Derivatives of 5-Aminolevulinic Acid for Photodynamic Therapy: Enzymatic Conversion into Protoporphyrin

In recent years, 5-aminolevulinic acid (ALA) has become a widespread agent for photodynamic therapy (PDT). In nucleated cells, ALA is converted into the endogenous photosensitizer protoporphyrin IX (PpIX). A major drawback of ALA is its low bioavailability. As a result, high doses of ALA must be administered in order to reach clinically relevant levels of PpIX. Moreover, only superficially located lesions can be treated as a result of the poor penetration of ALA into tissues. A possible solution for this problem may be provided by the prod rug concept. In the present study, prodrugs of ALA have been synthesized. These ALA prodrugs are shown to result in higher PpIX levels in cells than does ALA itself. Of a range of ester prodrugs of ALA, the ALA-pentyl ester elicits the highest fluorescence. Further-more, the enzymatic conversion of the derivatives into ALA and PpIX has been studied in lysed cells. Under these circumstances, the esters with the shorter alkyl chains induce the highest fluorescence. The alcohols that arise as side products from enzymatic conversion of the prodrugs are shown to have no influence on the experiments.     

Comparative molecular dynamics study of ether- and ester-linked phospholipid bilayers

The lipid membranes found in archaea have high bilayer stability and low permeability. The molecular structure of their constituent lipids is characterized by ether-linked, branched hydrophobic chains, whereas the conventional lipids obtained from eukaryotic or eubacterial sources have ester linked straight chains. In order to elucidate the influence of the ether linkage, instead of an ester one, on the physical properties of the lipid bilayers, we have carried out comparative 10 ns molecular dynamics simulations of diphytanyl phosphatidylcholine (ether-DPhPC) and diphytanoyl phosphatidylcholine (ester-DPhPC) bilayers in water, respectively. We analyze bilayer structures, hydration of the lipids, membrane dipole potentials, and free energy profiles of water and oxygen across the bilayers. We observe that the membrane dipole potential for the ether-DPhPC bilayer, which arises mainly from the ether linkage, is about half of that of the ester-DPhPC. The calculated free energy barrier for a water molecule in the ether-DPhPC bilayer system is slightly higher than that in the ester-DPhPC counterpart, which is in accord with experimental data.     

Photodynamic therapy: regulation of porphyrin synthesis and hydrolysis from ALA esters

Photodynamic therapy (PDT) is a tool for the treatment of certain cancerous and pre-cancerous conditions. The natural precursor of porphyrins 5-aminolevulinic acid (ALA) has been extensively used as a pro-photosensitiser in PDT. ALA's poor permeability has been enhanced by chemical esterification with aliphatic alcohols. Some of the ALA esters proved to be more efficient than ALA for porphyrin synthesis. In the present work we studied the nature of porphyrin synthesis regulation from the ALA esters Hexyl-ALA (He-ALA) and R,S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester (THP-ALA) in an adenocarcinoma cell line. We found that He-ALA is incorporated into the cells at a higher rate, followed by THP-ALA and ALA, whereas ALA and ALA esters efflux at the same rate mediated by passive diffusion. Although ALA entrance to the cell might be regulatory at low concentrations, ALA derivative uptake is not a limiting factor. At high concentrations, the regulation of ALA conversion into porphyrins is driven by the enzyme porphobilinogenase, whereas ALA esters hydrolysis is regulated by esterases. The key conclusion of this contribution is that the use of ALA esters has to be limited to low concentrations where no regulation on porphyrin synthesis takes place.     

Topical application of 5-aminolevulinic acid hexyl ester and 5-aminolevulinic acid to normal nude mouse skin: differences in protoporphyrin IX fluorescence kinetics and the role of the stratum corneum

