Application of Accelerated Predictive Stability Studies in Extemporaneously Compounded Formulations of Chlorhexidine to Assess the Shelf Life.

Industrially fabricated medicines have a well-defined shelf life supported by rigorous studies before their approval for commercialization. However, the shelf life of extemporaneous compounding topical formulations prepared at hospitals tends to be shorter, especially when no data are available to prove a longer stability period. Also, the storage conditions are unknown in many circumstances. Accelerated Predictive Stability (APS) studies have been shown to be a useful tool to predict in a faster and more accurate manner the chemical stability of extemporaneously compounded formulations requiring a minimum amount of formulation, thereby reducing the chemical drug waste per study. Shelf life will be allocated based on scientific data without compromising drug efficacy or safety. In this work, the APS approach was applied to the commercially available Cristalmina® (CR) and an extemporaneously compounded formulation of chlorhexidine (DCHX). A different degradation kinetic was found between DCHX and CR (Avrami vs. zero-order kinetics, respectively). This can explain the different shelf life described by the International Council for Harmonisation of Technical Requirements Registration Pharmaceuticals Human Use (ICH) conditions between both formulations. A predicted stability for the DCHX solution was obtained from the extrapolation of the degradation rate in long-term conditions from the Arrhenius equation. The estimated degradation from the Arrhenius equation for DCHX at 5 °C, 25 °C, and 30 °C at 365 days was 3.1%, 17.4%, and 25.9%, respectively. The predicted shelf life, in which the DCHX content was above 90%, was 26.67 months under refrigerated conditions and 5.75 and 2.24 months at 25 and 30 °C, respectively. Currently, the Spanish National Formulary recommends a shelf life of no longer than 3 months at room temperature for DCHX solution. Based on the predicted APS and confirmed by experimental long-term studies, we have demonstrated that the shelf life of DCHX extemporaneously compounded formulations could be prolonged by up to 6 months.

Co-Delivery of a High Dose of Amphotericin B and Itraconazole by Means of a Dry Powder Inhaler Formulation for the Treatment of Severe Fungal Pulmonary Infections.

Over the past few decades, there has been a considerable rise in the incidence and prevalence of pulmonary fungal infections, creating a global health problem due to a lack of antifungal therapies specifically designed for pulmonary administration. Amphotericin B (AmB) and itraconazole (ITR) are two antifungal drugs with different mechanisms of action that have been widely employed in antimycotic therapy. In this work, microparticles containing a high dose of AmB and ITR (20, 30, and 40% total antifungal drug loading) were engineered for use in dry powder inhalers (DPIs) with an aim to improve the pharmacological effect, thereby enhancing the existing off-label choices for pulmonary administration. A Design of Experiment (DoE) approach was employed to prepare DPI formulations consisting of AmB-ITR encapsulated within γ-cyclodextrin (γ-CD) alongside functional excipients, such as mannitol and leucine. In vitro deposition indicated a favourable lung deposition pattern characterised by an upper ITR distribution (mass median aerodynamic diameter (MMAD) ~ 6 µm) along with a lower AmB deposition (MMAD ~ 3 µm). This offers significant advantages for treating fungal infections, not only in the lung parenchyma but also in the upper respiratory tract, considering that Aspergillus spp. can cause upper and lower airway disorders. The in vitro deposition profile of ITR and larger MMAD was related to the higher unencapsulated crystalline fraction of the drug, which may be altered using a higher concentration of γ-CD.

Self-assembling, supramolecular chemistry and pharmacology of amphotericin B: Poly-aggregates, oligomers and monomers.

Antifungal drugs such as amphotericin B (AmB) interact with lipids and phospholipids located on fungal cell membranes to disrupt them and create pores, leading to cell apoptosis and therefore efficacy. At the same time, the interaction can also take place with cell components from mammalian cells, leading to toxicity. AmB was selected as a model antifungal drug due to the complexity of its supramolecular chemical structure which can self-assemble in three different aggregation states in aqueous media: monomer, oligomer (also known as dimer) and poly-aggregate. The interplay between AmB self-assembly and its efficacy or toxicity against fungal or mammalian cells is not yet fully understood. To the best of our knowledge, this is the first report that investigates the role of excipients in the supramolecular chemistry of AmB and the impact on its biological activity and toxicity. The monomeric state was obtained by complexation with cyclodextrins resulting in the most toxic state, which was attributed to the greater production of highly reactive oxygen species upon disruption of mammalian cell membranes, a less specific mechanism of action compared to the binding to the ergosterol located in fungal cell membranes. The interaction between AmB and sodium deoxycholate resulted in the oligomeric and poly-aggregated forms which bound more selectively to the ergosterol of fungal cell membranes. NMR combined with XRD studies elucidated the interaction between drug and excipient to achieve the AmB aggregation states, and ultimately, their diffusivity across membranes. A linear correlation between particle size and the efficacy/toxicity ratio was established allowing to modulate the biological effect of the drug and hence, to improve pharmacological regimens. However, particle size is not the only factor modulating the biological response but also the equilibrium of each state which dictates the fraction of free monomeric form available. Tuning the aggregation state of AmB formulations is a promising strategy to trigger a more selective response against fungal cells and to reduce the toxicity in mammalian cells.

