Influence of protein on mannitol polymorphic form produced during co-spray drying.

The stabilizing ability of the excipient on pharmaceutically relevant proteins for potential therapeutic use is an extensive area of research but the effect the protein has on the excipient is rarely reported. The influence of two model proteins on the polymorphic behaviour of mannitol during spray drying was therefore investigated. Spray dried mannitol/protein blends were characterised structurally using X-ray powder diffraction (XRPD) and Fourier transform Raman spectroscopy (FT-Raman) and thermally by differential scanning calorimetry (DSC) and also thermogravimetric analysis (TGA). To assess the long term storage stability, samples were subjected to conditions of elevated temperature and relative humidity (RH). Structural and thermal analysis of the samples showed that upon spray drying mannitol could be completely amorphous or crystalline dependent on the protein co-spray dried. Upon storage at elevated temperature and RH different polymorphic forms of mannitol (beta and delta) were evident again dependent on the protein co-spray dried. Under the conditions employed there was a polymorph directing effect on mannitol dependent on the protein with which it was co-spray dried with co-solute effects on relative water levels being indicated as a major factor in directing the polymorph.

The characterization and comparison of spray-dried mannitol samples.

BACKGROUND: Following the production of spray-dried mannitol powders, it is essential that the polymorphic content of each individual product is completely characterized. The implications of the polymorphic behavior of mannitol are immense. The appearance or disappearance of a crystalline form within a dosage form can have costly repercussions and lead to a dosage form being withdrawn. METHOD: In this study, commercially available and laboratory-produced spray-dried mannitol products were characterized to establish the polymorphic content of each. Their polymorphic behavior was also characterized after laboratory scale pharmaceutical processes. Thermal analysis employed differential scanning calorimetry, thermogravimetric analysis, and isothermal microcalorimetry. Structural analysis of the samples was obtained using X-ray powder diffraction and Fourier transform Raman spectroscopy. RESULTS: Structural analysis revealed that alpha- and beta- polymorphic forms were present in the commercial samples and some contained a mixture of polymorphs. Reprocessing employing spray drying indicated alpha- to beta- polymorphic transitions occurred within some of the samples. CONCLUSION: It is essential that preformulation studies where spray-dried mannitol products are to be employed must take into account its polymorphic behavior upon supply, processing, and subsequent storage.

Do co-spray dried excipients offer better lysozyme stabilisation than single excipients?

The inherent instability of proteins when isolated from their native conditions creates the necessity of suitable stabilisation techniques. Because of the instability of proteins in solution it is often necessary to produce them as solid formulations. A method of producing relatively stable, solid protein pharmaceuticals is to incorporate them with a suitable excipient into an amorphous matrix by dehydration. The use of spray dried multiple excipient/single protein blends was compared to single excipient/protein systems using lysozyme as a model protein to establish the stabilising ability of such systems. Unprocessed controls and spray dried samples were characterised structurally by X-ray powder diffraction and Fourier transform Raman spectroscopy and also thermally by differential scanning calorimetry and thermogravimetric analysis. Retained lysozyme activity was assayed enzymatically. To assess long-term stability, samples were subjected to conditions of elevated temperature and relative humidity (RH) 40 degrees C/75% RH. Structural and thermal analysis of samples revealed that mannitol/trehalose spray dried excipient/lysozyme blends were completely amorphous upon production but partially recrystallised upon storage at elevated temperature and RH. Biological activity assays revealed that samples containing trehalose retained the highest percentage activity. Under the conditions employed mannitol/trehalose systems stabilise lysozyme more effectively than single excipient systems due to their ability to form amorphous products.

The influence of drug partition coefficient on follicular penetration: in vitro human skin studies.

The aim of this study was to employ the novel skin sandwich system in order to quantify the influence of the octanol-water partition coefficient on follicular drug absorption in human skin. To this end, seven different drugs - estradiol, corticosterone, hydrocortisone, aldosterone, cimetidine, deoxyadenosine and adenosine - exhibiting a wide range of log octanol-water partition coefficients (logK(o/w)) but relatively similar molecular weights were selected as candidate solutes. Application of the skin sandwich technique yielded an interesting relationship between % follicular contribution and logK(o/w). The follicular contribution to total flux was small (4 and 2%) for the two most lipophilic drugs but varied between 34 and 60% for the remaining drugs of intermediate and low logK(o/w) values. Lipophilicity seems to be an important modulator of drug absorption into follicular orifices only above a critical logK(o/w) threshold. Below this critical logK(o/w) value, lipophilicity does not apparently influence the follicular contribution in an obvious way and the process is probably governed by other molecular properties. Identification of these other active properties would require performing this kind of a study on a much larger set of candidate drugs.

An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride.

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbamyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 microm ODS (C18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min-1 and the column temperature was maintained at 30 degrees C. Galactosamine hydrochloride (Gal-HCl) was used as an internal standard. Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32+/-1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15+/-0.1 cm2. The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1% v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 microg ml-1. The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) <12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was

Transfollicular drug delivery--is it a reality?