An important limitation of topical 5-aminolevulinic acid (ALA)-based photodetection and photodynamic therapy is that the amount of the fluorescing and photosensitizing product protoporphyrin IX (PpIX) formed is limited. The reason for this is probably the limited diffusion of ALA through the stratum corneum. A solution to this problem might be found in the use of ALA derivatives, as these compounds are more lipophilic and therefore might have better penetration properties than ALA itself. Previous studies have shown that ALA hexyl ester (ALAHE) is more successful than ALA for photodetection of early (pre)malignant lesions in the bladder. However, ALA pentyl ester slightly increased the in vivo PpIX fluorescence in early (pre)malignant lesions in hairless mouse skin compared to ALA. The increased PpIX fluorescence is located in the stratum corneum and not in the dysplastic epidermal layer. In the present study, ALA- and ALAHE-induced PpIX fluorescence kinetics are compared in the normal nude mouse skin, of which the permeability properties differ from the bladder. Application times and ALA(HE) concentrations were varied, the effect of a penetration enhancer and the effect of tape stripping the skin before or after application were investigated. Only during application for 24 h, did ALAHE induce slightly more PpIX fluorescence than ALA. After application times ranging from 1 to 60 min, ALA-induced PpIX fluorescence was higher than ALAHE-induced PpIX fluorescence. ALA also induced higher PpIX production than ALAHE after 10 min of application with concentrations ranging from 0.5 to 40%. The results of experiments with the penetration enhancer and tape stripping indicated that the stratum corneum acts a barrier against ALA and ALAHE. Use of penetration enhancer or tape stripping enhanced the PpIX production more in the case of ALAHE application than in the case of ALA application. This, together with the results from the different application times and concentrations indicates that ALAHE diffuses more slowly across the stratum corneum than ALA.     

Stretching effects on the permeability of water molecules across a lipid bilayer

Using a coarse grained molecular dynamics model of a solvent-surfactant system, we study the effects of stretching on the permeability of water across a lipid bilayer. The density profile, free energy profile, diffusion profile, and tail ordering parameter were computed for a set of stretched membranes maintained at constant area. We computed the water permeability across each membrane using the inhomogeneous solubility-diffusion model first proposed by Marrink and Berendsen [J. Phys. Chem. 98, 4155 (1994)]. We find that even though the resistance to permeation profile shows a great deal of qualitative change as the membranes are stretched, the overall permeability remains nearly constant within the relevant range of stretching. This is explained by the fact that the main barrier to permeation, located in the densest section of the tails, is insensitive to increased area per lipid, as a result of competing effects. Expansion leads to thinning and a higher density in the tail region, the latter leading to an increase in the free energy barrier. However, this is compensated by the reduction in the transverse distance to cross and a larger diffusion coefficient due to increased disordering in the chains.     

Passive transport of C60 fullerenes through a lipid membrane: a molecular dynamics simulation study

To investigate the implications of the unique properties of fullerenes on their interaction with and passive transport into lipid membranes, atomistic molecular dynamics simulations of a C60 fullerene in a fully hydrated di-myristoyl-phoshatidylcholine lipid membrane have been carried out. In these simulations the free energy and the diffusivity of the fullerene were obtained as a function of its position within the membrane. These properties were utilized to calculate the permeability of fullerenes through the lipid membrane. Simulations reveal that the free energy decreases as the fullerene passes from the aqueous phase, through the head group layer and into the hydrophobic core of the membrane. This decrease in free energy is not due to hydrophobic interactions but rather to stronger van der Waals (dispersion) interactions between the fullerene and the membrane compared to those between the fullerene and (bulk) water. It was found that there is no free energy barrier for transport of a fullerene from the aqueous phase into the lipid core of the membrane. In combination with strong partitioning of the fullerenes into the lipidic core of the membrane, this "barrierless" penetration results in an astonishingly large permeability of fullerenes through the lipid membrane, greater than observed for any other known penetrant. When the strength of the dispersion interactions between the fullerene and its surroundings is reduced in the simulations, thereby emulating a nanometer sized hydrophobic particle, a large free energy barrier for penetration of the head group layer emerges, indicating that the large permeability of fullerenes through lipid membranes is a result of their unique interaction with their surrounding medium.     

In vitro fluorescence, toxicity and phototoxicity induced by delta-aminolevulinic acid (ALA) or ALA-esters

Synthesis of delta-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 micrograms/mL), E1 and E2 (10 micrograms/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 micrograms/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm2) applied 6 h after 5 min incubation with 168 micrograms/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.     

