Quality control of pharmaceutical ingredients:developing a caking test for industry

Objectives - Powdered and granulated particulate materials make up most of the ingredients of pharmaceuticals and are often at risk of undergoing unwanted agglomeration, or caking, during transport or storage. This is particularly acute when bulk powders are exposed to extreme swings in temperature and relative humidity, which is now common as drugs are produced and administered in increasingly hostile climates and are stored for longer periods of time prior to use. This study explores the possibility of using a uniaxial unconfined compression test to compare the strength of caked agglomerates exposed to different temperatures and relative humidities. This is part of a longer-term study to construct a protocol to predict the caking tendency of a new bulk material from individual particle properties. The main challenge is to develop techniques that provide repeatable results yet are presented simply enough to be useful to a wide range of industries. Methods - Powdered sucrose, a major pharmaceutical ingredient, was poured into a split die and exposed to high and low relative humidity cycles at room temperature. The typical ranges were 20–30% for the lower value and 70–80% for the higher value. The outer die casing was then removed and the resultant agglomerate was subjected to an unconfined compression test using a plunger fitted to a Zwick compression tester. The force against displacement was logged so that the dynamics of failure as well as the failure load of the sample could be recorded. The experimental matrix included varying the number of cycles, the amount between the maximum and minimum relative humidity, the height and diameters of the samples, the number of cycles and the particle size. Results - Trends showed that the tensile strength of the agglomerates increased with the number of cycles and also with the more extreme swings in relative humidity. This agrees with previous work on alternative methods of measuring the tensile strength of sugar agglomerates formed from humidity cycling (Leaper et al 2003). Conclusions - The results show that at the very least the uniaxial tester is a good comparative tester to examine the caking tendency of powdered materials, with a simple arrangement and operation that are compatible with the requirements of industry. However, further work is required to continue to optimize the height/ diameter ratio during tests.

Crystal engineering strategies against antimicrobial resistance: a solid contribution to a contemporary problem

This PhD thesis sets its goal in the application of crystal engineering strategies to the design, formulation, synthesis, and characterization of innovative materials obtained by combining well established biologically active molecules and/or GRAS (generally recognized as safe) compounds with co-formers able to modulate specific properties of the molecule of interest. The solid-state association, via non-covalent interactions, of an active ingredient with another molecular component, a metal salt or a complex, may alter in a useful way the physicochemical properties of the active ingredient and/or may allow to explore new ways to enhance, in a synergistic way, the overall biological performance. More specifically this thesis will address the threat posed by the increasing antimicrobial resistance (AMR) developed by microorganisms, which call for novel therapeutic strategies. Crystal engineering provides new tools to approach this crisis in a greener and cost-effective way. This PhD work has been developed along two main research lines aiming to contribute to the search for innovative solutions to the AMR problem. Design, preparation and characterization of novel metal-based antimicrobials, whereby organic molecules with known antimicrobial properties are combined with metal atoms also known to exert antimicrobial action. Design, preparation and characterization of co-crystals obtained by combining antibacterial APIs (active pharmaceutical ingredients) with natural antimicrobials.

Increasing the symmetry of drug crystals: a monoclinic conformational polymorph of the platelet antiaggregating agent ticlopidine hydrochloride

Ticlopidine hydrochloride (TICLID (R)) is a platelet antiaggregating agent whose use as a potent antithrombotic pharmaceutical ingredient is widespread, even though this drug has not been well characterized in the solid state. Only the crystal phase used for drug product manufacturing is known. Here, a new polymorph of ticlopidine hydrochloride was discovered and its structure was determined. While the antecedent polymorph crystallizes in the triclinic space group P (1) over bar, the new crystal phase was solved in the monoclinic space group P2(1)/c. Both polymorphs crystallize as racemic mixtures of enantiomeric (ticlopidine)(+) cations. Detailed geometrical and packing comparisons between the crystal structures of the two polymorphs have allowed us to understand how different supramolecular architectures are assembled. It was feasible to conclude that the main difference between the two polymorphs is a rotation of about 120 degrees on the bridging bond between the thienopyridine and o-chlorobenzyl moieties. The differential o-chlorobenzyl conformation is related to changeable patterns of weak intermolecular contacts involving this moiety, such as edge-to-face Cl center dot center dot center dot pi and C-H center dot center dot center dot pi interactions in the new polymorph and face-to-face pi center dot center dot center dot pi contacts in the triclinic crystal phase, leading to a symmetry increase in the ticlopidine hydrochloride solid state form described for the first time in this study. Other conformational features are slightly different between the two polymorphs, such as the thienopyridine puckerings and the o-chlorophenyl orientations. These conformational characteristics were also correlated to the crystal packing patterns.

