Encapsulation of lipopeptides within liposomes: Effect of number of lipid chains, chain length and method of liposome preparation

The purpose of this study was to systematically investigate the effect of lipid chain length and number of lipid chains present on lipopeptides on their ability to be incorporated within liposomes. The peptide KAVYNFATM was synthesized and conjugated to lipoamino acids having acyl chain lengths of C-8, C-12 and C-16. The C-12 construct was also prepared in the monomeric, dimeric and trimeric form. Liposomes were prepared by two techniques: hydration of dried lipid films (Bangham method) and hydration of freeze-dried monophase systems. Encapsulation of lipopeptide within liposomes prepared by hydration of dried lipid films was incomplete in all cases ranging from an entrapment efficiency of 70% for monomeric lipoamino acids at a 5% (w/w) loading to less than 20% for di- and trimeric forms at loadings of 20% (w/w). The incomplete entrapment of lipopeptides within liposomes appeared to be a result of the different solubilities of the lipopeptide and the phospholipids in the solvent used for the preparation of the lipid film. In contrast, encapsulation of lipopeptide within liposomes prepared by hydration of freeze-dried monophase systems was high, even up to a loading of 20% (w/w) and was much less affected by the acyl chain length and number than when liposomes were prepared by hydration of dried lipid films. Freeze drying of monophase systems is better at maintaining a molecular dispersion of the lipopeptide within the solid phospholipid matrix compared to preparation of lipid film by evaporation, particularly if the solubility of the lipopeptide in solvents is markedly different from that of the polar lipids used for liposome preparation. Consequently, upon hydration, the lipopeptide is more efficiently intercalated within the phospholipid bilayers. (C) 2005 Elsevier B.V. All rights reserved.

Screening, characterization and flocculating property of carbohydrate polymer from newly isolated Enterobacter cloacae WD7

A total of 188 carbohydrate polymer-producing bacterial strains were isolated from recycled sludge of five seafood processing plants. Among three selected isolates, identified as Enterobacter cloacae WD7, Enterobacter agglomerans WD50 and Pseudomonas alcaligenes WD22. E. cloacae WD7 generated a viscous culture broth exhibiting the highest flocculating activity and a crude polymer yield of 2.27 g/L after 3 days cultivation. Partial purification of this polymer was performed by precipitation with 95% ethanol, dialysis and freeze-drying. It was characterized as an acidic heteropolysaccharide, composed of neutral sugars (29.4%), uronic acids (14.2%) and amino sugars (0.93%). The functional group analysis by FT-IR spectroscopy showed the presence of hydroxyl, carboxyl, carbonyl and methoxyl groups. Thermal analysis by DSC showed the crystalline transition and the crystalline melting point (T-m) at 300 degrees C. This polysaccharide was soluble in water and insoluble in any organic solvents tested; gelation occurred under alkaline conditions in the presence of divalent cations in which copper as CuSO4 gave the best result. Studies on the flocculation property revealed that this polysaccharide was stable at 4-60 degrees C and pH 5-7. The optimal concentrations for the flocculating activity were 2 mg/L polysaccharide and 40 mM CaCl2 which played the synergistic effect on kaolin flocculation. Moreover, this polysaccharide could flocculate the kaolin suspension over a wide range of pH (pH 2-8) and temperature (4-50 degrees C) tested in the presence of CaCl2. (c) 2006 Elsevier Ltd. All rights reserved.