Once regarded as merely evolutionary remnants, the hair follicles and sebaceous glands are increasingly recognised as potentially significant elements in the percutaneous drug delivery paradigm. Interest in pilosebaceous units has been directed towards their use as depots for localised therapy, particularly for the treatment of follicle-related disorders such as acne or the alopecias. Furthermore, considerable attention has also been focused on exploiting the follicles as transport shunts for systemic drug delivery. This paper reviews various key facets of this field including; relevant aspects of pilosebaceous anatomy and physiology, the design and efficacy of follicle-targeting formulations and the emergence of quantitative modeling systems. Several novel developments in this area promise to greatly expand our understanding of this field in the near future.

Electrically assisted skin delivery of liposomal estradiol; phospholipid as damage retardant.

This work investigated transdermal penetration of a model lipophilic drug (estradiol) through human epidermis from phosphatidylcholine (PC)-based liposomes and saturated aqueous estradiol solution (control). Representative examples of cholate-containing ultradeformable (Transfersomes), non-rigid (pure PC) and membrane-stabilized (cholesterol-containing) vesicles were used. The unilamellar vesicles' diameters and zeta potentials were determined. Transdermal penetration studies involved occluded passive penetration for 12 h and cathodic iontophoresis (0.8 mA/cm(2)) for 8 h for all systems. Combined electroporation (5 pulses, 100 V, 100 ms, 1 min spacing) and iontophoresis (0.8 mA/cm(2), for 2 h) was also employed for ultradeformable vesicles and control. Estradiol penetration parameters (flux and skin deposition) from different formulations were compared. All vesicles had essentially the same particle size, with ultradeformable liposomes showing the highest negative zeta potential (-29 mV). Occluded passive penetration improved estradiol skin penetration from liposomes relative to control. Iontophoretic studies revealed the superiority of ultradeformable vesicles regarding drug skin penetration and deposition compared to traditional liposomes. Combination of electroporation and iontophoresis did not markedly improve estradiol penetration for ultradeformable vesicles. The combination results implied repair of the skin barrier due to the penetration retarding effect of PC monomers released from liposomes.

Electroporation and ultradeformable liposomes; human skin barrier repair by phospholipid.

This work investigated the effect of electroporation on human epidermal penetration of a model neutral lipophilic compound (estradiol) from saturated aqueous solution and when encapsulated in ultradeformable liposomes. Total amount penetrated and skin deposition were compared with values obtained from passive diffusion. The effect of electrical pulsing on liposome size was investigated. The action of phosphatidylcholine on skin that was structurally altered by such pulses was determined. Electroporation did not affect liposome size. Skin pulsing considerably increased estradiol penetration and skin deposition from solution, relative to passive delivery, with subsequent partial recovery of skin resistance to molecular penetration. Surprisingly, with liposomes, electroporation did not markedly affect estradiol skin penetration. Importantly, liposomal phosphatidylcholine applied during or after pulsing accelerated skin barrier repair, i.e. provided an anti-enhancer or retardant effect.

Human skin sandwich for assessing shunt route penetration during passive and iontophoretic drug and liposome delivery.

This work explored the role of skin appendages (shunt route) in passive and iontophoretic drug and liposome penetration. The technique used an epidermis and stratum corneum sandwich from the same skin donor with the additional stratum corneum forming the top layer of the sandwich. Penetration was monitored during occluded passive and iontophoretic (0.5 mA cm(-2)) delivery of mannitol and estradiol solutions, and ultradeformable liposomes containing estradiol. The shunt route had a significant role during passive penetration of mannitol (hydrophilic compound), but was negligible during penetration of estradiol (lipophilic drug) and liposomes. In iontophoresis, the shunt route significantly contributed to the overall flux of all preparations, being highest for mannitol. However, shunts were not the only pathway for iontophoretic drug delivery and evidence was observed for the creation of new aqueous pathways via disorganization of the intercellular lipid domain of stratum corneum. The skin sandwich technique should prove valuable for general studies on routes of skin penetration.

Iontophoretic estradiol skin delivery and tritium exchange in ultradeformable liposomes.

This work evaluated the in vitro transdermal iontophoretic delivery of tritiated estradiol from ultradeformable liposomes compared with saturated aqueous solution (control). Effects of current density and application time on tritium exchange with water were also determined. Penetration studies used three Protocols. Protocol I involved occluded passive steady state estradiol penetration from ultradeformable liposomes and control. The effect of current densities on drug penetration rates was also assessed (Protocol II). In Protocol III, three consecutive stages of drug penetration (first passive, iontophoresis and second passive) through the same human epidermal membranes were monitored. Such an experimental design investigated the possible effect of high current density (0.8 mA/cm(2)) on skin integrity. The tritium exchange study showed that extent of exchange correlated well with current density and time of application, with some shielding of estradiol by the liposomal structure. Liposomes enhanced estradiol passive penetration after occlusion. Protocol II showed that estradiol flux increased linearly with current density, although being delivered against electroosmotic flow. In Protocol III, reduction in flux of the second passive stage to near that of the first reflected a reversibility of the structural changes induced in skin by current.