Biomedical subjects
B D Anderson
Publications and source records attributed to B D Anderson.
Permeability of acetic acid across gel and liquid-crystalline lipid bilayers conforms to free-surface-area theory.
Solubility-diffusion theory, which treats the lipid bilayer membrane as a bulk lipid solvent into which permeants must partition and diffuse across, fails to account for the effects of lipid bilayer chain order on the permeability coefficient of any given permeant. This study addresses the scaling factor that must be applied to predictions from solubility-diffusion theory to correct for chain ordering. The effects of bilayer chemical composition, temperature, and phase structure on the permeability coefficient (Pm) of acetic acid were investigated in large unilamellar vesicles by a combined method of NMR line broadening and dynamic light scattering. Permeability values were obtained in distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dilauroylphosphatidylcholine bilayers, and their mixtures with cholesterol, at various temperatures both above and below the gel-->liquid-crystalline phase transition temperatures (Tm). A new scaling factor, the permeability decrement f, is introduced to account for the decrease in permeability coefficient from that predicted by solubility-diffusion theory owing to chain ordering in lipid bilayers. Values of f were obtained by division of the observed Pm by the permeability coefficient predicted from a bulk solubility-diffusion model. In liquid-crystalline phases, a strong correlation (r = 0.94) between f and the normalized surface density sigma was obtained: in f = 5.3 - 10.6 sigma. Activation energies (Ea) for the permeability of acetic acid decreased with decreasing phospholipid chain length and correlated with the sensitivity of chain ordering to temperature, [symbol: see text] sigma/[symbol: see text](1/T), as chain length was varied. Pm values decreased abruptly at temperatures below the main phase transition temperatures in pure dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers (30-60-fold) and below the pretransition in dipalmitoylphosphatidylcholine bilayers (8-fold), and the linear relationship between in f and sigma established for liquid-crystalline bilayers was no longer followed. However, in both gel and liquid-crystalline phases in f was found to exhibit an inverse correlation with free surface area (in f = -0.31 - 29.1/af, where af is the average free area (in square angstroms) per lipid molecule). Thus, the lipid bilayer permeability of acetic acid can be predicted from the relevant chain-packing properties in the bilayer (free surface area), regardless of whether chain ordering is varied by changes in temperature, lipid chain length, cholesterol concentration, or bilayer phase structure, provided that temperature effects on permeant dehydration and diffusion and the chain-length effects on bilayer barrier thickness are properly taken into account.
Prospective multicenter study of sulfonylurea ingestion in children.
OBJECTIVE: Sixty-eight percent of pediatric sulfonylurea ingestions reported to poison centers do not result in laboratory or behavioral effects. Consequently, if all exposed children are admitted overnight or for 24 hours for these exposures, it will result in 600 to 700 hospital admissions per year of children who will remain free of symptoms. We prospectively studied exposures reported to 10 regional poison centers to determine if it were possible to differentiate those patients who would have symptoms from those who would remain symptom free. METHODS: We analyzed all sulfonylurea exposures in children < or = 12 years old reported to the participating poison centers. Hypoglycemia was defined as blood glucose (BG) concentration < 60 mg/dl. RESULTS: Hypoglycemia developed in 56 (30%) of 185 exposed patients. Fifty-four of the 56 (96%) hypoglycemic patients had development of hypoglycemia within 8 hours of ingestion. Eighty-seven of the patients were initially managed with oral supplementation only; in 13 cases, treatment advanced to intravenous administration of glucose or glucagon with the onset of hypoglycemia. There was no statistical difference in medical outcome between patients monitored during oral supplementation versus during intravenous infusion of dextrose. Ingestions analyzed by time of day did not predict risk of hypoglycemia. Sufficient data were available for 103 (58%) of the 177 patients who ingested glyburide or glipizide to calculate a toxic dose/weight ratio. Of these 103 patients, 31 of 36 patients who ingested < or = 0.3 mg/kg remained symptom free, whereas 31 of 67 who ingested more than 0.3 mg/kg had BG concentrations < 60 mg/dl (p < 0.005, 95% confidence interval 0.05 to 0.58; sensitivity 86%, specificity 46%). CONCLUSION: A lack of onset of hypoglycemia (BG > 60 mg/dl) in the first 8 hours after ingestion is predictive of a benign outcome in accidental pediatric sulfonylurea ingestion. Clinical observation of children for onset of hypoglycemia during oral feeding alone appears safe. Some children with symptoms of hypoglycemia need to receive intravenous dextrose therapy. Time of day of ingestion is not predictive of risk of hypoglycemia. Finally, at this time it appears inappropriate to use a milligram per kilogram body weight dose as a guide for management decisions.