Novel formulation strategies for the fabrication of lyophilised orally disintegrating tablets

Orally disintegrating Tablets (ODTs), also known as fast-disintegrating, fast-melt or fast-dissolving tablets, are a relatively novel dosage technology that involves the rapid disintegration or dissolution of the dosage form into a solution or suspension in the mouth without the need for water. The solution containing the active ingredients is swallowed, and the active ingredients are then absorbed through the gastrointestinal epithelium to reach the target and produce the desired effect. Formulation of ODTs was originally developed to address swallowing difficulties of conventional solid oral dosage forms (tablets and capsules) experienced by wide range of patient population, especially children and elderly. The current work investigates the formulation and development of ODTs prepared by freeze drying. Initial studies focused on formulation parameters that influence the manufacturing process and performance of lyophilised tablets based on excipients used in commercial products (gelatin and saccharides). The second phase of the work was followed up by comprehensive studies to address the essential need to create saccharide free ODTs using naturally accruing amino acids individually or in combinations. Furthermore, a factorial design study was carried out to investigate the feasibility of delivering multiparticulate systems of challenging drugs using a novel formulation that exploited the electrostatic associative interaction between gelatin and carrageenan. Finally, studies aimed to replace gelatin with ethically and morally accepted components to the end users were performed and the selected binder was used in factorial design studies to investigate and optimise ODT formulations that incorporated drugs with varies physicochemical properties. Our results show that formulation of elegant lyophilised ODTs with instant disintegration and adequate mechanical strength requires carful optimisation of gelatin concentration and bloom strength in addition to saccharide type and concentration. Successful formulation of saccharides free lyophilised ODTs requires amino acids that crystallise in the frozen state or display relatively high Tg', interact and integrate completely with the binder and, also, display short wetting time with the disintegrating medium. The use of an optimised mixture of gelatin, carrageenan and alanine was able to create viscous solutions to suspend multiparticulate systems and at the same time provide tablets with short disintegration times and adequate mechanical properties. On the other hand, gum arabic showed an outstanding potential for use as a binder in the formulation of lyophilised ODTs. Compared to gelatin formulations, the use of gum arabic simplified the formulation stages, shortened the freeze drying cycles and produced tablets with superior performance in terms of the disintegration time and mechanical strength. Furthermore, formulation of lyophilised ODTs based on gum arabic showed capability to deliver diverse range of drugs with advantages over commercial products.

Formulation and characterisation of lyophilised rapid disintegrating tablets using amino acids as matrix forming agents

Despite recent advances in the formulation of lyophilised rapid disintegrating tablets (RDTs), the inclusion of matrix supporting/disintegration enhancing agents has been limited to the use of saccharides and polyols. In this study, the feasibility of using amino acids as matrix forming agents in lyophilised RDTs was investigated. Twelve amino acids were chosen (alanine, arginine, threonine, glycine, cysteine, serine, histidine, lysine, valine, asparagine, glutamine and proline), and the suitability for freeze drying, mechanical properties and disintegration time after inclusion of the amino acids at varied concentration were studied. In addition, the porosity of the RDTs and wettability profile of the amino acids were investigated to understand the mechanisms of disintegration. The results suggest the suitability of these amino acids for the lyophilisation regime, as they displayed satisfactory safety margin between the glass transition and shelf temperature (-40 degrees C), except proline-based formulations. Moreover, the crystallisation behavior of alanine, glycine, cysteine and serine at high concentration increased the stability of the formulation. The characterisation of the RDTs suggests that high concentration of the amino acids is required to enhance the mechanical properties, whereas only optimum concentrations promote the disintegration. Moreover, wetting time of the amino acid and porosity of the tablet are the two factors that control the disintegration of RDTs.

Recent patents and trends in orally disintegrating tablets

Orally disintegrating tablets (ODTs) offer many advantages over the conventional oral dosage forms in terms of convenience and ease of use. Over the last decade, substantial advances in the formulation of ODTs have been achieved in academia and industry that resulted in the emerging of a large number of patents. The aim of this review is to summarise the most recent patents in ODT formulations and highlight their motivations, inventive steps and significances in the development of ODT formulations. Five major techniques have been applied in manufacturing of ODTs, namely conventional tablet press, moulding, freeze drying, tablet loading and pulverization, with majority of the patents dedicated to the use of conventional tablet pressing. The patents have addressed various issues concerning the manufacturing of robust and practical ODT formulations by disclosing new manufacturing techniques, advantageous materials, and innovative formulation steps. However, future developments are required to reduce the cost and widening the application of the new manufacturing techniques, while simplifying and shortening the formulation steps will be crucial in the well established ones.