Cromatografia preparativa: caracterização e purificação de produtos e impurezas com aplicação na indústria de APIs

Neste trabalho efectuou-se o desenvolvimento de métodos de purificação de princípios activos farmacêuticos utilizando a cromatografia preparativa em coluna (eluição batch). Por motivos de confidencialidade as amostras tiveram a denominação de Amostra Projecto A, Amostra Projecto B, Amostra Projecto C e Amostra 2 - Projecto A. Estudou-se a performance de coluna para cada projecto utilizando colunas pré-preparadas e colunas preparadas com a tecnologia DAC (Dynamical Axial Compression). Elaborou-se o procedimento experimental de enchimento de colunas preparativas sob compressão axial. As impurezas isoladas por cromatografia preparativa foram sujeitas a caracterização HPLC e LC-MS. Com colunas pré-preparadas, obteve-se para a amostra do projecto A um rendimento de 70,59% e relativamente à amostra projecto B obteve-se u, rendimento de 15,39%. Relativamente à impureza isolada da amostra do projecto A, o estudo por HPLC revelou 98,69% de pureza, quanto à impureza da amostra projecto B obteve-se 88,80% de pureza. A impureza da amostra do projecto A exibiu por LC-MS a massa de 467,15 enquanto que a impureza da amostra do projecto B obteve 503,31. O enchimento de colunas preparativas sob pressão axial dinâmica foi realizado numa coluna de dimensão 260 X 25 mm, onde 76,60% da coluna foi compactada com o material de enchimento SepTech ST150-C18, 10 µm. A coluna foi testada com a impureza isolada do projecto A. Isolou-se o pico referente à impureza, e por LC-MS verificou-se que a impureza foi isolada eficientemente com 97,20& de pureza e massa 467,15 como indicado anteriormente. Em relação à preparação de colunas com a tecnologia DAC concluímos que, neste caso, é mais vantajoso realizar o enchimento das colunas do que efectuar a compra de colunas pré-preparadas. Com estes resultados pode-se concluir que a cromatografia preparativa é um método eficaz no isolamento de impurezas de APIs (Active Pharmaceutical Ingredients) face a métodos de purificação clássicos que foram realizados previamente noutros estudos. Com futuros desenvolvimentos e investimentos, facilmente poderá tornar-se num método de purificação indispensável na Indústria de Química Fina.

Synthesis, characterization and antiproliferative activity on cancer cell lines of new ionic liquids from ampicillin

Ionic Liquids (ILs) are ionic compounds that possess melting temperature below 100ºC and they have been a topic of great interest since the mid-1990s due to their unique properties. The range of IL uses has been broadened, due to a significant increase in the variety of physical, chemical and biological ILs properties. They are now used as Active Pharmaceutical Ingredients (APIs) and recent interests are focused on their application as innovative solutions in new medical treatment and delivery options.1 In this work, our principal objective was the synthesis and investigation of physicochemical and medical properties of ionic liquids (ILs) and organic salts from ampicillin. This approach is of huge interest in pharmaceutical industry as cation and anion composition of ILs and organic salts can greatly alter their desired properties, namely the melting temperature and even synergistic effects can be obtained.2,3 For the synthesis of these compounds we used a recently developed method proposed by Ohno et al.4 for the preparation of quaternary ammonium and phosphonium hydroxides, that were neutralized by ampicillin. After purification we obtained pure ILs and salts in good yields. These ILs shows good antimicrobial and antifungal activities. As it is well known that some ionic liquids containing phosphonium and ammonium cation also shows anti-cancer activity1,5 we also decided to study these compounds against some cancer cell lines.

Development and biological evaluation of API-ILs based on anti-bacterial and anti-fungal drugs