Solid-state stability of human insulin. II. Effect of water on reactive intermediate partitioning in lyophiles from pH 2-5 solutions: stabilization against covalent dimer formation.
Previous studies have established that at low pH human insulin decomposition proceeds through a two-step mechanism involving rate-limiting intramolecular formation of a cyclic anhydride intermediate at the C-terminal AsnA21 followed by intermediate partitioning to various products, most notably desamido insulin and covalent dimers, in both aqueous solution and in the amorphous (lyophilized) solid state. This study examines the product distribution resulting from insulin degradation in lyophilized powders as a function of water content and the phase behavior of the solid (glassy versus rubbery) between pH 3 and 5. In amorphous solids at low water content (glassy state), the cyclic anhydride intermediate of insulin reacts predominantly with water to form deamidated insulin, whereas the intermolecular reaction with another insulin molecule to form a covalent dimer accounts for < or = 15% of the total degradation. Increasing water content reduces the glass transition temperature of insulin to < 35 degrees C, and covalent dimer formation becomes increasingly favored relative to deamidation. An increase in solid-state pH also favors dimerization as deprotonation of the terminal amino groups of insulin renders them more nucleophilic. Covalent dimerization was almost totally suppressed by incorporation into a glassy matrix of trehalose, which both minimizes molecular mobility and physically separates the insulin molecules. The kinetics and product distribution of human insulin in lyophilized powders between pH 3 and 5 illustrate the differential sensitivities of various solid-state reaction types to the effects of water activity and solid-phase behavior. The intramolecular cyclization at the AsnA21 position requires only short-range conformational flexibility and thus is only modestly restricted even in the glassy state. On the other hand, the competing bimolecular reactions involving either water or another molecule of insulin combining with the intermediate anhydride are dependent on molecular mobility of the reactants, in accord with predictions of free volume theory. In the glassy state, deamidation (reaction with water) is favored because of the restricted molecular mobility of proteins in rigid matrices. Increasing plasticization with increasing water content favors covalent aggregate formation because of the higher dependence of protein mobility on free volume within the solid matrix.
Role of brain tissue localized purine metabolizing enzymes in the central nervous system delivery of anti-HIV agents 2'-beta-fluoro-2',3'-dideoxyinosine and 2'-beta-fluoro-2',3'-dideoxyadenosine in rats.
PURPOSE: This study examines the central nervous system (CNS) delivery of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA) and 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), acid stable analogues of dideoxyadenosine (ddA) and dideoxyinosine (ddI) having reduced susceptibility to purine salvage pathway enzymes important in the metabolism of ddA and ddI, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), respectively. Their CNS delivery compared to that for ddI provides insight into the role of brain tissue ADA and PNP in these processes. METHODS: Brain and cerebrospinal fluid (CSF) concentration-time profiles were obtained for F-ddI during and after intravenous infusions of F-ddI, and for both F-ddA and F-ddI after F-ddA infusions in normal rats or rats pre-treated with the ADA inhibitor 2'-deoxycoformycin (DCF). Rate constants for CNS entry, efflux and metabolism were estimated by computer fits using plasma concentration-time profiles as the driving force functions. RESULTS: The CNS delivery of F-ddI did not differ significantly from that for ddI. F-ddA, which is more lipophilic than F-ddI, provided higher brain (approximately 8x) and CSF (approximately 11x) concentrations of total dideoxynucleoside (F-ddA and F-ddI) compared to F-ddI. Deamination by brain tissue ADA to form F-ddI reduced CNS levels of intact F-ddA but provided higher brain parenchyma (5x) and CSF/plasma (3x) ratios of F-ddI relative to F-ddI controls. Thus, F-ddA functions in part as a CNS-activated prodrug of F-ddI. DCF pre-treatment inhibited brain tissue ADA, abolishing the prodrug effect, and enhancing F-ddA concentrations in both brain parenchyma (5x) and CSF (6x). CONCLUSIONS: PNP metabolism does not appear to play a role in the low CNS delivery of ddI. On the other hand, deamination of F-ddA by brain tissue ADA is an important process, such that F-ddA functions in part as a CNS-activated prodrug of F-ddI. Enhanced CNS uptake of intact F-ddA can be achieved with ADA inhibition.
