Thermodynamic considerations of solvent/enhancer uptake into a model membrane.

The aim of this study was to conduct a thermodynamic analysis of the uptake of solvents into a model membrane as a precursor to skin transport studies. The investigation was designed so that the methodology may be applied to analyse data produced from measurement of the uptake of enhancers into skin. The uptake of a series of alcohols into polydimethylsiloxane (silicone) membranes in the temperature range 5-45 degrees C was examined. A thermodynamic analysis of the data was performed to provide fundamental insight into the uptake process. A simple structure activity relationship was found to exist for the uptake of alcohols with a carbon chain length greater than four, with additional methylene groups exponentially decreasing the equilibrium uptake. Two separate straight lines were observed in the van't Hoff plot for the equilibrium solvent uptake above and below 16 degrees C. The two separate straight lines in the van't Hoff plot suggest a change in the mechanism of solvent uptake and solvent structure in the membrane above and below 16 degrees C. This is likely to have implications for the effect of the solvents on the partitioning of drugs into the membrane and will be used to provide insight into dynamic measurements of the effect of temperature on the transport of drug molecules in the same vehicles, across the membrane. The analysis described here should provide a useful methodology for investigating the uptake of solvents into model membranes.

Inter- and intralaboratory variation of in vitro diffusion cell measurements: an international multicenter study using quasi-standardized methods and materials.

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. "Standardized" calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 microg cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 microg cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required.

Thermodynamic considerations of microgel swelling behavior.

A simple but novel thermodynamic model is presented, based upon van't Hoff analysis, for the reversible swelling behavior of colloidal microgels. The swelling, as a function of temperature, of poly(N-isopropylacrylamide/N,N'-methylenebisacrylamide) as well as poly(N-isopropylacrylamide/vinylpyridine/N,N'-methylenebisacrylamide) and poly(N-isopropylacrylamide/acrylic acid/N,N'-methylenebisacrylamide) microgel dispersions in H2O and D2O has been studied by photon correlation spectroscopy (PCS). PCS data was used to obtain the hydrodynamic diameter and hence the volume of the microgels (before and after reconstitution following freeze-drying) as a function of temperature. The choice of standard reference states, for analyzing the data attained, is discussed, and the one selected is that of the volume of the microgels at 333 K in H2O. For all microgels examined the volume, at this temperature, is shown to be independent of solvent (H2O, D2O). The derived data has allowed the exploration of a novel thermodynamic approach to the study of the swelling behavior of the microgels. The constant volume, at 333 K, for each of the polymer systems constituting the microgels is suggested to be an intrinsic property of the polymers themselves.

UV-spectrophotometry study of membrane transport processes with a novel diffusion cell.

A novel diffusion cell has been constructed which allows study of membrane diffusion processes without the need for sampling of the receiver compartment, that is highly sensitive and, being based around a diode array spectrophotometer also allows for continuous, real-time recording of multi-species concentration changes in the receiving compartment. The system is controlled to operate isothermally (via a Peltier control system) at temperatures between 15 and 85 degrees C. To examine the performance of this novel design, the transfer of tetracaine from a preparation in PEG 400 (20% tetracaine in PEG 400) has been studied. The results have been used to determine flux, lag time and related parameters. The performance of the novel cell is compared with results from traditional Franz cell diffusion studies.

The stability of benzoyl peroxide by isothermal microcalorimetry.

Isothermal microcalorimetry may be used to determine kinetic and thermodynamic parameters for chemical reactions. This paper reports rate constants, determined as a function of temperature, and the activation enthalpy for the degradation of solid benzoyl peroxide as determined by isothermal microcalorimetry. Studies were conducted on aqueous suspension phase, solid benzoyl peroxide. In addition, supporting evidence is cited from work carried out in this laboratory on the solution phase degradation of benzoyl peroxide using UV-visible spectrophotometry. The activation energy obtained by microcalorimetry was E(a)=137.8+/-6.6 kJ mol(-1) and the activation energy obtained from UV-visible spectrophotometry was E(a)=112.7+/-4.2 kJ mol(-1).

The stability of benzoyl peroxide formulations determined from isothermal microcalorimetric studies.

Recent developments in the analysis of microcalorimetric data output allow the possibility of determining both thermodynamic and kinetic parameters for complex reaction systems. Such experiments routinely take around 50 h, hence qualifying for the description rapid. The methods have earlier been applied to a study of the stability of benzoyl peroxide itself in aqueous suspension. This paper reports the results of isothermal microcalorimetric study of the stability of benzoyl peroxide in the presence of a wide range of excipients and in formulated materials. The results are shown to assist in formulation design, are achieved rapidly and are derived from direct experimental study of the complex systems themselves. That is, no ancillary information is required nor are the studies invasive or destructive.