In recent years Ionic Liquids (ILs) are being applied in life sciences. ILs are being produce with active pharmaceutical drugs (API) as they can reduce polymorphism and drug solubility problems [1] Also ILs are being applied as a drug delivery device in innovative therapies What is appealing in ILs is the ILs building up platform, the counter-ion can be carefully chosen in order to avoid undesirable side effects or to give innovative therapies in which two active ions are paired. This work shows ILs based on ampicillin (an anti-bacterial agent) and ILs based on Amphotericin B. Also we show studies that indicate that ILs based on Ampicillin could reverse resistance in some bacteria. The ILs produced in this work were synthetized by the neutralization method described in Ferraz et. al. [2] Ampicillin anion was combined with the following organic cations 1-ethyl-3-methylimidazolium, [EMIM]; 1-hydroxy-ethyl-3-methylimidazolium, [C2OHMIM]; choline, [cholin]; tetraethylammonium, [TEA]; cetylpyridinium, [C16pyr] and trihexyltetradecylphosphonium, [P6,6,6,14]. Amphotericin B was combined with [C16pyr], [cholin] and 1-metohyethyl-3-methylimidazolium, [C3OMIM]. The ILs-APIs based on ampicillin[2] were tested against sensitive Gram-negative bacteria Escherichia coli ATCC 25922 and Klebsiella pneumonia (clinical isolated), as well as on Gram positive Staphylococcus Aureus ATCC 25923, Staphylococcus epidermidis and Enterococcus faecalis. The arising resistance developed by bacteria to antibiotics is a serious public health threat and needs new and urgent measures. We study the bacterial activity of these compounds against a panel of resistant bacteria (clinical isolated strains): E. coli CTX M9, E. coli TEM CTX M9, E. coli TEM1, E. coli CTX M2, E. coli AmpC Mox2. In this work we demonstrate that is possible to produce ILs from anti-bacterial and anti-fungal compounds. We show here that the new ILs can reverse the bacteria resistance. With the careful choice of the organic cation, it is possible to create important biological and physic-chemical properties. This work also shows that the ion-pair is fundamental in ampicillin mechanism of action.

QuEChERS: a new sample preparation approach for the determination of ibuprofen and its metabolites in soils

Ibuprofen is one of the most used active pharmaceutical ingredients worldwide. A new method for the analysis of ibuprofen and its metabolites, hydroxyibuprofen and carboxyibuprofen, in soils is presented. The extraction of these compounds from the soil matrices was performed by using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. The method involves a single extraction of the investigated compounds with purified water (acidified at pH 2.5 with hydrochloric acid), and a slow and continuous addition of the QuEChERS content, followed by the addition of acidified acetonitrile (1% acetic acid), prior to the determination by liquid chromatography coupled with fluorescence detection (LC–FLD). Validation studies were carried out using soil samples with a range of organic carbon contents. Recoveries of the fortified samples ranged from 79.5% to 101%. Relative standard deviations for all matrix–compound combinations did not exceed 3%. The method quantification limits were ≤22.4 μg kg−1 in all cases. The developed method was applied to the analysis of sixteen real samples.

Bioatividade de Líquidos Iónicos derivados do Ácido Valpróico em bactérias Gram-negativa

Os Líquidos Iónicos (LIs) são sais orgânicos constituídos exclusivamente por iões e possuem pontos de fusão inferiores a 100ºC. As suas propriedades únicas e o facto de ser possível ajustar as suas propriedades físicas, químicas e biológicas, de acordo com o objetivo pretendido, tornam esta classe de compostos, um grande objeto de estudo de inúmeros investigadores. Desde os inícios da sua aplicação até à atualidade, a investigação nesta área expandiu o seu raio de ação, estando já descrito o seu potencial como agentes antimicrobianos e, mais recentemente, como compostos farmacêuticos ativos. Atualmente muitas das suas aplicações são baseadas nas suas propriedades biológicas. Esta Tese teve como objetivo avaliar a influência que os LIs podem exercer a nível do crescimento bacteriano e estudar alternativas de combater a resistência bacteriana. Todos os LIs utilizados neste trabalho tinham como anião o ácido valpróico, sendo utilizados catiões orgânicos de amónio e de imidazólio. Foram utilizadas 4 bactérias e avaliou-se a atividade biológica e a respetiva taxa de crescimento. O estudo da sua atividade biológica foi feito através da determinação da Concentração Mínima Inibitória (CMI) e a análise das suas curvas de crescimentos na presença e ausência de composto. Com este trabalho foi possível verificar que dentro dos compostos em estudo, LIs derivados do valproato, o Valproato com o cetilperidínio [valp] [cetylpir] foi o que influenciou o crescimento de todas as bactérias estudadas. Este estudo demonstrou o potencial antibacteriano de alguns compostos, podendo desta forma vir a ser utilizados para fins farmacêuticos

Development of electrospun Ion jelly fibers® for drug delivery

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Application of OSN in membrane cascade for purification of the API Amoxicillin

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Development of composite particles for pulmonary delivery

