Formulation and process engineering of freeze-dried orally disintegrating tablets

Orally disintegrating tablets (ODTs) which are also referred to as orodispersible and fast disintegrating tablets, are solid oral dosage forms which upon placing on the tongue, disperse/disintegrate rapidly before being swallowed as a suspension or solution. ODTs are therefore easier and more convenient to administer than conventional tablets and are particularly beneficial for paediatric and geriatric patients, who generally have difficulty swallowing their medication. The work presented in this thesis involved the formulation and process development of ODTs, prepared using freeze-drying. Gelatin is one of the principal excipients used in the formulation of freeze-dried ODTs. One of the studies presented in this thesis investigated the potential modification of the properties of this excipient, in order to improve the performance of the tablets. As gelatin is derived from animal sources, a number of ethical issues surround its use as an excipient in pharmaceutical preparations. This was one of the motivations, Methocel™ and Kollicoat® IR were evaluated as binders as alternative materials to gelatin. Polyox™ was also evaluated as a binder together with its potential uses as a viscosity increasing and mucoadhesive agent to increase the retention of tablets in the mouth to encourage pre-gastric absorption of active pharmaceutical ingredients (APIs). The in vitro oral retention of freeze-dried ODT formulations was one property which was assessed in a design of experiments – factorial design study, which was carried out to further understand the role that formulation excipients have on the properties of the tablets. Finally, the novel approach of incorporating polymeric nanoparticles in freeze-dried ODTs was investigated, to study if the release profile of APIs could be modified, which could improve their therapeutic effect. The results from these studies demonstrated that the properties of gelatin-based formulations can be modified by adjusting pH and ionic strength. Adjustment of formulation pH has shown to significantly reduce tablet disintegration time. Evaluating Methocel™, in particular low viscosity grades, and Kollicoat® IR as binders has shown that these polymers can form tablets of satisfactory hardness and disintegration time. Investigating Polyox™ as an excipient in freeze-dried ODT formulations revealed that low viscosity grades appear suitable as binders whilst higher viscosity grades could potentially be utilised as viscosity increasing and mucoadhesive agents. The design of experiments – factorial design study revealed the influence of individual excipients in a formulation mix on resultant tablet properties and in vitro oral retention of APIs. Novel methods have been developed, which allows the incorporation of polymeric nanoparticles in situ in freeze-dried ODT formulations, which allows the modification of the release profile of APIs.

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.

The formulation of artificial reference standards for use within the ELISPOT assay

Whether to assess the functionality of equipment or as a determinate for the accuracy of assays, reference standards are essential for the purposes of standardisation and validation. The ELISPOT assay, developed over thirty years ago, has emerged as a leading immunological assay in the development of novel vaccines for the assessment of efficacy. However, with its widespread use, there is a growing demand for a greater level of standardisation across different laboratories. One of the major difficulties in achieving this goal has been the lack of definitive reference standards. This is partly due to the ex vivo nature of the assay, which relies on cells being placed directly into the wells. Thus, the aim of this thesis was to produce an artificial reference standard using liposomes, for use within the assay. Liposomes are spherical bilayer vesicles with an enclosed aqueous compartment and therefore are models for biological membranes. Initial work examined pre-design considerations in order to produce an optimal formulation that would closely mimic the action of the cells ordinarily placed on the assay. Recognition of the structural differences between liposomes and cells led to the formulation of liposomes with increased density. This was achieved by using a synthesised cholesterol analogue. By incorporating this cholesterol analogue in liposomes, increased sedimentation rates were observed within the first few hours. The optimal liposome formulation from these studies was composed of 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Chol) and brominated cholesterol (Brchol) at a 16:4:12 µMol ratio, based on a significantly higher (p<0.01) sedimentation (as determined by a percentage transmission of 59 ± 5.9 % compared to the control formulation at 29 ± 12 % after four hours). By considering a range of liposome formulations ‘proof of principle’ for using liposomes as ELISPOT reference standards was shown; recombinant IFN? cytokine was successfully entrapped within vesicles of different lipid compositions, which were able to promote spot formation within the ELISPOT assay. Using optimised liposome formulations composed of phosphatidylcholine with or without cholesterol (16 µMol total lipid) further development was undertaken to produce an optimised, scalable protocol for the production of liposomes as reference standards. A linear increase in spot number by the manipulation of cytokine concentration and/or lipid concentrations was not possible, potentially due to the saturation that occurred within the base of wells. Investigations into storage of the formulations demonstrated the feasibility of freezing and lyophilisation with disaccharide cryoprotectants, but also highlighted the need for further protocol optimisation to achieve a robust reference standard upon storage. Finally, the transfer of small-scale production to a medium lab-scale batch (40 mL) demonstrated this was feasible within the laboratory using the optimised protocol.