Oral Fixed-Dose Combination Pharmaceutical Products: Industrial Manufacturing Versus Personalized 3D Printing.

Fixed-dose combination (FDC) products containing at least two different active pharmaceutical ingredients are designed to treat more effectively different pathologies as they have demonstrated to enhance patient compliance. However, the combination of multiple drugs within the same dosage form can bring many physicochemical and pharmacodynamic interactions. The manufacturing process of FDC products can be challenging, especially when it is required to achieve different drug release profiles within the same dosage form to overcome physicochemical drug interactions. Monolithic, multiple-layer, and multiparticulate systems are the most common type of FDCs. Currently, the main manufacturing techniques utilized in industrial pharmaceutical companies rely on the use of combined wet and dry granulation, hot-melt extrusion coupled with spray coating, and compression of bilayered tablets. Nowadays, personalized medicines are gaining importance in clinical settings and 3D printing is taking a highlighted role in the manufacturing of complex and personalized 3D solid dosage forms that could not be manufactured using conventional techniques. In this review, it will be discussed in detail current marketed FDC products and their application in several diseases with an especial focus on antimicrobial drugs. Current industrial conventional techniques will be compared with 3D printing manufacturing of FDCs. Graphical Abstract.

Transferosomes as nanocarriers for drugs across the skin: Quality by design from lab to industrial scale.

Transferosomes, also known as transfersomes, are ultradeformable vesicles for transdermal applications consisting of a lipid bilayer with phospholipids and an edge activator and an ethanol/aqueous core. Depending on the lipophilicity of the active substance, it can be encapsulated within the core or amongst the lipid bilayer. Compared to liposomes, transferosomes are able to reach intact deeper regions of the skin after topical administration delivering higher concentrations of active substances making them a successful drug delivery carrier for transdermal applications. Most transferosomes contain phosphatidylcholine (C18) as it is the most abundant lipid component of the cell membrane, and hence, it is highly tolerated for the skin, decreasing the risk of undesirable effects, such as hypersensitive reactions. The most common edge activators are surfactants such as sodium deoxycholate, Tween® 80 and Span® 80. Their chain length is optimal for intercalation within the C18 phospholipid bilayer. A wide variety of drugs has been successfully encapsulated within transferosomes such as phytocompounds like sinomenine or apigenin for rheumatoid arthritis and leukaemia respectively, small hydrophobic drugs but also macromolecules like insulin. The main factors to develop optimal transferosomal formulations (with high drug loading and nanometric size) are the optimal ratio between the main components as well as the critical process parameters for their manufacture. Application of quality by design (QbD), specifically design of experiments (DoE), is crucial to understand the interplay among all these factors not only during the preparation at lab scale but also in the scale-up process. Clinical trials of a licensed topical ketoprofen transferosomal gel have shown promising results in the alleviation of symptons in orthreothritis with non-severe skin and subcutaneous tissue disorders. However, the product was withdrawn from the market which probably was related to the higher cost of the medicine linked to the expensive manufacturing process required in the production of transferosomes compared to other conventional gel formulations. This example brings out the need for a careful formulation design to exploit the best properties of this drug delivery system as well as the development of manufacturing processes easily scalable at industrial level.

Oral particle uptake and organ targeting drives the activity of amphotericin B nanoparticles.

There are very few drug delivery systems that target key organs via the oral route, as oral delivery advances normally address gastrointestinal drug dissolution, permeation, and stability. Here we introduce a nanomedicine in which nanoparticles, while also protecting the drug from gastric degradation, are taken up by the gastrointestinal epithelia and transported to the lung, liver, and spleen, thus selectively enhancing drug bioavailability in these target organs and diminishing kidney exposure (relevant to nephrotoxic drugs). Our work demonstrates, for the first time, that oral particle uptake and translocation to specific organs may be used to achieve a beneficial therapeutic response. We have illustrated this using amphotericin B, a nephrotoxic drug encapsulated within N-palmitoyl-N-methyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycol chitosan (GCPQ) nanoparticles, and have evidenced our approach in three separate disease states (visceral leishmaniasis, candidiasis, and aspergillosis) using industry standard models of the disease in small animals. The oral bioavailability of AmB-GCPQ nanoparticles is 24%. In all disease models, AmB-GCPQ nanoparticles show comparable efficacy to parenteral liposomal AmB (AmBisome). Our work thus paves the way for others to use nanoparticles to achieve a specific targeted delivery of drug to key organs via the oral route. This is especially important for drugs with a narrow therapeutic index.