Lipids out of equilibrium: energetics of desorption and pore mediated flip-flop

The potential of mean force (PMF) of a phospholipid in a bilayer is a key thermodynamic property that describes the energetic cost of localized lipid defects. We have calculated the PMF by umbrella sampling using molecular dynamics simulations. The profile has a deep minimum at the equilibrium position in the bilayer and steeply rises for displacements both deeper into the bilayer and moving away from the bilayer. As the lipid loses contact with the bilayer, the profile abruptly flattens without a significant barrier. The calculated free energy difference of 80 kJ/mol between the minimum of the PMF and the value in water agrees well with the free energy difference calculated from the experimentally measured critical micelle concentration. Significant water/lipid defects form when a lipid is forced into the bilayer interior, in the form of a small water pore that spans the membrane. The energy required to form such a water pore is also found to be 80 kJ/mol. On the basis of this energy, we estimate the lipid flip-flop rate and permeability rate of sodium ions. The resulting rates are in good agreement with experimental measurements, suggesting lipid flip-flop and basal permeability of ions are pore mediated.     

Comparison of the pharmacokinetics and phototoxicity of protoporphyrin IX metabolized from 5-aminolevulinic acid and two derivatives in human skin in vivo

Our novel approach was to compare the pharmacokinetics of 5-aminolevulinic acid (ALA), ALA-n-butyl and ALA-n-hexylester induced protoporphyrin IX (PpIX), together with the phototoxicity after photodynamic therapy (PDT) in human skin in vivo, using iontophoresis as a dose-control system. A series of four increasing doses of each compound was iontophoresed into healthy skin of 10 volunteers. The kinetics of PpIX metabolism (n = 4) and the response to PDT (n = 6) performed 5 h after iontophoresis, were assessed by surface PpIX fluorescence and post-irradiation erythema. Whilst ALA-induced PpIX peaked at 7.5 h, highest PpIX fluorescence induced by ALA-n-hexylester was observed at 3-6 h and no clear peak was seen with ALA-n-butylester. With ALA-n-hexylester, more PpIX was formed after 3 (P < 0.05) and 4.5 h, than with ALA or ALA-n-butylester. All compounds showed a linear correlation between logarithm of dose and PpIX fluorescence/phototoxicity at 5 h, with R-values ranging from 0.87 to 1. In addition, the ALA-n-hexylester showed the tendency to cause greater erythema than ALA and ALA-n-butylester. Fluorescence microscopy (n = 2) showed similar PpIX distributions and penetration depths for the three drugs, although both ALA esters led to a more homogeneous PpIX localization. Hence, ALA-n-hexylester appears to have slightly more favorable characteristics for PDT than ALA or ALA-n-butylester.     

On the role of iron and one of its chelating agents in the production of protoporphyrin IX generated by 5-aminolevulinic acid and its hexyl ester derivative tested on an epidermal equivalent of human skin

Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) or its derivatives as precursors of protoporphyrin IX (PPIX) is routinely used in dermatology for the treatment of various pathologies. However, this methodology suffers to some extent from a limited efficacy. Therefore, the main goal of this study was to investigate the modulation and pharmacokinetics of PPIX buildup after a 5 h incubation with ALA (1.5 mM) and one of its derivatives, the hexyl ester of ALA (h-ALA) (1.5 mM), on the human epidermal equivalent Epidex. PPIX production was modulated with (L+) ascorbic acid iron (II) salt (LAI) or the iron (II)-specific chelating agent deferoxamine (DFO). PPIX fluorescence from the Epidex layers was measured up to 150 h after the precursor administration using a microspectrofluorometer (lambda(ex): 400 +/- 20 nm; lambda(det): 635 nm). The maximum PPIX fluorescence intensity induced by h-ALA was about 1.7 x larger than that induced by ALA. The addition of DFO resulted in a more than 50% increase in PPIX fluorescence for both precursors. The decay half life measured for PPIX fluorescence is 30 and 42.5 h, respectively, for ALA and h-ALA. These half lives are doubled when the samples contain DFO. In the samples with the highest fluorescence intensity, a modified fluorescence spectrum was observed after 10 h, with the emergence of a peak at 590 nm, which is attributed to zinc protoporphyrin IX (Zn PPIX).     