Grewia polysaccharide gum as a pharmaceutical excipient

Grewia gum is obtained from the inner stem bark of the edible plant Grewia mollis Juss (Fam. Tiliaceae) which grows widely in the middle belt region of Nigeria, and is also cultivated. The dried and pulverised inner stem bark is used as a thickening agent in some food delicacies in that region of the country. This ability of the material to increase solution viscosity has generated a lot of interest and is the catalysing momentum for this research. Such materials have been used as stabilizers or suspending agents in cosmetics, foods and liquid medications, and as mucoadhesives and controlled release polymeric matrices in solid dosage forms. The physicochemical characterization of candidate excipients forms an essential step towards establishing suitability for pharmaceutical application. For natural gums, this usually requires isolation of the gum from the storage site by extraction processes. Grewia polysaccharide gum was extracted and dried using techniques such as air-drying, freeze-drying or spray-drying. Component analysis of the gum showed that it contains five neutral sugars: glucose, galactose, rhamnose, arabinose and xylose. The gum contains traces of elements such as zinc, magnesium, calcium and phosphorus. At low substance weight, the gum hydrates in aqueous medium swelling and dispersing to give a highly viscous dispersion with pseudoplasmic flow behaviour. The method by which drying is achieved can have significant effect on some physicochemical properties of the gum. Consequently, the intrinsic viscosity and molecular weight, and parameters of powder flow were shown to differ with the method of drying. The gum has good thermal stability. In comparison with established excipients, grewia gum may be preferable to gum Arabic or sodium carboxymethylcellulose as a suspending agent in ibuprofen suspension formulations. The release retardant property of the gum was superior to guar and Metolose® in ibuprofen matrices. Similarly, carboxy methylcellulose, Methocel®, gum Arabic or Metolose® may not be preferable to grewia gum when controlled release of a soluble drug like cimetidine is indicated. The mucoadhesive performance of the gum compared favourably with excellent mucoadhesives such as hydroxypropyl methylcellulose, carboxymethylcellulose, guar and carbopol 971 P.

Cyclodextrin complexes of antimicrobial agents

Poorly water-soluble drugs show an increase in solubility in the presence of cyclodextrins (CyD) due to the formation of a water-soluble complex between the drug and dissolved CyD. This study investigated the interactions of -Cyd and hydroxypropyl--CyD (HP--CyD, M.S. = 0.6) with antimicrobial agents of limited solubility in an attempt to increase their microbiological efficacy. The agents studied were chlorhexidine dihydrochloride (CHX), p-hydroxybenzoic acid esters (methyl, ethyl, proply and butyl) and triclosan. The interactions between the antimicrobials and CyDs were studied in solution and solid phases. Phase solubility studied revealed an enhancement in the aqueous drug solubility in the presence of the CyD and also gave an indication of the complex stability constant (Ks). The temperature-dependence of the stability constant of the complex was modelled by the van't Hoff plot which yielded the thermodynamic parameters for complexation. Further confirmation of the inclusion of the antimicrobials within the cavity of the CyDs in aqueous solution was obtained from proton magnetic resonance and ultraviolet absorption spectroscopies. The former method indicated that the chlorophenyl moiety of the CHX was included within the -CyD cavity and the stoichiometry of the complex formed was 1:1. The solid-phase complexes were prepared by freeze-drying. The inclusion complex of triclosan with HP--CyD was obtained from aqueous solution with the addition of ammonia. Evidence to confirm complex formation was obtained from DSC, IR and X-ray powder diffraction studies. Dissolution studies of the solid inclusion complexes using the dispersed powder technique illustrated their superior solubilities as compared to the equimolar physical mix of the guest and CyD. It was shown that these solutions of the complex were supersaturated with respect to the free guest. This was further demonstrated by diffusion studies which showed the flux of free drug from donor solutions of the antimicrobial-CyD complex to be significantly greater than the flux from donor suspensions of drug alone.

