Comparison between DSC and TMDSC in the investigation into frozen aqueous cryoprotectants solutions.

PURPOSE: The influence of thermal parameters in the observation of thermal events and in the calculation of heat transformation in aqueous cryoprotectant solutions after freezing was investigated using conventional differential scanning calorimetry (DSC) and temperature-modulated DSC (TMDSC), respectively. METHODS: The systems under study were formed by pure water and diluted aqueous solutions of mannitol, trehalose, sucrose, sorbitol, and glycine. The influence of different combinations of frequency and amplitude was analyzed in heating-cooling and heating-iso TMDSC scans. RESULTS: Trehalose, sucrose, and sorbitol present a lesser critical temperature of primary drying than other cryoprotectants studied. The calorimetric variables selection is crucial to detect or not the thermal events, or to detect so with different numerical values. Then, the values of the calorimetric parameters determined are different if measured in a mode of heating-cooling or heating-iso. The TMDSC method-1 used in this study employs a higher number of cycles in each thermal event. The use of Lissajous figures and the study of the C(p in-phase) signal evolution will allow us to understand the complexity of the events detected. CONCLUSIONS: The comparative study of both techniques points to the selection of conventional or modulated technique depending on the type of system and the nature of the studied events.

Normal values in ambulatory oesophageal pH monitoring at two levels in Spain.

AIM: Upper oesophageal pH monitoring may play a significant role in the study of extra-oesophageal GERD, but limited normal data are available to date. Our aim was to develop a large series of normal values of proximal oesophageal acidification. METHODS: 155 healthy volunteers (74 male) participated in a multi-centre national study including oesophageal manometry and 24 hours oesophageal pH monitoring using two electrodes individually located 5 cm above the LOS and 3 cm below the UOS. RESULTS: 130 participants with normal manometry completed all the study. Twelve of them were excluded for inadequate pH tests. Twenty-seven subjects had abnormal conventional pH. The remaining 91 subjects (37 M; 18-72 yrs age range) formed the reference group for normality. At the level of the upper oesophagus, the 95th percentile of the total number of reflux events was 30, after eliminating the meal periods 22, and after eliminating also the pseudo-reflux events 18. Duration of the longest episodes was 5, 4 and 4 min, respectively (3.5 min in upright and 0.5 min in supine). The upper limit for the percentage of acid exposure time was 1.35, 1.05 and 0.95%, respectively. No reflux events were recorded in the upper oesophagus in 8 cases. CONCLUSION: This is the largest series of normal values of proximal oesophageal reflux that confirm the existence of acid reflux at that level in healthy subjects, in small quantity and unrelated to age or gender. Our data support the convenience of excluding pseudo-reflux events and meal periods from analysis.

A linear mixed-effects statistical model for in-vivo evaluation of recombinant human growth hormone implants in hypophysectomized rats.

We have used a linear mixed-effects statistical model to evaluate previously published results of body weight evolution in hypophysectomized (Hpx) rats after administration of two different controlled-release formulations of recombinant human growth hormone (rhGH). Using the linear mixed-effects model, it was possible to distinguish between maximal pharmacological response with time in different subjects and relate it to the structure of the different formulations, the release of the hormone from them and the time necessary to obtain a quantitative result as a consequence of the hormone activity, contrary to the multivariate variance analysis model (MANOVA) used in our earlier work. These results confirmed that the maximum body weight gain obtained with the controlled-release implants is similar to that with subcutaneous rhGH, but with the advantage that laminar biodegradable implants need to be administered only once every 2 weeks.

A mathematical model for interpreting in vitro rhGH release from laminar implants.

Recombinant human growth hormone (rhGH), used mainly for the treatment of growth hormone deficiency in children, requires daily subcutaneous injections. The use of controlled release formulations with appropriate rhGH release kinetics reduces the frequency of medication, improving patient compliance and quality of life. Biodegradable implants are a valid alternative, offering the feasibility of a regular release rate after administering a single dose, though it exists the slight disadvantage of a very minor surgical operation. Three laminar implant formulations (F(1), F(2) and F(3)) were produced by different manufacture procedures using solvent-casting techniques with the same copoly(D,L-lactic) glycolic acid (PLGA) polymer (Mw=48 kDa). A correlation in vitro between polymer matrix degradation and drug release rate from these formulations was found and a mathematical model was developed to interpret this. This model was applied to each formulation. The obtained results where explained in terms of manufacture parameters with the aim of elucidate whether drug release only occurs by diffusion or erosion, or by a combination of both mechanisms. Controlling the manufacture method and the resultant changes in polymer structure facilitates a suitable rhGH release profile for different rhGH deficiency treatments.