Systemic component of protoporphyrin IX production in nude mouse skin upon topical application of aminolevulinic acid depends on the application conditions

Topical application of 5-aminolevulinic acid (ALA) for protoporphyrin IX (PpIX)-based photodynamic therapy of skin cancer is generally considered not to induce systemic side effects because PpIX is supposed to be formed locally. However, earlier studies with topically applied ALA have revealed that in mice PpIX is not only produced in the application area but also in other organs including skin outside the application area, whereas esterified ALA does not. From these results, it was concluded that it is not redistribution of circulating PpIX that causes the fluorescence distant from the ALA application site, but rather, local PpIX production induced by circulating ALA. In the present study we investigate the effects of the ALA concentration in the cream, the application time, the presence of a penetration enhancer, the presence of the stratum corneum and esterification of ALA on the PpIX production in nude mouse skin outside the area where ALA is applied. For this purpose, ALA and ALA hexyl ester (ALAHE) were applied to one flank, and the PpIX fluorescence was measured in the contralateral flank. During a 24 h application of ALA, PpIX was produced in the contralateral flank. No PpIX could be detected in the contralateral flank after ALA application times ranging from 1 to 60 min. Tape-stripping the skin prior to short-term ALA application, but not the addition of a penetration enhancer, resulted in PpIX production in the contralateral flank. When ALAHE was applied, no PpIX fluorescence was measured in the contralateral flank under any application condition. The results suggest that the systemic component of PpIX production outside the ALA application area plays a minor or no role in relevant clinical situations, when the duration of ALA (ester) application is relatively short and a penetration enhancer is possibly added.     

Characterisation of liposomes containing aminolevulinic acid and derived esters

Liposomes of different compositions have been designed to improve delivery of aminolevulinic acid (ALA) and its esterified derivatives ALA-Hexyl ester (He-ALA) and ALA-Undecanoyl ester (Und-ALA) for its use in photodynamic therapy (PDT). Egg yolk phosphatidyl choline (PC), phosphatidic acid (PA) and phosphatidyl glycerol (PG) were employed in the preparation of the liposomes. Sonicated vesicles composed of PC, PC-PG (80:20) or PC-PA (80:20) containing ALA or derivatives were obtained and purified by a minicolumn centrifugation method. PC liposomes presented encapsulation percentages around 6% for 2 mM ALA, 13% for 2 mM He-ALA and 51% for 2 mM Und-ALA. The addition of PG or PA to the formulation, resulted in an increased entrapment: 19% for 2 mM ALA, 69% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC-PG liposomes and 21% for 2 mM ALA, 60% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC-PA liposomes. Higher concentrations of ALA or derivatives resulted in lower percentages of entrapment. The three formulations containing ALA or derivatives were stable up to 1 week upon storage at 4 degrees C. However, upon dilution with medium, ALA leaked from the liposomes, while on the contrary, He-ALA was highly retained, being therefore a good choice for its use in PDT. The stability of Und-ALA upon dilution could not be tested, but Und-ALA proved to have the highest entrapment efficacy.     

Porphyrin synthesis from aminolevulinic acid esters in endothelial cells and its role in photodynamic therapy

Photodynamic therapy (PDT) may cause tumour cell destruction by direct toxicity or by inducing microcirculatory shutdown. Protoporphyrin IX generated from 5-aminolevulinic acid (ALA) has been widely used as an endogenous photosensitiser in PDT. However, the hydrophilic nature of the ALA molecule limits its penetration through the stratum corneum of the skin and cell membranes and thus, ALA alkyl-esters have been developed to improve ALA permeation.The aim of this work was to study Protoporphyrin IX synthesis from ALA and its derivatives ALA methyl ester (Me-ALA) and ALA hexyl ester (He-ALA) in the microvascular endothelial cell line HMEC-1 derived from normal skin, and to evaluate their response to PDT.We found that lower light doses are required to photosensitise HMEC-1 endothelial cells than to photosensitise PAM212 transformed keratinocytes, showing some possible selectivity of ALA-PDT for vascularisation in skin.Employed at concentrations leading to equal Protoporphyrin IX synthesis, ALA, He-ALA and Me-ALA presented the same efficacy of HMEC-1 photosensitisation. However, He-ALA was a promising compound for the use in the enhancement of Protoporphyrin IX in HMEC-1 cells employed at low concentrations at both short and long time exposures whereas Me-ALA should be employed at high concentrations and longer time periods in order to surpass the Protoporphyrin IX levels obtained with ALA. The advantage of Me-ALA over ALA was based on its lower dark toxicity.This is the first work to report vascular cell photosensitisation employing alkyl-esters of ALA, and we demonstrated that these derivatives could exert the same effect as ALA and under certain conditions enhance photosensitisation of vasculature.     