Localization of purine metabolizing enzymes in bovine brain microvessel endothelial cells: an enzymatic blood-brain barrier for dideoxynucleosides?
PURPOSE: The specific activities of the purine and pyrimidine metabolizing enzymes, purine nucleoside phosphorylase (PNP), adenosine deaminase (ADA) and cytidine deaminase (CDA) were determined in bovine brain microvessel endothelial cells (BBMECs), whole cerebral tissue and erythrocytes. In addition, the substrate specificities (Km and Vmax) of purified calf spleen PNP for inosine and 2',3'-dideoxyinosine (ddI) and of purified calf intestinal ADA for 2',3'-dideoxyadenosine (ddA), 6-chloro-2',3'-dideoxypurine (6-Cl-ddP), and 2'-beta-fluoro-2', 3'-dideoxyadenosine (F-ddA) have been explored. METHODS: BBMECs were isolated from bovine cerebral cortex by a two step enzymatic dispersion treatment followed by centrifugation over 50% Percoll density gradients. Activities of alkaline phosphatase, gamma-glutamyl transpeptidase, ADA, PNP and CDA were determined in various tissue homogenates (cerebral cortex, BBMECs and erythrocytes). Enzyme kinetic studies were also conducted using commercially available enzymes and several nucleoside analogs of interest. RESULTS: The activities of ADA and PNP were 42-fold and 247-fold higher in the cerebral microvessels than in the cerebral cortex, respectively, while there was no detectable CDA activity in the microvessel fraction and very little overall activity in the cortex. CONCLUSIONS: ADA and PNP may serve as an enzymatic blood-brain barrier for some of the anti-HIV dideoxynucleosides. Simulations of brain availability for ddI, ddA, 6-Cl-ddP, and F-ddA demonstrated that the quantitative significance of enzyme localization may vary dramatically, however, depending on the membrane permeability of the drug and its bioconversion rate constant within the endothelial cell.
Solid-state stability of human insulin. I. Mechanism and the effect of water on the kinetics of degradation in lyophiles from pH 2-5 solutions.
PURPOSE: Previous studies have established that in aqueous solution at low pH human insulin decomposition proceeds through a cyclic anhydride intermediate leading to the formation of both deamidated and covalent dimer products. This study examines the mechanism and kinetics of insulin degradation in the amorphous solid state (lyophilized powders) as a function of water content over a similar pH range. METHODS: Solutions of 1.0 mg/mL insulin were adjusted to pH 2-5 using HCl, freeze-dried, then exposed to various relative humidities at 35 degrees C. The water content within the powders was determined by Karl Fischer titration, and the concentrations of insulin and its degradation products were determined by HPLC. Degradation kinetics were determined by both the initial rates of product formation and insulin disappearance. RESULTS: Semi-logarithmic plots of insulin remaining in lyophilized powders versus time were non-linear, asymptotically approaching non-zero apparent plateau values, mathematically describable by a reversible, first-order kinetic model. The rate of degradation of insulin in the solid state was observed to increase with decreasing apparent pH ('pH') yielding, at any given water content, solid-state 'pH'-rate profiles parallel to the solution pH-rate profile. This 'pH' dependence could be accounted for in terms of the fraction of the insulin A21 carboxyl in its neutral form, with an apparent pKa of approximately 4, independent of water content. Aniline trapping studies established that the mechanism of degradation of human insulin in lyophilized powders between pH 3-5 and at 35 degrees C involves rate-limiting intramolecular nucleophilic attack of the AsnA21 C-terminal carboxylic acid onto the side-chain amide carbonyl to form a reactive cyclic anhydride intermediate, which further reacts with either water or an N-terminal primary amino group (e.g., PheB1 and GlyA1) of another insulin molecule to generate either deamidated insulin (AspA21) or an amide-linked covalent dimer (e.g., [AspA21-PheB1] or [AspA21-GlyA1]), respectively. The rate of insulin degradation in lyophilized powders at 35 degrees C increases with water content at levels of hydration well below the suspected glass transition and approaches the rate in solution at or near the water content (20-50%) required to induce a glass transition. CONCLUSIONS: The decomposition of human insulin in lyophilized powders between pH 3-5 is a water induced solid-state reaction accelerated by the plasticization effect of sorbed water. The formation of the cyclic anhydride intermediate at A21 occurs readily even in the glassy state, presumably due to the conformational flexibility of the A21 segment even under conditions in which the insulin molecules as a whole are largely immobile.