A family of metrics for biopolymers based on counting independent sets.

We introduce a new family of metrics for graphs of fixed size, based on counting-independent sets. Our definition is simpler and easier to calculate than the edge ideal metric family defined by Llabrés and Rosselló without loosing any of its abstract properties. We contrast them on some examples with graphs that represent protein secondary and three-dimensional (3D) structures. We conclude that although the edge ideal metrics are faster to calculate on some sparse graphs, in general, the independent set metrics are more tractable.

In vivo-in vitro study of biodegradable methadone delivery systems.

Three one-week controlled-release methadone formulations: polylactic acid microspheres (F-PLA) and poly(lactide-co-glycolide) microspheres (F-PLGA) with 24 and 30% methadone content, respectively, and an implant of 50:50 poly(lactide-co-glycolide): methadone, were evaluated in vitro and in vivo. The implant released the total amount of methadone in vitro while microsphere formulations released the methadone incompletely, 63% from F-PLA and 85% from F-PLGA in a week. Methadone release in vivo was estimated by deconvolution, F-PLGA giving a bioavailability >99% (methadone was totally released in 48h), while the estimated bioavailability of F-PLA was lower than expected. The bioavailability of the implant by deconvolution was around 60%, but absence of methadone in the implant indicated its complete release. These differences are due to an increase in methadone clearance after 72 h of the in vivo experimental period had passed, disturbing a good in vivo-in vitro correlation. A linear correlation between in vitro methadone release and in vivo release calculated from the amount of drug remaining within the implant, was found until the drug was completely released.

Rheological properties of PLGA film-based implants: correlation with polymer degradation and SPf66 antimalaric synthetic peptide release.

This paper reports on the rheological properties of poly(D,L-lactic-co-glycolic acid) polymers (PLGA) dispersions used to form films and of the implants prepared by compression of SPf66 antimalaric peptide between several films, before application and during drug release. 25% PLGA (M(w)=48,000Da) dispersions in dichloromethane showed viscous Newtonian behaviour, being easy flowing and adaptable to the moulds. Evolution of viscoelastic properties, polymer molecular weight, and SPf66 release pattern from the implants immersed in various media was evaluated. Oscillatory shear test showed that freshly prepared implants have an elastic modulus, G', greater than the viscous modulus, G", being both practically independent of angular frequency. After 6 weeks immersion in a pH 7.4 phosphate buffer, G' and G" increased in almost one order of magnitude, despite of a significant polymer degradation. Polymer molecular weight decreased slowly during the first 10 days of immersion (a similar pattern was obtained at pHs 2 and 7.4) and then the degradation process accelerated (degradation index on day 7 equals to 0.89, and on day 14 equals to 16.5). SPf66 release profile followed a pattern similar to that of the polymer degradation index. These observations are explained in terms of changes in polymer structure and conformation that happen in the implant.

A new family of metrics for biopolymer contact structures.

Contact structures are simplified representations of biopolymers' three-dimensional structures. In this paper we introduce a new family of metrics (dm)m >/=3 for contact structures of a fixed length, based on their representation by means of edge ideals of a polynomial ring, that generalize Reidys and Stadler's subgroup metric for RNA secondary structures. We study some abstract properties of these metrics, and we obtain explicit descriptions of them for some values of m.

One-month sustained release microspheres of 125I-bovine calcitonin. In vitro-in vivo studies.

To obtain a 1-month release formulation of 125I-bovine calcitonin, microspheres were prepared with three different PLA copolymers, PLGA I (mol. wt. [MW]=30000), polyethyleneglycol (PEG)-PLGA (MW=34000) and PLGA II (MW=12000) using the double emulsion method. The release of 125I-bovine calcitonin was assayed in vitro using dialysis bags at 37 degrees C in isotonic phosphate buffer (pH 7.4). The in vitro release results indicated a very slow release rate for an optimal 1-month sustained release formulation. 125I-bovine calcitonin microspheres were administered under the skin on the back of Wistar rats and the radioactivity at the injection site was subsequently measured over a 4-week period. The in vitro and in vivo profiles were affected by the weight average molecular weight of the copolymers. The 125I-bovine calcitonin release rate was faster from microspheres prepared with PLGA II (MW=12000) than from microspheres prepared with higher molecular weight copolymers (PLGA I and PEG-PLGA). Microspheres prepared with PLGA II (MW=12000) release 100% of the dose in 1 month, in vivo release profiles presented two phases, during the first 2 weeks approximately 70% of the 125I-bovine calcitonin injected was released, followed by a second slower phase.