Strategic development and physicochemical analysis of oral preparations for unstable drugs

Oral liquid formulations are ideal dosage forms for paediatric, geriatric and patient with dysphagia. Dysphagia is prominent among patients suffering from stroke, motor neurone disease, advanced Alzheimer’s and Parkinson’s disease. However oral liquid preparations are particularly difficult to formulate for hydrophobic and unstable drugs. Therefore current methods employed in solving this issue include the use of ‘specials’ or extemporaneous preparations. In order to challenge this, the government has encouraged research into the field of oral liquid formulations, with the EMEA and MHRA publishing list of drugs of interest. The current work investigates strategic formulation development and characterisation of select API’s (captopril, gliclazide, melatonin, L-arginine and lansoprazole), each with unique obstacles to overcome during solubilisation, stabilisation and when developing a palatable dosage from. By preparing a validated calibration protocol for each of the drug candidates, the oral liquid formulations were assessed for stability, according to the ICH guidelines along with thorough physiochemical characterisation. The results showed that pH and polarity of the solvent had the greatest influence on the extent of drug solubilisation, with inclusion of antioxidants and molecular steric hindrance influencing the extent of drug stability. Captopril, a hydrophilic ACE inhibitor (160 mg.mL-1), undergoes dimerisation with another captopril molecule. It was found that with the addition of EDTA and HP-β-CD, the drug molecule was stabilised and prevented from initiating a thiol induced first order free radical oxidation. The cyclodextrin provided further steric hindrance (1:1 molar ratio) resulting in complete reduction of the intensity of sulphur like smell associated with captopril. Palatability is a crucial factor in patient compliance, particularly when developing a dosage form targeted towards paediatrics. L-arginine is extremely bitter in solution (148.7 g.L-1). The addition of tartaric acid into the 100 mg.mL-1 formulation was sufficient to mask the bitterness associated with its guanidium ions. The hydrophobicity of gliclazide (55 mg.L-1) was strategically challenged using a binary system of a co-solvent and surfactant to reduce the polarity of the medium and ultimately increase the solubility of the drug. A second simpler method was developed using pH modification with L-arginine. Melatonin has two major obstacles in formulation: solubility (100 μg.mL-1) and photosensitivity, which were both overcome by lowering the dielectric constant of the medium and by reversibly binding the drug within the cyclodextrin cup (1:1 ratio). The cyclodextrin acts by preventing UV rays from reaching the drug molecule and initiated the degradation pathway. Lansoprazole is an acid labile drug that could only be delivered orally via a delivery vehicle. In oral liquid preparations this involved nanoparticulate vesicles. The extent of drug loading was found to be influenced by the type of polymer, concentration of polymer, and the molecular weight. All of the formulations achieved relatively long shelf-lives with good preservative efficacy.

Unravelling the theories of pre-eclampsia:Are the protective pathways the new paradigm?

Pre-eclampsia is a vascular disorder of pregnancy where anti-angiogenic factors, systemic inflammation and oxidative stress predominate, but none can claim to cause pre-eclampsia. This review provides an alternative to the 'two-stage model' of pre-eclampsia in which abnormal spiral arteries modification leads to placental hypoxia, oxidative stress and aberrant maternal systemic inflammation. Very high maternal soluble fms-like tyrosine kinase-1 (sFlt-1 also known as sVEGFR) and very low placenta growth factor (PlGF) are unique to pre-eclampsia; however, abnormal spiral arteries and excessive inflammation are also prevalent in other placental disorders. Metaphorically speaking, pregnancy can be viewed as a car with an accelerator and brakes, where inflammation, oxidative stress and an imbalance in the angiogenic milieu act as the 'accelerator'. The 'braking system' includes the protective pathways of haem oxygenase 1 (also referred as Hmox1 or HO-1) and cystathionine-γ-lyase (also known as CSE or Cth), which generate carbon monoxide (CO) and hydrogen sulphide (H2S) respectively. The failure in these pathways (brakes) results in the pregnancy going out of control and the system crashing. Put simply, pre-eclampsia is an accelerator-brake defect disorder. CO and H2S hold great promise because of their unique ability to suppress the anti-angiogenic factors sFlt-1 and soluble endoglin as well as to promote PlGF and endothelial NOS activity. The key to finding a cure lies in the identification of cheap, safe and effective drugs that induce the braking system to keep the pregnancy vehicle on track past the finishing line.