A microcalorimetric comparison of the anti-Streptococcus mutans efficacy of plant extracts and antimicrobial agents in oral hygiene formulations.

AIMS: This study aimed to evaluate the efficacy of 'natural' putative antimicrobial agents against Streptococcus mutans and to compare these with synthetic agents using the flow microcalorimeter. Streptococcus mutans is one of the oral pathogens responsible for dental caries. METHODS AND RESULTS: Traditional microbiological techniques are invasive and destructive unlike flow microcalorimetry. This rapid technique was used to continuously monitor the power output (bioactivity) of Strep. mutans with reproducibility, precision and accuracy. The antibacterial agents found in oral hygiene products and all the natural agents tested showed anti-Strep. mutans ability. CONCLUSION: In this study microcalorimetry identified agents that had a biological effect and quantified the rate of kill achieved enabling four broad categories of antimicrobial agent to be defined. SIGNIFICANCE AND IMPACT OF THE STUDY: Microcalorimetric data are a better indication of antimicrobial efficacy than merely determining concentrations at which an antimicrobial agent is bacteriostatic or bactericidal.

QSAR based on biological microcalorimetry.

In this paper we describe a QSAR based on biological microcalorimetry for a set of antimicrobial hydrazides acting against Saccharomyces cerivisiae and Escherichia coli. Results show that an extrathermodynamic relationship exists based upon partitioning (log P(TA)) and microcalorimetrically measured biopotencies using the same cell systems. Moreover, the extrathermodynamic relationship between drug potencies for these two cell systems shows that both cellular systems appear to behave in the same way with respect to the importance of partitioning. This means that the same set of congeneric compounds experience a similar environment in the two systems. This represents a lateral validation of the method and discloses the validity of the QSAR model.

Pharmaceutical microcalorimetry: applications to long-term stability studies.

Calorimetry has been a mainstay of stability analyses for some time in the form of differential scanning microcalorimetry (DSC). This technique exploits high (relatively) temperature studies of pure materials and of formulations to accelerate any degradation or interactions. The behaviour of the material at storage or ambient conditions is then estimated via extrapolation from the Arrhenius equation. Recent developments in isothermal microcalorimetry allow the direct determination of both kinetic and thermodynamic parameters for long, slow reactions from studies conducted at appropriate temperatures and under designated environmental control (pH, pO2, RH etc.). This review introduces the kinetic analysis of microcalorimetric data and, through selected examples, shows applications of the method.

An acrylic ampoule represents a denture surface in a microcalorimetric biofilm problem.

An acrylic microcalorimetric ampoule with inserts represented a denture surface in a Streptococcus mutants biofilm study. The biofilm of micro-organisms did not contain as many micro-organisms as those previously studied in planktonic suspension. A more sensitive microcalorimeter (Thermometric's thermal activity monitor) was required to detect the metabolism of S. mutants adhered to an engineered acrylic flow-through ampoule. Biofilm heat outputs were obtained in the acrylic ampoule following standardized procedures with cryopreserved aliquots of a homogeneous suspension of S. mutants.

Determination of receptor-bound drug conformations by QSAR using flexible fitting to derive a molecular similarity index.

Results are presented for a QSAR analysis of bisamidines, using a similarity index as descriptor. The method allows for differences in conformation of bisamidines at the receptor site to be taken into consideration. In particular, it has been suggested by others that pentamidine binds in the minor groove of DNA in a so-called isohelical conformation, and our QSAR supports this suggestion. The molecular similarity index for comparison of molecules can be used as a parameter for correlating and hence rationalising the activity as well as suggesting the design of bioactive molecules. The studied compounds had been evaluated for potency against Leishmania mexicana amazonensis, and this potency was used as a dependent variable in a series of QSAR analyses. For the calculation of similarity indexes, each analogue was in turn superimposed on a chosen lead compound in a reference conformation, either extended or isohelical, maximising overlap and hence similarity by flexible fitting.

Different immunomodulator regulated patterns of effector-target cell interactions: a novel application of microcalorimetry.

Microcalorimetric studies of target/effector cell interactions are reported. Significant changes in the observed thermal profiles are seen upon pre-incubation of the effector cells with chemical immunomodulators. The results of these latter studies indicate that the microcalorimetric technique can reveal differences in the modes of action of different groups of immunomodulators. It is proposed that the technique can be used to screen drug activities and, following further analytical work, to contribute to an understanding of modes of action.