Biological activity of 5-aminolevulinic acid and its methyl ester after storage under different conditions

5-Aminolevulinic acid (ALA) is a natural precursor of protoporphyrin IX (PpIX) and heme in cells. Photodynamic therapy (PDT) utilizes a metabolic imbalance in cancer cells, leading to increased PpIX generation from exogenous ALA. Due to chemical instability of ALA in therapeutic concentrations at pH values larger than 5.0 and at high temperatures, it looses its activity by spontaneous dimerization to 2,5-dicarboxyethyl-3,6-dihydropyrazine (DHPY). ALA esters are now supplementing ALA in PDT, but little is known about their stability. We have studied the stability of ALA and its methyl ester (MAL) stored under different conditions (temperatures, pH values) by measuring their ability to generate PpIX. 100mM solutions of both compounds were found to be stable at pH 4 and at 4 degrees C. However, at pH 5.5 they lost almost 10% of the initial activity during 5days of storage at 4 degrees C. The fastest decay of ALA and MAL was seen at pH 7.4 and at 37 degrees C, and followed first order kinetics. At pH 7.4 and at 4 degrees C MAL lost its PpIX producing ability more slowly than at 37 degrees C. Our work shows that solutions should be prepared immediately before use and stored at low temperatures. The pH of stock solutions should not exceed 5.     

Theoretical study of 5-aminolevulinic acid tautomerization: a novel self-catalyzed mechanism

5-Aminolevulinic acid (5ALA) is the key synthetic building block in protoporphyrin IX (PpIX), the heme chromophore in mitochondria. In this study density functional theory calculations were performed on the tautomers of 5ALA and the tautomerization reaction mechanism from its enolic forms (5-amino-4-hydroxypent-3-enoic acid and 5-amino-4-hydroxypent-4-enoic acid) to the more stable 5ALA. The hydrated form 5-amino-4,4-dihydroxypentanoic acid was also studied. The lowest energy pathway of 5ALA tautomerization is by means of autocatalysis, in that an oxygen of the carboxylic group transfers the hydrogen atom as a "crane", with an activation energy of approximately 15 kcal/mol. This should be compared to the barriers of about 35 kcal/mol for water assisted tautomerization, and 60 kcal/mol for direct hydrogen transfer. For hydration of 5ALA, the water catalyzed activation barrier is found to be approximately 35 kcal/mol, approximately 5 kcal/mol lower than direct hydration.     

Enhanced hydrophobicity of fluorinated lipid bilayer: a molecular dynamics study

A molecular dynamics simulation of a partially fluorinated phospholipid bilayer has been carried out to understand the effects of fluorination of the hydrophobic chains on the structure and water permeability across the membrane. Fluorocarbon chains typically have an all-trans conformation, showing a highly ordered structure in the membrane core compared to ordinary hydrocarbon chains. The free energy profiles of water across the bilayers were successfully estimated by a revised cavity insertion Widom method. The fluorinated bilayer showed a higher free energy barrier than an ordinary nonfluorinated lipid bilayer by about 1.2 kcal/mol, suggesting a lower water permeability of the fluorinated bilayer membrane. A cavity distribution analysis elucidated the reduced free volume in the fluorinated membrane due to the neatly packed chains, which should account for the higher free energy barrier.     

Fluorescence kinetics of protoporphyrin-IX induced from 5-ALA compounds in rabbit postballoon injury model for ALA-photoangioplasty