Nanotechnology and microfluidics:formulation design and on-chip manufacture of nanoparticles

Nanoparticles offer an ideal platform for the delivery of small molecule drugs, subunit vaccines and genetic constructs. Besides the necessity of a homogenous size distribution, defined loading efficiencies and reasonable production and development costs, one of the major bottlenecks in translating nanoparticles into clinical application is the need for rapid, robust and reproducible development techniques. Within this thesis, microfluidic methods were investigated for the manufacturing, drug or protein loading and purification of pharmaceutically relevant nanoparticles. Initially, methods to prepare small liposomes were evaluated and compared to a microfluidics-directed nanoprecipitation method. To support the implementation of statistical process control, design of experiment models aided the process robustness and validation for the methods investigated and gave an initial overview of the size ranges obtainable in each method whilst evaluating advantages and disadvantages of each method. The lab-on-a-chip system resulted in a high-throughput vesicle manufacturing, enabling a rapid process and a high degree of process control. To further investigate this method, cationic low transition temperature lipids, cationic bola-amphiphiles with delocalized charge centers, neutral lipids and polymers were used in the microfluidics-directed nanoprecipitation method to formulate vesicles. Whereas the total flow rate (TFR) and the ratio of solvent to aqueous stream (flow rate ratio, FRR) was shown to be influential for controlling the vesicle size in high transition temperature lipids, the factor FRR was found the most influential factor controlling the size of vesicles consisting of low transition temperature lipids and polymer-based nanoparticles. The biological activity of the resulting constructs was confirmed by an invitro transfection of pDNA constructs using cationic nanoprecipitated vesicles. Design of experiments and multivariate data analysis revealed the mathematical relationship and significance of the factors TFR and FRR in the microfluidics process to the liposome size, polydispersity and transfection efficiency. Multivariate tools were used to cluster and predict specific in-vivo immune responses dependent on key liposome adjuvant characteristics upon delivery a tuberculosis antigen in a vaccine candidate. The addition of a low solubility model drug (propofol) in the nanoprecipitation method resulted in a significantly higher solubilisation of the drug within the liposomal bilayer, compared to the control method. The microfluidics method underwent scale-up work by increasing the channel diameter and parallelisation of the mixers in a planar way, resulting in an overall 40-fold increase in throughput. Furthermore, microfluidic tools were developed based on a microfluidics-directed tangential flow filtration, which allowed for a continuous manufacturing, purification and concentration of liposomal drug products.

Microfluidics based manufacture of liposomes simultaneously entrapping hydrophilic and lipophilic drugs

Despite the substantial body of research investigating the use of liposomes, niosomes and other bilayer vesicles for drug delivery, the translation of these systems into licensed products remains limited. Indeed, recent shortages in the supply of liposomal products demonstrate the need for new scalable production methods for liposomes. Therefore, the aim of our research has been to consider the application of microfluidics in the manufacture of liposomes containing either or both a water soluble and a lipid soluble drug to promote co-delivery of drugs. For the first time, we demonstrate the entrapment of a hydrophilic and a lipophilic drug (metformin and glipizide respectively) both individually, and in combination, using a scalable microfluidics manufacturing system. In terms of the operating parameters, the choice of solvents, lipid concentration and aqueous:solvent ratio all impact on liposome size with vesicle diameter ranging from ∼90 to 300 nm. In terms of drug loading, microfluidics production promoted high loading within ∼100 nm vesicles for both the water soluble drug (20–25% of initial amount added) and the bilayer embedded drug (40–42% of initial amount added) with co-loading of the drugs making no impact on entrapment efficacy. However, co-loading of glipizide and metformin within the same liposome formulation did impact on the drug release profiles; in both instances the presence of both drugs in the one formulation promoted faster (up to 2 fold) release compared to liposomes containing a single drug alone. Overall, these results demonstrate the application of microfluidics to prepare liposomal systems incorporating either or both an aqueous soluble drug and a bilayer loaded drug.