Improving diagnosis and oral vaccination strategies against bovine tuberculosis

In this work peptide antigens [ESAT-6,p45 in water (1ml, 1mg/ml)] have been adsorbed onto 10mg inorganic substrates (hydroxyapatite (MHA P201;P120, CHA), polystyrene, calcium carbonate and glass microspheres) and in vitro release characteristics were determined. The aim of formulation was to enhance the interaction of peptides with antigen presenting cells and to achieve rapid peptide release from the carrier compartment system in a mildly acidic environment. Hydroxyapatite microparticle P201 has a greater surface area and thus has the largest peptide adsorption compared to the P120. CHA gave a further higher adsorption due to larger surface area than that available on microparticles. These particles were incorporated into the BOVIGAMTM assay to determine if they improve the sensitivity. After overnight incubation the blood plasma was removed and the amount of IFN-g in each plasma sample was estimated. CHA and MHA P201 gave a significantly higher immune response at low peptide concentration compared to the free peptide, thus indicating that these systems can be used to evaluate Tuberculosis (TB) amongst cattle using the BOVIGAMTM assay. Badgers are a source of TB and pass infection to cattle. At the moment vaccination against TB in badgers is via the parenteral route and requires a trained veterinary surgeon as well as catching the badgers. This process is expensive and time consuming; consequently an oral delivery system for delivery of BCG vaccines is easier and cheaper. The initial stage involved addition of various surfactants and suspending agents to disperse BCG and the second stage involved testing for BCG viability. Various copolymers of Eudragit were used as enteric coating systems to protect BCG against the acidic environment of the stomach (SGF, 0.1M HCl pH 1.2 at 37oC) while dissolving completely in the alkaline environment of the small intestine (SIF, IM PBS solution pH 7.4 at 37oC). Eudragit L100 dispersed in 2ml PBS solution and 0.9ml Tween 80 (0.1%w/v) gave the best results remaining intact in SGF loosing only approximately 10-15% of the initial weight and dissolving completely within 3 hours. BCG was incorporated within the matrix formulation adjusted to pH 7 at the initial formulation stage containing PBS solution and Tween 80. It gave viability of x106 cfu/ml at initial formulation stage, freezing and freeze-drying stages. After this stage the matrix was compressed at 4 tons for 3 mins and placed in SGF for 2 hours and then in SIF until dissolved. The BCG viability dropped to x106 cfu/ml. There is potential to develop it further for oral delivery of BCG vaccine.

Use of amino acids as counterions improves the solubility of the BCS II model drug, indomethacin

The number of new chemical entities (NCE) is increasing every day after the introduction of combinatorial chemistry and high throughput screening to the drug discovery cycle. One third of these new compounds have aqueous solubility less than 20µg/mL [1]. Therefore, a great deal of interest has been forwarded to the salt formation technique to overcome solubility limitations. This study aims to improve the drug solubility of a Biopharmaceutical Classification System class II (BCS II) model drug (Indomethacin; IND) using basic amino acids (L-arginine, L-lysine and L-histidine) as counterions. Three new salts were prepared using freeze drying method and characterised by FT-IR spectroscopy, proton nuclear magnetic resonance ((1)HNMR), Differential Scanning Calorimetry (DSC) and Thermogravimetric analysis (TGA). The effect of pH on IND solubility was also investigated using pH-solubility profile. Both arginine and lysine formed novel salts with IND, while histidine failed to dissociate the free acid and in turn no salt was formed. Arginine and lysine increased IND solubility by 10,000 and 2296 fold, respectively. An increase in dissolution rate was also observed for the novel salts. Since these new salts have improved IND solubility to that similar to BCS class I drugs, IND salts could be considered for possible waivers of bioequivalence.