Microcalorimetric studies of the initial interaction between antimycobacterial drugs and Mycobacterium avium.

To further our understanding of the mechanisms by which ethambutol potentiates the effect of other antimycobacterial drugs on mycobacteria we have studied the initial physico-chemical interaction between ethambutol and the Mycobacterium avium cell envelope using batch reaction microcalorimetry. When strains of M. avium were exposed to ethambutol an immediate endothermic reaction was recorded. When the M. avium cells were pre-treated with ethambutol this strongly affected the initial interaction between streptomycin and the bacterial cell surface. When the M. avium cells were simultaneously exposed to a combination of ethambutol and streptomycin an altered initial interaction with streptomycin was seen. These data suggest that ethambutol may potentiate the effect of other antibacterial drugs on M. avium by increasing cell wall permeability.

Studies on the mechanisms of the synergistic effects of ethambutol and other antibacterial drugs on Mycobacterium avium complex.

Synergistic effects of combinations of anti-mycobacterial drugs on Mycobacterium avium complex (MAC) in vitro was studied by radiometric respirometry. Pronounced synergy was seen for several drug combinations where ethambutol was found to be the key drug in the synergistic potentiation. Microcalorimetric studies show that a very rapid physico-chemical interaction occurs between the cell-surface of MAC and ethambutol. When MAC cells were pretreated with ethambutol and then subjected to streptomycin the thermal response significantly differed from that seen with MAC cells which had not been pretreated. The typical thermal effects of the interaction of ethambutol with live and UV-killed MAC cells was not seen with heat-killed MAC cells. It is proposed that specific cell-surface protein(s) act as receptors in the initial interaction with ethambutol.

Flow microcalorimetric assay of antibiotics--I. Polymyxin B sulphate and its combinations with neomycin sulphate and zinc bacitracin on interaction with Bordetella bronchiseptica (NCTC 8344).

A flow microcalorimetric assay for polymyxin B sulphate has been developed which has a better reproducibility (relative standard deviation less than 3%) and sensitivity (0.35 micrograms ml-1) than conventional microbiological assays, and requires an assay time of ca. 4.5 h. The combinations with zinc bacitracin, with neomycin sulphate, and with both zinc bacitracin and neomycin sulphate indicate antagonism between these antibiotics upon interaction with Bordetella bronchiseptica (NCTC 8344). The combinations of all three antibiotics assayed were: (1) equimolar proportions; and (2) those proportions present in the commercial preparation TrisepR (ICI, Macclesfield, UK).

Flow microcalorimetric assay of antibiotics--II. Neomycin sulphate and its combinations with polymyxin B sulphate and zinc bacitracin on interaction with Bacillus pumilus (NCTC 8241).

A flow microcalorimetric assay for Neomycin has been developed which is monitored through interaction of the antibiotic with Bacillus pumilus as the test organism. The assay has better reproducibility (relative standard deviation 2.3%) and is more sensitive than conventional microbiological bioassay (0.5-2 micrograms ml-1). The effects of combinations with zinc bacitracin, with polymyxin B sulphate, and with both zinc bacitracin and polymyxin B sulphate (both in equimolar proportions), and in those proportions present in the commercial preparation TrisepR (ICI, Macclesfield, UK) have also been investigated. Synergy was observed for the combinations of Neomycin with the other two antibiotics in binary mixtures at the relative proportions found in TrisepR. The addition of all three antibiotics at the levels used in TrisepR did not show synergy. However, addition of all three antibiotics at equimolar concentrations did show synergy. It is suggested that microcalorimetry may be useful in in vitro experiments for exploring the relative proportions required for maximal effect in antibiotic combinations.

Flow microcalorimetric assay of antibiotics--III. Zinc bacitracin and its combinations with polymyxin B sulphate and neomycin sulphate on interaction with Micrococcus luteus.

A flow microcalorimetric assay for zinc bacitracin has been developed which has better reproducibility (relative standard deviation less than 2%) and sensitivity (0.02 micrograms ml-1) than conventional microbiological assays, and requires an assay time of between 7.5-9 h. The assay is not suitable for zinc bacitracin determinations in the presence of equimolar concentrations of polymyxin B sulphate or neomycin sulphate, or of these antibiotics in the proportions in which they occur in the commercial preparation Trisep (ICI, Macclesfield, UK).