Effect of aggregation state on the toxicity of different amphotericin B preparations.

The aim of this work was to study the effect of aggregation of amphotericin B (AMB) in their toxicity. The aggregation of AMB depends on different formulation factors such as pH and excipients, therefore three formulations with different AMB aggregation states were prepared: a monomeric form (M-AMB), a dimeric form (D-AMB) and a poly-aggregated form (P-AMB). The predominant aggregation state of each AMB formulation was characterized by spectrophotometry and their size by dynamic laser light scattering. Toxicity was evaluated by lethality in mice and hemolysis test from human erythrocytes and the experimental AMB formulations were compared with reference formulations of AmBisome, Fungizone and heated Fungizone. The less toxic aggregation form of AMB was the poly-aggregated one which was similar to AmBisome. Moreover, the P-AMB and heated Fungizone were centrifuged to isolate different size fractions. The toxicity of these two heterogeneous formulations was related to their size, so the smaller the aggregation size fraction the higher the toxicity determined by hemolysis. It can be concluded that the aggregation state of AMB and their size affects critically the toxicity of AMB. The low toxic P-AMB formulations contain a different poly-aggregated state to that of AmBisome, heated Fungizone and the other studied AMB aggregation states.

Comparative pharmacokinetics and safety of a novel lyophilized amphotericin B lecithin-based oil-water microemulsion and amphotericin B deoxycholate in animal models.

Amphotericin B (AmB) has been a most effective systemic antifungal agent, but its use is circumscribed by the dose-limiting toxicity of the conventional micellar dispersion formulation Fungizone (D-AmB). To lower AmB-associated toxicity, AmB may be integrated into oil-in-water lecithin-based microemulsions. The present study compares the pharmacokinetic characteristics of D-AmB with the alternative formulation of AmB in microemulsion (M-AmB), which has proved effective in a murine candidiasis model. Both formulations were given by intravenous bolus: D-AmB 1 mg/kg, and M-AmB 0.5, 1 or 2 mg/kg. The pharmacokinetics of D-AmB and M-AmB have several differences, specifically with regard to the respective Cmax and AUC0- infinity values. Elimination of AmB from serum was biphasic for both M-AmB and D-AmB. Single-dose D-AmB (1 mg/kg) achieved a Cmax of 3.89 +/- 0.48 mg/L and an AUC0- infinity of 32.28 +/- 7.31 mg.h/L, whereas single-dose M-AmB (1 mg/kg) by comparison achieved a lower Cmax (2.92 +/- 0.54 mg/L) and a lower AUC0- infinity (21.89 +/- 5.17 mg.h/L). To evaluate the safety of M-AmB, a multiple-dose toxicity study was performed in groups of 10 mice, each receiving D-AmB 1 mg/kg, or M-AmB 1, 1.5, 2 or 3 mg/kg. The findings suggest that, in comparison with D-AmB, M-AmB produces no histologically demonstrable renal lesions, or changes in clinical chemistry.

Lecithin-based oil-in-water microemulsions for parenteral use: pseudoternary phase diagrams, characterization and toxicity studies.

Pseudoternary phase diagrams have been constructed to evaluate the phase behavior of systems containing water/lecithin/polysorbate 80/isopropyl myristate at different polysorbate 80:lecithin weight ratios (K(m)). Oil-in-water microemulsion regions were accurately determined and the influence of the K(m) on the area of existence of such disperse systems was also examined. Viscosity studies as well as particle size analysis by dynamic light scattering were carried out on oil-water microemulsions, and the influence of the oil phase content, the total amount of surfactants and K(m) on the rheological behavior, viscosity, and droplet size of such disperse systems was evaluated. All systems studied showed a water-rich isotrope region (oil-in-water microemulsion area), that was seen to be highly dependent upon the surfactant/cosurfactant weight ratio. Most of the microemulsions analyzed showed a non-Newtonian rheological behavior and both, droplet size, and viscosity of the disperse systems, were found to be much more influenced by the total content of oil phase and surfactants present in the microemulsion than by the K(m). The selected system underwent both stability and in vivo acute toxicity studies, and seemed to be highly stable, even at extreme conditions, and very low toxic according to the results obtained.