Intrinsic solubility estimation and pH-solubility behavior of cosalane (NSC 658586), an extremely hydrophobic diprotic acid.
PURPOSE: The selection of cosalane (NSC 658586) by the National Cancer Institute for further development as a potential drug candidate for the treatment of AIDS led to the exploration of the solubility behavior of this extremely hydrophobic drug, which has an intrinsic solubility (S0 approaching 1 ng/ml. This study describes attempts to reliably measure the intrinsic solubility of cosalane and examine its pH-solubility behavior. METHODS: S0 was estimated by 5 different strategies: (a) direct determination in an aqueous suspension: (b) facilitated dissolution; (c) estimation from the octanol/water partition coefficient and octanol solubility (d) application of an empirical equation based on melting point and partition coefficient; and (e) estimation from the hydrocarbon solubility and functional group contributions for transfer from hydrocarbon to water. RESULTS: S0 estimates using these five methods varied over a 5 x 107-fold range Method (a) yielded the highest values, two-orders of magnitude greater than those obtained by method (b) (facilitated dissolution. 1.4 +/- 0.5 ng/ml). Method (c) gave a value 20-fold higher while that from method (d) was in fair agreement with that from facilitated dissolution. Method (e) yielded a value several orders-of-magnitude lower than other methods. A molecular dynamics simulation suggests that folded conformations not accounted for by group contributions may reduce cosalane's effective hydrophobicity. Ionic equilibria calculations for this weak diprotic acid suggested a 100-fold increase in solubility per pH unit increase. The pH-solubility profile of cosalane at 25 degrees C agreed closely with theory. CONCLUSIONS: These studies highlight the difficulty in determining solubility of very poorly soluble compounds and the possible advantage of the facilitated dissolution method. The diprotic nature of cosalane enabled a solubility enhancement of > 107-fold by simple pH adjustment.
Dynamics of virus versus host interaction in children with human immunodeficiency virus type 1 infection.
To investigate the dynamic interplay between human immunodeficiency virus type I (HIV-1) replication and the extent of immune destruction in HIV-1-infected children, virus burden in lymphoid tissues (LN) and peripheral blood was compared with changes in LN architecture and cytokine levels constitutively expressed in LN. In agreement with results of a preliminary study, the plasma HIV-1 RNA level correlated with the amount of provirus in LN. However, the level was also associated with a degree of destruction of lymphoid follicular architecture and an alteration of immune cytokine expression. Expression of interleukin (IL)-4 was higher in LN with higher virus replication. Reduction of plasma viremia was associated with an increase in IL-2 mRNA levels in LN. These findings suggest that measurable virus burden in the peripheral blood is not a simple reflection of viral replication in LN but is also influenced by the extent of progressive immune destruction.
Quantitative assessment of blood-brain barrier damage during microdialysis.
In view of the increasing use of microdialysis for monitoring drug uptake into the brain, the consequences of tissue/blood-brain barrier (BBB) damage that occurs on microdialysis probe insertion on the extent and rate of solute uptake need to be more carefully examined. In this study, both microdialysis and a classic method were used to compare the apparent brain uptake of two polar permeants, [3H]sucrose and [14C]urea. The blood-to-brain transfer constants of these compounds differ significantly, with the value of urea exceeding that of sucrose by a factor of approximately 20 when compared by the classic one-point-per-animal method. The BBB selectivity to these nonmetabolized permeants as assessed by microdialysis provides a sensitive measure of the integrity of the BBB to polar nonelectrolytes within the molecular size range of most drugs. The following evidence for blood-brain barrier damage during microdialysis sampling was obtained: (1) the loss of [3H]sucrose from the extracellular fluid in brain on termination of an intravenous infusion is biphasic, with the initial phase evident immediately on termination by the infusion, suggesting that a fraction of the microdialysis probe resides in a region in rapid equilibrium with plasma; (2) complete loss of selectivity in the rate constants for CNS entry of sucrose vs. urea and (3) there were substantially higher area under the concentration vs. time curve AUCECF/AUCplasma ratios for both sucrose and urea generated by microdialysis than the corresponding ratios (AUCCSF/AUCplasma or AUCbrain/AUCplasma) obtained by classic methods. These results suggest that the BBB to small molecule transport likely remains compromised for some time after microdialysis probe insertion.
