucose metabolism by 25% at both 8 and 27.5 mM. The acute adaptive changes in islet sensitivity to moderate glucose and mannose concentrations during short term fasting (24 h) cannot be explained by an altered usage of the added hexoses.

Cure of acromegaly by operative removal of an islet cell tumor of the pancreas.

We studied a 30 year old woman in whom acromegaly was cured by operative removal of a large cystic beta cell adenoma of the pancreas. We detected substantial amounts of immunoreactive human growth hormone (hGH)-like activity in a tumor tissue extract. Extracts of the tumor and a normal human pituitary gland eluted from a Sephadex G-75 column in two identical peaks. Serial dilutions of the tumor extract displaced radioactive 125I hGH parallel to a standard curve. Surprisingly, an extract of a normal human pancreas contained large amounts of hGH-like activity and gave results similar to those of the tumor extract on gel chromatography and on serial dilution displacement in the growth hormone immunoassay. Paper electrophoretic studies of 125I hGH after incubation with normal pancreatic and tumor extracts with and without enzyme inhibitors suggested that pancreatic proteolytic enzymes damaged the 125I hGH used in growth hormone radioimmunoassay and produced a false detection of hGH.