Vicky L. Kett

The relevance of the amorphous state to pharmaceutical dosage forms: glassy drugs and freeze dried systems

Many pharmaceuticals, either by accident or design, may exist in a total or partially amorphous state. Consequently, it is essential to have an understanding of the physico-chemical principles underpinning the behaviour of such systems. In this discussion, the nature of the glassy state will be described, with particular emphasis on the molecular processes associated with glass transitional behaviour and the use of thermal methods for characterising the glass transition temperature, Tg. The practicalities of such measurements, the significance of the accompanying relaxation endotherm and plasticization effects are considered. The advantages and difficulties associated with the use of amorphous drugs will be outlined, with discussion given regarding the problems associated with physical and chemical stability. Finally, the principles of freeze drying will be described, including discussion of the relevance of glass transitional behaviour to product stability.

An evaluation of the use of modulated temperature DSC as a means of assessing the relaxation behaviour of amorphous lactose.

AbstractPurpose. To evaluate the use of Modulated Temperature DSC(MTDSC) as a means of assessing the relaxation behaviour ofamorphous lactose via measurement of the heat capacity, glasstransition (Tg) and relaxation endotherm. Methods. Samples of amorphous lactose were prepared by freezedrying. MTDSC was conducted using a TA Instruments 2920 MDSCusing a heating rate of 2°C/minute, a modulation amplitude of ±0.3°Cand a period of 60 seconds. Samples were cycled by heating to 140°Cand cooling to a range of annealing temperatures between 80°C and100°C, followed by reheating through the Tg region. Systems werethen recooled to allow for correction of the Tg shift effect. Results. MTDSC enabled separation of the glass transition from therelaxation endotherm, thereby facilitating calculation of the relaxationtime as a function of temperature. The relative merits of using MTDSCfor the assessment of relaxation processes are discussed. In addition,the use of the fictive temperature rather than the experimentally derivedTg is outlined. Conclusions. MTDSC allows assessment of the glass transitiontemperature, the magnitude of the relaxation endotherm and the valueof the heat capacity, thus facilitating calculation of relaxation times.Limitations identified with the approach include the slow scanningspeed, the need for careful choice of experimental parameters and theTg shift effect.

Development of liposome gel based formulations for intravaginal delivery of the recombinant HIV-1 envelope protein CN54gp140

Mucosally-administered vaccine strategies are widely investigated as a promising means of preventing HIV infection. This study describes the development of liposomal gel formulations, and novel lyophilised variants, comprising HIV-1 envelope glycoprotein, CN54gp140, encapsulated within neutral, positively charged or negatively charged liposomes. The CN54gp140 liposomes were evaluated for mean vesicle diameter, polydispersity, morphology, zeta potential and antigen encapsulation efficiency before being incorporated into hydroxyethyl cellulose (HEC) aqueous gel and subsequently lyophilised to produce a rod-shaped solid dosage form for practical vaginal application. The lyophilised liposome-HEC rods were evaluated for moisture content and redispersibility in simulated vaginal fluid. Since these rods are designed to revert to gel form following intravaginal application, mucoadhesive, mechanical (compressibility and hardness) and rheological properties of the reformed gels were evaluated. The liposomes exhibited good encapsulation efficiency and the gels demonstrated suitable mucoadhesive strength. The freeze-dried liposome-HEC formulations represent a novel formulation strategy that could offer potential as stable and practical dosage form.

Spray congealed lipid microparticles with high protein loading: Preparation and solid state characterisation

The spray-congealing technique, a solvent-free drug encapsulation process, was successfully employed to obtain lipid-based particulate systems with high (10-20% w/w) protein loading. Bovine serum albumin (BSA) was utilised as model protein and three low melting lipids (glyceryl palmitostearate, trimirystin and tristearin) were employed as carriers. BSA-loaded lipid microparticles were characterised in terms of particle size, morphology and drug loading. The results showed that the microparticles exhibited a spherical shape, mean diameter in the range 150-300 μm and an encapsulation efficiency higher than 90%. Possible changes in the protein structure as a result of the manufacturing process was then investigated for the first time using UV spectrophotometry in fourth derivative mode and FT-Raman spectroscopy. The results suggested that the structural integrity of the protein was maintained within the particles. Thermal analysis indicated that the effect of protein on the thermal properties of the carriers could be detected. Spray-congealing could thus be considered a suitable technique to produce highly BSA-loaded microparticles preserving the structure of the protein.