Role of altered metabolism in dideoxynucleoside pharmacokinetics. Studies of 2'-beta-fluoro-2',3'-dideoxyinosine and 2'-beta-fluoro-2',3'-dideoxyadenosine in rats.
Enzymes of the purine salvage pathway play an important role in altering the in vivo pharmacokinetics of 2',3'-dideoxypurine nucleosides. This study examines the pharmacokinetics of enzyme-resistant 2'-beta-fluoro analogues of 2',3'-dideoxyinosine (ddI) and 2',3'-dideoxyadenosine (ddA). 2'-beta-Fluoro-2',3'-dideoxyinosine (F-ddI) is an acid-stable analogue of ddI that is highly resistant to purine nucleoside phosphorylase, the principal enzyme in ddI metabolism. 2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA), an acid-stable and purine nucleoside phosphorylase-resistant analogue of ddA, is converted in vivo to F-ddI by adenosine deaminase (ADA) but is a much poorer substrate for this enzyme than is ddA. Both F-ddA and F-ddI have been shown to have activity against human immunodeficiency virus in vitro, and F-ddA has been selected by the National Cancer Institute for clinical trials as a new human immunodeficiency virus reverse transcriptase inhibitor. The pharmacokinetics of F-ddI and ddI were compared at equivalent doses in chronically catheterized rats. Because ddI and F-ddI are isosteres having nearly identical lipophilicity, this comparison is likely to reflect primarily metabolic differences. The clearance of F-ddI was substantially reduced, in comparison with that of ddI (27.3 ml/min/kg vs. 90.9 ml/min/kg), resulting in higher systemic concentrations at steady state and prolonged retention of F-ddI after termination of infusions, consistent with a significant metabolic component in the clearance of ddI. Concentrations of F-ddA and F-ddI during and after infusions of F-ddA were determined in both untreated and 2'-deoxycoformycin-pretreated rats. In untreated rats, F-ddA was rapidly eliminated from plasma, with a total clearance of 68.5 ml/kg/min. Metabolic clearance of F-ddA to F-ddI accounted for 58% of this value (bioconversion t1/2 = 9.8 +/- 1.9 min). Pretreatment with 2'-deoxycoformycin, an ADA inhibitor, reduced the clearance of F-ddA to 23.8 ml/min/kg, leading to 2.9 +/- 0.4-fold higher steady-state plasma concentrations of F-ddA, in agreement with a 2.5-fold enhancement predicted by a compartmental model assuming complete ADA inhibition.
Noninvasive clearance of airway secretions.
Airway clearance techniques are indicated for specific diseases that have known clearance abnormalities (Table 2). Murray and others have commented that such techniques are required only for patients with a daily sputum production of greater than 30 mL. The authors have observed that patients with diseases known to cause clearance abnormalities can have sputum clearance with some techniques, such as positive expiratory pressure, autogenic drainage, and active cycle of breathing techniques, when PDPV has not been effective. Hasani et al has shown that use of the forced exhalatory technique in patients with nonproductive cough still resulted in movement of secretions proximally from all regions of the lung in patients with airway obstruction. It is therefore reasonable to consider airway clearance techniques for any patient who has a disease known to alter mucous clearance, including CF, dyskinetic cilia syndromes, and bronchiectasis from any cause. Patients with atelectasis from mucous plugs and hypersecretory states, such as asthma and chronic bronchitis, patients with pain secondary to surgical procedures, and patients with neuromuscular disease, weak cough, and abnormal patency of the airway may also benefit from the application of airway clearance techniques. Infants and children up to 3 years of age with airway clearance problems need to be treated with PDPV. Manual percussion with hands alone or a flexible face mask or cup and small mechanical vibrator/percussors, such as the ultrasonic devices, can be used. The intrapulmonary percussive ventilator shows growing promise in this area. The high-frequency oscillator is not supplied with vests of appropriate sizes for tiny babies and has not been studied in this group. Young patients with neuromuscular disease may require assisted ventilation and airway oscillations can be applied. CPAP alone has been shown to improve achievable flow rates that will increase air-liquid interactions for patients with these diseases or airway malacia. Use of positive pressure to maintain airway patency in these children allows cephalad clearance of secretions. Patients with segmental atelectasis, particularly related to asthma, may benefit from intrapulmonary percussive ventilator, positive expiratory pressure, or PDPV. Prevention of postoperative atelectasis is particularly well suited to positive expiratory pressure, which is not as painful as techniques using oscillations. Neurologically abnormal patients who are unable to cooperate with any active method are also treated using intrapulmonary percussive ventilator, PDPV, and suctioning, if necessary. Musculoskeletal abnormalities, muscular