Methadone implants for methadone maintenance treatment. In vitro and in vivo animal studies.

Methadone implant formulations elaborated with polylactide-co-glycolide (PLGA) and polylactic acid (PLA) for 1 week and 1 month release duration, respectively, were evaluated in vitro and in vivo. One-week implants prepared with methadone clorhydrate, methadone clorhydrate/methadone base blend or methadone base were tested in vitro. Results showed that the methadone release rate decreased as the methadone base increased. The best release profile was achieve when the methadone base implants, made by compression of a 50:50 PLGA (12 kDa) and methadone base mix, were coated with PLA (30 kDa). For 1-month implants, the methadone base load was increased to 65% and PLA of 30 kDa was used as a matrix component. In this case the implants were coated with the same polymer. Deconvolution methods could not be used for in vivo release estimation because an increase in methadone clearance was observed with methadone clorhydrate solution multiple-dose treatment. Therefore the amount of drug remaining within the implants was evaluated and the deconvolution was only used to establish the release profile range. The upper limit was estimated applying the absorption-disposition function obtained after multiple-dose administrations while the lower curve was estimated using the single-dose function. Methadone serum levels were maintained around 200 ng/ml during 1 week and approximately 5 weeks with the optimised implants. In vivo-in vitro correlations were always very good with slopes near 1.

Design and evaluation of sustained-release tablets of lithium in a fat matrix and its bioavailability in humans.

The development of sustained-release lithium (Li) tablets, intended to release the active principle at a rate of 1.0 mM/h for 10 h, was undertaken. The parameters used for the control of the release were the glyceril palmite-stearate content, the carboxypolymethylene content, and the compression force. The experimental design is based on Hadamard's matrices and is of the adaption by stages type. The formulation seen as optimal from in vitro assays was later assessed in vivo by a crossover study of six subjects. The parameters used to measure the bioavailability were the total amount of Li excreted in the urine in the 96 h following ingestion, the maximum urinary excretion rate, and the time at which this rate was reached. The acceptability interval for the first two parameters was established from the theoretical curve of urinary excretion, which was calculated by convolution of the desired in vivo release variable (1.0 mM/h for 10 h) by the absorption-disposition variable obtained after administering the preparation in Li carbonate capsules. The results obtained show that the bioavailability of the formulation is 75% of the immediate-release formulation used as control and that the release rate, although close to the desired value, lasts only 7 or 8 h; these results agree with those given by numerical deconvolution using the mean urinary excretion curves.

Quantification of the effect of excipients on bioavailability by means of response surfaces II: Amoxicillin in fat-silica matrix.

This report studies the bioavailability of amoxicillin in different fat-silica matrixes. A urinary excretion study was carried out on four formulations containing fat and silica excipients. The formulations were administered to 24 healthy volunteers according to a Latin-square design. The following percent proportions of fat-silica were used: 15:3.75, 15:7.50, 30:3.75, and 30:7.50. The urinary excretion curves were characterized using the quantity of unchanged drug excreted between 0-2 and 0-12 hr postadministration, respectively as parameters. The ANOVA results showed that both excipients had an additive effect on the quantity of drug excreted between 0 and 2 hr, whereas the effect on the quantity of drug excreted between 0 and 12 hr was also one of interaction between both excipients. Quantification of the ANOVA results in terms of excipient content was conducted by means of the adequate linear functions. At the same time, a dissolution study was carried out using the quantity of drug dissolved in 30 and 180 min as parameters. The behavior was similar to that encountered for the in vivo parameters.

Quantification of the effect of excipients on bioavailability by means of response surfaces I: Amoxicillin in fat matrix.

A study was carried out to determine the effect of a fat excipient on the bioavailability of amoxicillin tablets. Three formulations with a fat excipient content of 10, 20, and 30%, respectively, were administered to 15 healthy volunteers according to a Latin-square design. The excretion curves were characterized with the help of two parameters, namely, the quantity of drug excreted between 0-2 and 0-12 hr postadministration, respectively. The effect of the fat excipient content was quantified with the use of polynomials whose corresponding orders were given by the ANOVA. In the case of both parameters, a quadratic response to the fat excipient content was found. At the same time, a dissolution study was carried out using a previously established method. Here, the parameters used to characterize the dissolution curves were the quantities of dissolved drug in 30 and 180 min, respectively. Again, a quadratic response to the fat excipient content was observed in the case of both parameters.