Preparation, optimisation and characterisation of lyophilised rapid disintegrating tablets based on gelatin and saccharide

Despite recent success, rapidly disintegrating lyophilized tablets still face problems of low mechanical strength and higher disintegration times resulting in poor patient compliance. The aim of the current work was to carry out a systematic study to understand the factors controlling mechanical properties of these formulations. The work investigated the influence of two bloom strengths of gelatin: low (60 bloom) and high (225 bloom) at different stock solution concentrations (2, 5, 7.5, and 10 %w/w). This was followed by investigation of addition of five saccharides (xylitol, glucose, trehalose, maltotriose and mannitol) at varied concentration range (10-80 %w/w) to decipher their influence on disintegration time, mechanical and thermal properties of the formulation. The results indicated that the disintegration time of the tablets dramatically decreased by decreasing the concentration and bloom strength of gelatin in the stock solution. However the mechanical properties of the tablets were mainly influenced by the concentration of gelatin rather than the bloom strength. The addition of saccharides resulted in enhancement of tablet properties and was concentration dependent. All the saccharides improved the fractubility of the tablets significantly at high concentration (equal or higher than 40% w/w). However, only high concentration (equal or higher than 40% w/w) of trehalose, maltotriose and mannitol significantly enhanced the hardness. Additionally, mannitol crytallised during freeze drying and consequently produced elegant tablets, whilst the other saccarides exhibited lyoprotectant activity as they were able to retain amorphous status. Based on the above findings, an optimized formulation was also successfully developed and characterized to deliver 100 microg dose of Clonidine HCl.

Poly (D,L-Lactide) based microspheres for pulmonary delivery of proteins

Initial work focused on the preparation, optimisation and characterisation of poly (D,L-lactide) (PLA) microspheres with the aim of optimising their formulation based on minimizing the particle size into the range suitable for pulmonary delivery to alveoli. In order to produce dry powders and to enhance their long-term physico-chemical stability, microspheres were prepared as a dry powder via freeze-drying. Optimisation studies showed that using appropriate concentrations of polymer 3% (w/v) in organic phase and emulsifier 10% (w/v) in external aqueous phase, the double solvent evaporation method produced high protein loading microspheres (72 ± 0.5%) with an appropriate particle size for pulmonary drug delivery. Combined use of trehalose and leucine as cyroprotectants (6% and 1% respectively, w/v) produced freeze-dried powders with the best aerosolisation profile among those tested. Although the freeze-dried PLA microsphere powders were not particularly respirable in dry powder inhalation, nebulisation of the rehydrated powders using an ultrasonic nebuliser resulted in improved aerosilisation performance compared to the air-jet nebuliser. When tested in vitro using a macrophage cell line, the PLA microspheres system exhibited a low cytotoxicity and the microspheres induced phagocytic activity in macrophages. However, interestingly, the addition of an immunomodulator to the microsphere formulations (4%, w/w of polymer) reduced this phagocytic activity and macrophage activation compared to microspheres formulated using PLA alone. This suggested that the addition of trehalose dibehenate may not enhance the ability of these microspheres to be used as vaccine delivery systems.

Vaccine entrapment in liposomes

The use of liposomes as carriers of peptide, protein, and DNA vaccines requires simple, easy-to-scale-up technology capable of high-yield vaccine entrapment. Work from this laboratory has led to the development of techniques that can generate liposomes of various sizes, containing soluble antigens such as proteins and particulate antigens (e.g., killed or attenuated bacteria or viruses), as well as antigen-encoding DNA vaccines. Entrapment of vaccines is carried out by the dehydration-rehydration procedure which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free vaccines. On rehydration, the large multilamellar vesicles formed incorporate up to 90% or more of the vaccine used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped vaccine still associated with the vesicles. A similar technique applied for the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 microm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 30% or more of the spores used are associated with generated giant liposomes of similar mean size.