dystrophies, myasthenia gravis, poliomyelitis, or other similar diseases require stabilization of bellows function. Optimizing ventilation in patients with such abnormalities may require positive pressure ventilation either during sleep or continuously. Externally applied pressure, such as with the In-Exsufflator or the cyclically inflated pneumatic belt, can augment the patient's own efforts and is sometimes helpful. Normalizing the vital capacity and functional residual capacity typically helps to improve the ability to cough and clear secretions. Assisted cough devices or maneuvers are described in other papers by Bach and Hill. Not all patients who have weak muscles require nocturnal or continuous support, and may benefit from positive expiratory pressure mask treatments. Further studies are sorely needed for this population. Long-term controlled trials are urgently needed to help establish the best types of treatment for patients with CF and bronchiectasis. Such studies will become more complicated by the introduction of new treatments, such as DNase and other therapies that alter secretions, and may begin to change mucociliary or cough clearance. The selection of appropriate outcome measures is central to studying these questions, and it is unclear which are the most important. (ABSTRACT TRUNCATED)
Evidence for a common intermediate in insulin deamidation and covalent dimer formation: effects of pH and aniline trapping in dilute acidic solutions.
The effects of pH and aniline trapping on the partitioning of the A-21 cyclic anhydride intermediate of human insulin into deamidated insulin and covalent dimer were investigated at low pH and 35 degrees C. Characterization of the covalent dimer was achieved by proteolytic cleavage and electrospray mass spectrometry and indicated that the deamidated A-21 asparagine of one insulin molecule and the B-1 phenylalanine residue of another are involved. Anhydride trapping with aniline at pH 4.0 provided evidence that the rate-limiting generation of a cyclic anhydride intermediate is involved in the formation of both deamidated and dimeric insulin. In the presence of aniline at pH 4.0 insulin formed two anilide products, A-21 N delta 2-phenylasparagine and N delta 2-phenylasparagine and N gamma 2-phenylaspartic acid human insulin at the expense of both desamido A-21 and covalent dimer formation, consistent with the formation of a common intermediate. At 35 degrees C and under conditions where the insulin monomer predominates, the fraction of insulin reacting to form [desamidoA-21] insulin decreased with a concurrent increase in formation of [desamidoA-21-PheB-1] dimer with an increase in pH from 2.0 to 5.0. The pH dependence of insulin product distribution could not be quantitatively rationalized solely in terms of the fraction of the PheB-1 amine group in un-ionized form. Rather, consideration of the charge states of ionizable residues near the reacting groups was necessary to fully account for the observed pH effects on product formation.
Human skin permeability enhancement by lauric acid under equilibrium aqueous conditions.
An in vitro method was developed to investigate the enhancement of hydrocortisone transport across human stratum corneum (SC) by a model enhancer, lauric acid, in aqueous solutions under equilibrium conditions with respect to the enhancer. In contrast to classical (i.e., nonequilibrium) loading techniques, in which the enhancer is applied only to the donor side of SC either in pure form or in an organic solvent while enhancer-free aqueous buffers are placed in the receptor phase, this method allowed the investigation of pH effects, concentration effects, and reversibility of both enhancer uptake and enhancement of drug transport under thermodynamically well-defined conditions. The SC-buffer partition coefficients for lauric acid were linear with concentration and sigmoidal with pH, suggesting that both the neutral species and laurate anion partition into SC. Comparisons of partition coefficients in delipidized and untreated SC as a function of pH indicated that the uptake of lauric acid in neutral form is governed primarily by the lipid domain, whereas the protein domain accounts for anion uptake. The effects of lauric acid on skin permeability were > 80% reversible upon extraction of the enhancer from the membrane. However, the degree of enhancement of hydrocortisone permeability was nonlinearly dependent on the equilibrium concentration of lauric acid in either the aqueous buffer or the membrane, exhibiting thresholds in the appearance of enhancement with concentration. The enhancer concentration necessary to achieve isoenhancement of about 6-fold varied from approximately 1 x 10(-5) M at pH < pKa to approximately 1 x 10(-2) M at high pH (pH > 8) demonstrating the higher influence of the free acid species.(ABSTRACT TRUNCATED AT 250 WORDS)
Development of a combined NMR paramagnetic ion-induced line-broadening/dynamic light scattering method for permeability measurements across lipid bilayer membranes.