Intravaginal immunization using the recombinant HIV-1 clade-C trimeric envelope glycoprotein CN54gp140 formulated within lyophilized solid dosage forms

Vaccine-mediated prevention of primary HIV-1 infection at the heterosexual mucosal portal of entry may be facilitated by highly optimised formulations or drug delivery devices for intravaginal (i.vag) immunization. Previously we described hydroxyethylcellulose (HEC)-based rheologically structured gel vehicles (RSVs) for vaginal immunization of an HIV-1 vaccine candidate, a soluble recombinant trimeric HIV-1 clade-C envelope glycoprotein designated CN54gp140. Here we investigated the efficacy of lyophilized solid dosage formulations (LSDFs) for prolonging antigen stability and as i.vag delivery modalities. LSDFs were designed and developed that upon i.vag administration they would reconstitute with the imbibing of vaginal fluid to mucoadhesive, site-retentive semi-solids. Mice were immunized with lyophilized equivalents of (i) RSVs, (ii) modified versions of the RSVs more suited to lyophilization (sodium carboxymethyl cellulose (NaCMC)-based gels) and (iii) Carbopol® gel, all containing CN54gp140. NaCMC-based LSDFs provided significantly enhanced antigen stability compared to aqueous-based RSVs. Rheological analysis indicated the NaCMC-based LSDFs would offer enhanced vaginal retention in woman compared to more conventional vaginal gel formulations. All LSDFs were well tolerated in the mouse model. Following i.vag administration, all LSDFs boosted systemic CN54gp140-specific antibody responses in sub-cutaneously primed mice. Induction of CN54gp140-specific antibody responses in the female genital tract was evident. Of all the LSDFs the fastest releasing which was lyophilized Carbopol® gel elicited immune responses comparable to buffer instillation of antigen suggesting that rather than slower sustained release, initial high burst release from the LSDFs may suffice. The boosting of specific immune responses upon i.vag administration indicates that LSDFs are viable mucosal vaccine delivery modalities promoting antigen stability and facilitating intimate exposure of CN54gp140 to the mucosal-associated lymphoid tissue of the female genital tract.

Characterisation of the Interaction of Lactate Dehydrogenase With Tween-20 Using Isothermal Titration Calorimetry, Interfacial Rheometry and Surface Tension Measurements

In this study the nature of the interaction between Tween-20 and lactate dehydrogenase (LDH) was investigated using isothermal titration calorimetry (ITC). In addition the effects of the protein and surfactant on the interfacial properties were followed with interfacial rheology and surface tension measurements in order to understand the mechanism by which the surfactant prevents protein adsorption to the air-water interface. Comparisons were made with Tween-40 and Tween-80 in order to further investigate the mechanism. ITC measurements indicated a weak, probably hydrophobic, interaction between Tween-20 and LDH. Prevention of LDH adsorption to the air-water interface by the Tween surfactants was correlated with surface energy rather than surfactant CMC. While surface pressure appears to be the main driving force for the displacement of LDH from the air-water interface by Tween-20 a solubilisation mechanism may exist for other protein molecules. More generally the results of this study highlight the value of the use of ITC and interfacial measurements in characterising the surface behaviour of mixed surfactant and protein systems.

The measurement of small quantities of amorphous material - Should we be considering the rigid amorphous fraction?

There has been considerable interest in recent years concerning the measurement of small quantities of amorphous material within otherwise crystalline samples. This interest has arisen as a result of the suggestion that many observed phenomena such as anomalous water sorption behavior may be interpreted in terms of a surface layer of glassy material. While constituting only a small percentage of the entire mass of the sample, this layer could nevertheless constitute a significant proportion of the surface and hence have a profound effect on product performance (1). The emphasis to date in both academia and industry regarding this issue has been to attempt to quantify the proportion of amorphous material present with techniques such as microcalorimetry and vapour sorption measurements being, particularly, widely used (2). This approach does, however, carry the concomitant assumption that the amorphous fraction of these semi-crystalline systems is essentially comparable to wholly amorphous material prepared by spray- or freeze-drying. Indeed, this assumption is central not only to the concept of the “quantity” of amorphous material being a definable (and indeed useful) parameter but also to the methods by which the aforementioned techniques are calibrated. Clearly, in theory, an infinite number of amorphous states may be generated by supercooling a material below its melting temperature under different experimental conditions. However, for most pharmaceutical systems, there does appear to be general acceptance of the two-phase model of a semi-crystalline material containing discrete crystalline and amorphous regions of uniform behaviour equivalent to that of “perfect” crystals and “perfect” glasses. Examination of the polymer science literature suggests that there may be alternative approaches to this issue. More specifically, it is now recognized that a proportion of amorphous material in semi-crystalline polymers may exist in a distinct state whereby molecular mobility is restrained to a greater extent than in the “perfect” glass. This material, known as the rigid amorphous fraction, is believed to be associated with the interface between the crystalline and mobile amorphous phases and has properties that are intermediate between the two (3‐8). The formation of the rigid amorphous fraction is shown schematically in Fig. 1 for a polymer that