Quantification of the effect of excipients on bioavailability by means of response surfaces III: In vivo--in vitro correlations.

This study compares one of the previously studied formulations with commercial amoxicillin capsules. The results indicate that the percentage of the dose absorbed is similar in both formulations; nevertheless, the amoxicillin capsules present a higher absorption rate. The in vivo--in vitro correlations in terms of response surfaces, and the general correlation among all the formulations studied in the three articles of this series is discussed. The quantity of drug excreted in 2 hr and the quantity of drug dissolved in 30 min presents a correlation coefficient r = 0.9458 (p less than 0.01) and the quantity of amoxicillin excreted in 12 hr and the quantity dissolved in 180 min presents a correlation coefficient r = 0.9761 (p less than 0.01).

Statistical assessment of between batch stability equivalence.

A statistical method for testing the equivalence between batches regarding their stability is proposed. This method is based on the statistical linear model making use of a set of dummy variables to code the different batches. The method gives us the point estimates of the slope and zero intercept of one batch, and the differences and the corresponding confidence intervals with the remaining batches. In a second step, zero intercepts and slopes are estimated for all the batches. Stability equivalence assessment is based on the comparison of the confidence intervals for the differences between batches with the maximum difference allowable. The main advantages of this method are the possibility to compare several batches, to disclose the equivalence stability criteria from the statistical hypothesis about the equality between slopes, and the joint estimated of the residual variance whatever the decision to pool or not the data from different batches. This method is illustrated with two data set; the first one, previously published by other authors, involved six batches; the second data set include two batches and arose in a stability study of a commercial human insulin conducted in our laboratory.

Comparative study of protein molecular weights by size-exclusion chromatography and laser-light scattering.

High-performance size-exclusion chromatography (SEC) based on UV-Vis detection is a relative technique for molecular weight determination whereas procedure based on multi-angle laser light scattering (MALLS) is both rapid and absolute. The two methods using recombinant human growth hormone (rHGH) and beta-lactoglobulin samples were compared. A calibration curve for the chromatographic system was generated based on standard proteins and the data were fitted by least squares to a third order polynomial model. The molecular weight from the conventional SEC method for both proteins was higher than the reported values. The molecular weight of rHGH from MALLS was 23.1+/-0.57 and 21.2+/-0.80 kDa using differential refractive index (SEC-MALLS/RI) and UV (SEC-MALLS/UV-Vis) detectors as mass detectors. Both values agree, within experimental error with the molecular weight sequence of rHGH, 22.1 kDa. In contrast, the molecular weight from LS for beta-lactoglobulin was 22.5+/-0.55 kDa by SEC-MALLS/RI and 23.0+/-1.22 kDa by SEC-MALLS/UV-Vis, respectively, values always higher than those supplied by the manufacturer, 18.4 kDa. The reproducibility of the SEC-MALLS/UV-Vis method versus the SEC-MALLS/RI method was performed using the concordance correlation coefficient. The method's reproducibility was accepted by assuming a precision of 98% and a 1% loss in precision.

Stability study of human serum albumin pharmaceutical preparations.

The influence of temperature on the stability of human serum albumin (HSA) pharmaceutical preparations has been studied by size-exclusion high-performance liquid chromatography with multi-angle laser-light-scattering detection and by particle-size analysis. The behaviour of HSA in two pharmaceutical preparations stored at different temperatures (40, 55 and 70 degrees C) followed the same pattern--an increase in the relative percentage of dimer (MW 132 000 Da) and aggregate (MW > 200 000 Da), and then a decrease in the concentration of all species and, finally, sudden protein coagulation. These results suggest a time- and temperature-dependent process. At 70 degrees C, monomer only was detected for both preparations; the amount remaining was 83 and 72% for formulations A and B, respectively. Analysis of size-distribution curves also seems to confirm these results. Initially, three distributions were observed with length-volume mean diameters (d1,v) of 1.67, 10.6 and 57 microm. After 80 days at 55 degrees C, only two distributions were observed, with d1,v of 3.07 and 76 microm. An additional study using pure HSA at different concentrations (0.3, 2.5, 5 and 10% w/v) and storage at 75 degrees C was performed to determine the influence of the concentration of auxiliary substances and of the HSA. Only when the HSA concentration was 0.3% w/v did the remaining fraction of HSA fit a Prout-Thompkins nucleation model. Initially three distributions with mean sizes of 2, 20 and 40 microm were observed whereas at the end of the assay only one distribution, mean size 129 microm, was seen. The methodology used enabled us to separate the HSA degradation products and to determine the absolute molecular weight of albumin monomer and dimer. It is possible to conclude that the degradation mechanism for the formulations studied is complex, and that it is possible to fit the degradation data to Prout-Thompkins kinetics only when the concentration of HSA is low enough (0.3% w/v).