A combined method using NMR line-broadening for permeant lifetime determination and dynamic light scattering for vesicle size determination has been developed for the measurement of permeability coefficients of ionizable permeants across phospholipid:cholesterol large unilamellar vesicles. The method has been validated by examining its reproducibility and the influence of various factors that might affect the permeability measurements. The vesicle hydrodynamic diameter was varied between 0.1 and 0.2 micron by extruding multilamellar vesicles through polycarbonate membranes with different pore sizes (0.03-0.2 microns). For these large unilamellar vesicles, the normalized size distributions analyzed by the CONTIN method had standard deviations < 0.36, which led to errors in permeability coefficients < 10% as predicted from a theoretical model developed here. The permeability coefficient for acetic acid is independent of its concentration, vesicle hydrodynamic diameter, the concentration of Pr3+, and ionic strength over the ranges 0.01-0.2 M, 0.1-0.2 microns, 0.004-0.04 M, and 0.03-0.3, respectively. Membrane/water and decane/water partition coefficient measurements of acetic acid indicate that the effects of permeant binding onto the bilayer membrane and self-association are negligible within the permeant concentration range 0.01-0.2 M. The addition of Pr3+ ions induces vesicle fusion with rates increasing with temperature and decreasing with cholesterol concentration in the membranes. While the intravesicular resonance intensity for acetic acid decreases continuously with time due to vesicle fusion under certain conditions, the corresponding line width and chemical shift remain constant over the same period, highlighting an important advantage of this NMR method over those based on detecting net flux in response to a concentration gradient as there is no means in the latter experiments of discerning vesicle leakiness from passive diffusion rates. The effective chemical nature of a dimristoylphosphatidylcholine:cholesterol bilayer barrier microenvironment was explored by comparing the transport of two permeants, D-(-)-mandelic acid and phenylacetic acid, to their relative bulk solvent/water partition coefficients using three reference solvents (n-decane, 1,9-decadiene, and isoamyl alcohol). Using the NMR line-broadening method, the permeability coefficients for these two permeants were determined to be (2.9 +/- 0.4) x 10(-4) cm/s and (3.9 +/- 0.7) x 10(-2) cm/s, respectively, at 294 K and Xchol = 0.3. The incremental free energy of transport for the additional OH group in D-(-)-mandelic acid, delta delta G0 = +2.9 kcal/mol, resembles most closely that for the transfer of this group from water to 1,9-decadiene, suggesting that the barrier domain resides in the acyl chain region and is slightly more polar/polarizable than a saturated hydrocarbon, possibly due to the presence of a double bond in cholesterol and/or the proximity of the barrier domain to the hydrophilic interface.
Phospholipid surface density determines the partitioning and permeability of acetic acid in DMPC:cholesterol bilayers.
Relationships between the permeability coefficient (PHA) and partition coefficient (K m/w) of acetic acid and the surface density of DMPC:cholesterol bilayers have been investigated. Permeability coefficients were measured in large unilamellar vesicles by NMR line broadening. Bilayer surface density, sigma, was varied over a range of 0.5-0.9 by changing cholesterol concentration and temperature. The temperature dependence of PHA for acetic acid exhibits Arrhenius behavior with an average apparent activation energy (Ea) of 22 +/- 3 kcal/mole over a cholesterol mole fraction range of 0.00-0.40. This value is much greater than the enthalpy change for acetic acid partitioning between bulk decane and water (delta H degree = 4.8 +/- 0.8 kcal/mole) and the calculated Ea (= 8.0 kcal/mole) assuming a "bulk phase" permeability model which includes the enthalpy of transfer from water to decane and the temperature dependence of acetic acid's diffusion coefficient in decane. These results suggest that dehydration, previously considered to be a dominant component, is a minor factor in determining Ea. Values of 1n PHA decrease linearly with the normalized phospholipid surface density with a slope of kappa = -12.4 +/- 1.1 (r = 0.90). Correction of PHA for those temperature effects considered to be independent of lipid chain order (i.e., enthalpy of transfer from water to decane and activation energy for diffusion in bulk hydrocarbon) yielded an improved correlation (kappa = -11.7 +/- 0.5 (r = 0.96)). The temperature dependence of Km/w is substantially smaller than that for PHA and dependent on cholesterol composition. Values of 1n K m/w decrease linearly with the surface density with a slope of kappa = -4.6 +/- 0.3 (r = 0.95), which is 2.7-fold smaller than the slope of the plot of 1n PHA vs. sigma. Thus, chain ordering is a major determinant for molecular partitioning into and transport across lipid bilayers, regardless of whether it is varied by lipid composition or temperature.