Investigation Into The Effect of Varying L-leucine Concentration on the product characteristics of Spray-dried Liposome Powders

Objectives  Spray‐dried formulations offer an attractive delivery system for administration of drug encapsulated into liposomes to the lung, but can suffer from low encapsulation efficiency and poor aerodynamic properties. In this paper the effect of the concentration of the anti‐adherent l‐leucine was investigated in tandem with the protectants sucrose and trehalose.

Development of a method to quantify the DNA content in cationic peptide-DNA nanoparticles.

Gene therapy has the potential to provide safe and targeted therapies for a variety of diseases. A range of intracellular gene delivery vehicles have been proposed for this purpose. Non-viral vectors are a particularly attractive option and among them cationic peptides have emerged as promising candidates. For the pharmaceutical formulation and application to clinical studies it is necessary to quantify the amount of pDNA condensed with the delivery system. There is a severe deficiency in this area, thus far no methods have been reported specifically for pDNA condensed with cationic peptide to form nanoparticles. The current study seeks to address this and describes the evaluation of a range of disruption agents to extract DNA from nanoparticles formed by condensation with cationic fusogenic peptides RALA and KALA. Only proteinase K exhibited efficient and reproducible results and compatibility with the PicoGreen reagent based quantification assay. Thus we report for the first time a simple and reliable method that can quantify the pDNA content in pDNA cationic peptide nanoparticles.

Intravaginal immunisation using a novel antigen-releasing ring device elicits robust vaccine antigen-specific systemic and mucosal humoral immune responses

&NA; The generation of effective levels of antigen‐specific immunity at the mucosal sites of pathogen entry is a key goal for vaccinologists. We explored topical vaginal application as an approach to initiate local antigen‐specific immunity, enhance previously existing systemic immunity or re‐target responses to the mucosae. To deliver a protein vaccine formulation to the vaginal mucosal surface, we used a novel vaginal ring device comprising a silicone elastomer body into which three freeze‐dried, rod‐shaped, hydroxypropylmethylcellulose inserts were incorporated. Each rod contained recombinant HIV‐1 CN54gp140 protein (167 &mgr;g) ± R848 (167 &mgr;g) adjuvant. The inserts were loaded into cavities within each ring such that only the ends of the inserts were initially exposed. Sheep received a prime‐boost vaccination regime comprising intramuscular injection of 100 &mgr;g CN54gp140 + 200 &mgr;g R848 followed by three successive ring applications of one week duration and separated by one month intervals. Other sheep received only the ring devices without intramuscular priming. Serum and vaginal mucosal fluids were sampled every two weeks and analysed by CN54gp140 ELISA and antigen‐specific B cells were measured by flow cytometry at necropsy. Vaccine antigen‐specific serum antibody responses were detected in both the intramuscularly‐primed and vaginal mucosally‐primed groups. Those animals that received only vaginal vaccinations had identical IgG but superior IgA responses. Analysis revealed that all animals exhibited mucosal antigen‐specific IgG and IgA with the IgA responses 30‐fold greater than systemic levels. Importantly, very high numbers of antigen‐specific B cells were detected in local genital draining lymph nodes. We have elicited local genital antigen‐specific immune responses after topical application of an adjuvanted antigen formulation within a novel vaginal ring vaccine release device. This regimen and delivery method elicited high levels of antigen‐specific mucosal IgA and large numbers of local antigen‐reactive B cells, both likely essential for effective mucosal protection. Graphical abstract Figure. No caption available.