Abstract Protoporphyrin IX (PpIX) is one of the photodynamically active substances that are endogenously synthesized in the metabolic pathway for heme as a precursor. Aminolevulinic acid-esters are more lipophilic than conventional 5-aminolevulinic acid (ALA) and some of them are currently being approved as new drugs for photodynamic diagnosis (PDD) and photodynamic therapy (PDT). In order to investigate the pharmacokinetics of ALA and ALA-ethyl ester (ALA-ethyl) in the atheromatous plaque and normal aortic wall of rabbit postballoon injured artery, each 60 mg kg(-1) of ALA or ALA-ethyl was injected intravenously followed by serial detection of PpIX fluorescence of harvested arteries at 0-48 h post-injection. Maximum PpIX build-up in the atheromatous plaque was seen at 2 h after injecting ALA. In contrast, it occurred at 9 h after injecting ALA-ethyl. In addition, the selective build-up of ALA in the atheromatous plaque compared to normal vessel wall was much higher (10 times) than that of ALA-ethyl. The time of maximum fluorescence intensity of PpIX was employed as drug-light-interval for subsequent PDT treatment of the atheromatous plaque with 50-150 J cm(-1) of light dose. Significant reduction in plaque was observed without damage of the medial wall at both groups, but smooth muscle cell (SMC) was still present in the media region below the PDT-treated atheromatous plaque. In conclusion, ALA may be a more effective compound for endovascular PDT treatment of the atheromatous plaque compared with ALA-ethyl based on their pharmacokinetics, but further optimization of PDT methodology remains to remove completely residual SMC in the media for preventing potential restenosis.     

New 5-aminolevulinic acid esters--efficient protoporphyrin precursors for photodetection and photodynamic therapy

Photodetection (PD) and photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) accumulation are approaches to detect and treat dysplasia and early cancer in the gastrointestinal tract and in the urinary bladder. Because ALA-induced PPIX production is limited, we synthesized ALA ester hydrochlorides 3-22 and tested them in two different in vitro models (gastrointestinal tract: HT29-CCD18; urinary bladder: J82-UROTSA). PPIX accumulation after incubation with 0.12 mmol/L for 3 h and PPIX accumulation as a function of different incubation times were measured using flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to check cellular dark toxicity. Phototoxicity after irradiation was tested. ALA nonafluorohexylester hydrochloride 11, ALA thiohexylester hydrochloride 13 and ALA dibenzyldiester dihydrochloride 19 induced appreciably increased PPIX levels and showed improved phototoxicity compared with the references ALA hydrochloride 1, ALA hexylester hydrochloride 3 and ALA benzylester hydrochloride 4. Thus, the new compounds 11, 13 and 19 are promising compounds for PD and PDT.     

Simulating the Physiological Phase of Hydrated DPPC Bilayers: The Ester Moiety

Abstract

The charge on the ester oxygen of the sn2 group of the dipalmitoylphosphatidylcholine (DPPC) has a remarkable effect on the square area per lipid in simulations of a hydrated bilayer. This is in contrast to simulations of nonpolar, neutral lipids, where it has been found to have little effect. The charges associated with the GROMOS96 45A3 and 45A4 biomolecular force fields have been previously shown to cause significant membrane shrinkage. We find that the use of larger charges at the ester groups alone (as opposed to on all the polar moieties in the head group) remedies the shrinkage. The source of this effect in DPPC lies in the fact that the charge distribution of this polar group profoundly influences its free energy of hydration and, correspondingly, the water distribution around it. In an attempt to rationally tune the ester parameters, the repulsive Lennard–Jones parameters that represent the van der Waals interaction have been refined to reproduce the experimental density and heat of vaporization, and the charges of the ester groups have been tuned to reproduce the experimental free energies of hydration of a series of alkane esters. The new parameters form part of the GROMOS96 53A5 and 53A6 force fields. However, with the new force‐field parameters, the area per lipid in simulations of hydrated DPPC bilayers lies below that of the physiological liquid‐crystalline phase, the implications of which are discussed.     

Thermodynamic study of benzocaine insertion into different lipid bilayers

Despite the general consensus concerning the role played by sodium channels in the molecular mechanism of local anesthetics, the potency of anaesthetic drugs also seems to be related with their solubility in lipid bilayers. In this respect, this work represents a thermodynamic study of benzocaine insertion into lipid bilayers of different compositions by means of molecular dynamics simulation. Thus, the free energy profiles associated with benzocaine insertion into symmetric lipid bilayers composed of different proportions of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine were studied. From the simulation results, a maximum in the free energy (ΔG) profile was measured in the region of the lipid/solution interface. This free energy barrier appears to be very much dependent on the lipid composition of the membrane. On the other hand, the minimum free energy (ΔG) within the bilayer remained almost independent of the lipid composition of the bilayer. By repeating the study at different temperatures, it was seen how the spontaneity of benzocaine insertion into the lipid bilayer is due to an increase in the entropy associated with the process.