Effects of insulin concentration and self-association on the partitioning of its A-21 cyclic anhydride intermediate to desamido insulin and covalent dimer.
PURPOSE: In the pH range 2-5, human insulin degrades via deamidation at the A-21 asn and covalent dimerization. Both products form via a common cyclic anhydride intermediate, a product of intramolecular neucleophilic attack by the A-21 carboxyl terminus. This study examines the influence of [insulin] and self-association on the partitioning of the intermediate to products. METHODS: Insulin self-association was characterized (pH 2-4) by concentration difference spectroscopy. Deamination rates (pH 2-4) and concurrent rates of covalent dimer formation (pH 4) were determined versus [insulin] at 35 degrees C by initial rates. A mathematical model was developed to account for the overall rate and product composition profile versus pH and [insulin]. RESULTS: Between pH 2-4, insulin self-associates to form non-covalent dimers with a pH independent association constant of 1.8 x 10(4) M-1. The overall rate of degradation is governed by intermediate formation, while product distribution is determined by competition between water and the phe B-1 amino group of insulin for the anhydride. In dilute solutions, deamidation is first-order in [insulin] while covalent dimerization is second-order. Thus, deamidation predominates in dilute solutions but the fraction of covalent dimer formed increases with [insulin]. At high [insulin], self-association inhibits covalent dimer formation, preventing exclusive degradation via this pathway. The model accurately predicts a maximum in covalent dimer formation near pH 4. CONCLUSIONS: A mechanism is described which accounts for the complex dependence of insulin's degradation rate and product distribution profile on pH (between 2-5) and [insulin]. If these results can be generalized, they suggest that covalent aggregation in proteins may be inhibited by self-association.
Enhanced oral bioavailability of DDI after administration of 6-Cl-ddP, an adenosine deaminase-activated prodrug, to chronically catheterized rats.
PURPOSE: 6-Cl-2',3'-dideoxypurine (6-Cl-ddP), an adenosine deaminase (ADA) activated prodrug of ddI, may be an effective antiretroviral agent for the treatment of AIDS dementia due to its ability to deliver increased concentrations of ddI to brain tissue. To examine the feasibility of administering this drug orally, the oral and hepatic portal bioavailabilities of 6-Cl-ddP were determined. In addition, the oral and portal bioavailabilities of ddI after administration of the prodrug were compared to those from administration of ddI itself. METHODS: Pharmacokinetic and bioavailability studies were conducted in fully conscious, chronically catheterized rats in a randomized crossover design. Plasma ddI and 6-Cl-ddP concentration-time profiles were determined by HPLC. RESULTS: 6-Cl-ddP has poor apparent oral bioavailability (7% +/- 3%, n = 3) but high bioavailability after portal administration (97% +/- 11%), suggesting either poor absorption or extensive gut wall metabolism. The appearance of > 50% of the dose as ddI in the systemic circulation after an oral dose of 6-Cl-ddP rules out poor absorption of the prodrug, and confirms expectations of high ADA activity in the gastrointestinal tract. Gastric administration of 6-Cl-ddP resulted in a > 10-fold increase in the oral bioavailability of ddI, from 3-7% to > 50%, and a significant decrease in the variability in apparent bioavailability. CONCLUSIONS: These data indicate that lipophilic adenosine deaminase activated prodrugs of dideoxypurine nucleosides may have limited utility for improving CNS delivery after oral administration but may be useful in enhancing the oral bioavailability of highly polar and therefore poorly absorbed dideoxynucleosides.