Amino acids in oral drug delivery:salts, ion-pairs and transcriptomics

Oral drug delivery is considered the most popular route of delivery because of the ease of administration, availability of a wide range of dosage forms and the large surface area for drug absorption via the intestinal membrane. However, besides the unfavourable biopharmaceutical properties of the therapeutic agents, efflux transporters such as Pglycoprotein (P-gp) and multiple resistance proteins (MRP) decrease the overall drug uptake by extruding the drug from the cells. Although, prodrugs have been investigated to improve drug partitioning by masking the polar groups covalently with pre-moieties promoting increased uptake, they present significant challenges including reduced solubility and increased toxicity. The current work investigates the use of amino acids as ion-pairs for three model drugs: indomethacin (weak acid), trimethoprim (weak base) and ciprofloxacin (zwitter ion) in an attempt to improve both solubility and uptake. Solubility was studied by salt formation while creating new routes for uptake across the membranes via amino acids transporter proteins or dipeptidyl transporters was the rationale to enhance absorption. New salts were prepared for the model drugs and the oppositely charged amino acids by freeze drying and they were characterised using FTIR, 1HNMR, DSC, SEM, pH solubility profile, solubility and dissolution. Permeability profiles were assessed using an in vitro cell based method; Caco-2 cells and the genetic changes occurring across the transporter genes and various pathways involved in the cellular activities were studied using DNA microarrays. Solubility data showed a significant increase in drug solubility upon preparing the new salts with the oppositely charged counter ions (ciprofloxacin glutamate salt exhibiting 2.9x103 fold enhancement when compared to the free drug). Moreover, permeability studies showed a 3 fold increase in trimethoprim and indomethacin permeabilities upon ion-pairing with amino acids and more than 10 fold when the zwitter ionic drug was paired with glutamic acid. Microarray data revealed that trimethoprim was absorbed actively via OCTN1 transporters while MRP7 is the main transporter gene that mediates its efflux. The absorption of trimethoprim from trimethoprim glutamic acid ion-paired formulations was affected by the ratio of glutamic acid in the formulation which was inversely proportional to the degree of expression of OCTN1. Interestingly, ciprofloxacin glutamic acid ion-pairs were found to decrease the up-regulation of ciprofloxacin efflux proteins (P-gp and MRP4) and over-express two solute carrier transporters; (PEPT2 and SLCO1A2) suggesting that a high aqueous binding constant (K11aq) enables the ion-paired formulations to be absorbed as one entity. In conclusion, formation of ion-pairs with amino acids can influence in a positive way solubility, transfer and gene expression effects of drugs.

Challenges and emerging solutions in the development of compressed orally disintegrating tablets

Areas covered: The review discusses the main challenges of ODT manufacturing process and the emerging solutions featured at early drug development stages. The research specifically describes the methods reported for taste masking/assessment and solubilisation of unpalatable and poorly soluble drugs, respectively. Furthermore, this review highlights the techniques used for developing modified-release ODTs, an emerging area in the field. In addition, it also discusses the poor flowability and segregation problems of directly compressed powders. Moreover, the review describes the tests reported in the literature for ODT disintegration time assessment since a universal technique is still non-existent. Expert opinion: The approaches used to overcome the manufacturing challenges often have a bearing on the price of the end product. However, despite the technical and regulatory challenges, ODTs can offer many advantages over the conventional dosage forms if accompanied by suitable adjuvant technologies and in vitro analytical tools. © 2014 Informa UK, Ltd. Introduction: Orally disintegrating tablets (ODTs) provide several advantages over conventional tablets such as suitability for patients with swallowing difficulties and faster onset of action. The manufacture of ODTs by compression/tableting offers a practical and cost-effective strategy over the freeze drying (lyophilisation) method. Nonetheless, the FDA recommends a disintegration time of 30 s and a maximum weight of 500 mg for a tablet to be labelled as an ODT. These requirements, alongside other desirable product properties, have created a number of challenges for the formulator to overcome while developing compressed ODTs.

Short cycle times for cost-efficient processing in lyophilized formulations

Pharmaceutical scientists who bulk freeze dry need to foremost identify what quality factors are of a priority during cycle development since the economics of freeze-drying do not allow for both the cost-efficient production and the ability to obtain the highest quality score across all quality factors. Consider; morphology, activity, dissolution, long-term storage, packaging and cost.