[The bioavailability of drugs administered via a minimal jejunostomy catheter. A clinico-experimental study]. / Biodisponibilidad de los fármacos administrados por yeyunostomía mínima a catéter. Estudio clínico-experimental.

For the evaluation of the bioavailability of drugs administered through a minimal jejunostomy catheter (MJC) postoperatively, ampicillin and metronidazol were administered via this route. The study with ampicillin consisted of a first cross-over experimental trial, in which six dogs were used, and a second based on the experience with four human volunteers. As a measure of the absorption of the drug, the serum levels of the lower plasma area are monitored. The statistical analysis was done by means of logarithmic transformation of the areas, and two-way analysis of variance. The posterior distribution of the relative bioavailability of the drug was obtained by applying a bayesian method. The results showed an important decrease of the ampicillin absorption, while the expected bioavailability when administered through MJC was 0.24, and the 95% interval (0.19, 0.30). The expected bioavailability of metronidazol when administered through MJC was 0.84, and the 95% interval (0.66, 1.07). This new route of drug administration in patients who undergo abdominal surgery, is shown to be of great importance from a clinical point of view, and should be kept in mind.

Study of quality and stability of ursodeoxycholic acid formulations for oral pediatric administration.

This paper describes a rational method of characterizing the biopharmaceutical stability of two oral suspensions of ursodeoxycholic acid (UDCA) used in pediatrics. Because there is no commercial presentation of UDCA that can administer appropriate doses for infants and children, an active pharmaceutical ingredient (API) formulation is required. Due to its very low solubility and low dose in the formula (1.5%), two different suspensions with minimal use of excipients were studied, avoiding the use of complex additives and those not recommended by the European Medicines Agency (EMA). Adherence to Standard Operating Procedure (SOP) allows the preparation of formulations with appropriately sized and stable particles, and suitable rheological behavior in withdrawing the dose after stirring. Dose uniformity, expressed as mass and content variability, was determined using the criteria of the European and the United States Pharmacopoeia. Additionally, dose content variation of every mass determined was studied. A rational method was developed for determining the dose uniformity of UDCA in suspensions, whether freshly prepared or after storage under different conditions for 30 and 60 days. This method permits detection of differences between doses taken at different heights in the vessel at various times and storage conditions. UDCA was stable under all conditions studied, requiring the presence of glycerol in the formulation to obtain the declared API value after stirring. Storage of UDCA suspensions in a refrigerator increased variability between doses.

Evaluation of cholecystokinin (CCK-8) peptide thermal stability for use as radiopharmaceutical by means isothermal and nonisothermal approaches.

The purpose of this research was to study the thermal stability of cholecystokinin octapeptide (CCK-8) in aqueous solution at pH 12 and ionic strength 0.01 M, which were kept as constants, by using isothermal and nonisothermal methods. The isothermal decomposition of CCK-8 was investigated as a function of temperature (40 degrees C to 70 degrees C). Nonisothermal stability studies were performed using a linear increasing temperature program. Two different nonisothermal studies were carried out at 0.25 degrees K and 0.5 degrees K per hour, and the temperature interval varied from 40 degrees C to 82 degrees C. The degradation of CCK-8 followed first-order kinetics, obeying the Arrhenius equation in the experimental temperature range. This indicated that the degradation mechanism of CCK-8 could be the equal within the temperature range studied. The nonisothermal approach resulted in activation energy (Ea) and shelf-life (t90%) values that agree well with those obtained by the isothermal method. The level of uncertainty in the estimates of t90% and Ea values is determined mainly by the extent of drug degradation and temperature change during the experiment. Therefore, nonisothermal experiments save time, labor and materials (i.e. the amount of drugs necessary to conduct the experiment) compared to the classic isothermal experiments, if they are performed using a suitable experimental design and a precise analytical method.