In this work, biocompatible and biodegradable poly(D-L-lactide-co-glycolide) (PLGA) microparticles with the potential for use as a controlled release system of vaccines and other drugs to the lung were manufactured using supercritical CO2, through the Supercritical Assisted Atomization (SAA) technique. After performing a controlled variance in production parameters (temperature, pressure, CO2/solution flow ratio) PLGA microparticles were characterized and later used to encapsulate active pharmaceutical ingredients (API). Bovine serum albumin (BSA) was chosen as model protein and vaccine, while sildenafil was the chosen drug to treat pulmonary artery hypertension and their effect on the particles characteristics was evaluated. All the produced formulations were characterized in relation to their morphology (Morphologi G3 and scanning electronic microscopy (SEM)), to their physical-chemical properties (X-ray diffraction (XRD, differential scanning calorimetry (DSC), Fourier transform infrared (FTIR)) and aerodynamic performance using an in vitro aerosolization study – Andersen cascade impactor (ACI) - to obtain data such as the fine particle fraction (FPF) and the mass median aerodynamic diameter (MMAD). Furthermore, pharmacokinetic, biodegradability and biocompatibility tests were performed in order to verify the particle suitability for inhalation. The resulting particles showed aerodynamic diameters between the 3 and 5 μm, yields up to 58% and FPF percentages rounding the 30%. Taken as a whole, the produced microparticles do present the necessary requests to make them appropriate for pulmonary delivery.

Fiber functionalization for biomedical applications

Deep-eutectic solvents (DES) are considered novel renewable and biodegradable solvents, with a cheap and easy synthesis, without waste production. Later it was discovered a new subclass of DES that even can be biocompatible, since their synthesis uses primary metabolites such as amino acids, organic acids and sugars, from organisms. This subclass was named natural deep-eutectic solvents (NADES). Due to their properties it was tried to study the interaction between these solvents and biopolymers, in order to produce functionalized fibers for biomedical applications. In this way, fibers were produced by using the electrospinning technique. However, it was first necessary to study some physical properties of NADES, as well as the influence of water in their properties. It has been concluded that the water has a high influence on NADES properties, which can be seen on the results obtained from the rheology and viscosity studies. The fluid dynamics had changed, as well as the viscosity. Afterwards, it was tested the viability of using a starch blend. First it was tested the dissolution of these biopolymers into NADES, in order to study the viability of their application in electrospinning. However the results obtained were not satisfactory, since the starch polymers studied did not presented any dissolution in any NADES, or even in organic solvents. In this way it was changed the approach, and it was used other biocompatible polymers. Poly(ethylene oxide), poly(vinyl alcohol) and gelatin were the others biopolymers tested for the electrospinning, with NADES. All polymers show good results, since it was possible to obtain fibers. However for gelatin it was used only eutectic mixtures, containing active pharmaceutical ingredients (API’s), instead of NADES. For this case it was used mandelic acid (antimicrobial properties), choline chloride, ibuprofen (anti-inflammatory properties) and menthol (analgesic properties). The polymers and the produced fibers were characterized by scanning electron microscope (SEM), Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With the help of these techniques it was possible to conclude that it was possible to encapsulate NADES within the fibers. Rheology it was also study for poly(ethylene oxide) and poly(vinyl alcohol), in a way to understand the influence of polymer concentration, on the electrospinning technique. For the gelatin, among the characterization techniques, it was also performed cytotoxicity and drug release studies. The gelatin membranes did not show any toxicity for the cells, since their viability was maintained. Regarding the controlled release profile experiment no conclusion could be drawn from the experiments, due to the rapid and complete dissolution of the gelatin in the buffer solution. However it was possible to quantify the mixture of choline chloride with mandelic acid, allowing thus to complete, and confirm, the information already obtained for the others characterization technique.

POZylation: a new approach to enhance nanoparticle diffusion through mucosal barriers

The increasing use of nanoparticles in the pharmaceutical industry is generating concomitant interest in developing nanomaterials that can rapidly penetrate into, and permeate through, biological membranes to facilitate drug delivery and improve the bioavailability of active pharmaceutical ingredients. Here, we demonstrate that the permeation of thiolated silica nanoparticles through porcine gastric mucosa can be significantly enhanced by their functionalization with either 5 kDa poly(2-ethyl-2-oxazoline) or poly(ethylene glycol). Nanoparticle diffusion was assessed using two independent techniques; Nanoparticle Tracking Analysis, and fluorescence microscopy. Our results show that poly(2-ethyl-2-oxazoline) and poly(ethylene glycol) have comparable abilities to enhance diffusion of silica nanoparticles in mucin dispersions and through the gastric mucosa. These findings provide a new strategy in the design of nanomedicines, by surface modification or nanoparticle core construction, for enhanced transmucosal drug delivery.

Preparação e carcterização de complexos multicomponentes contendo ciclodextrinas e benznidazol

The benznidazole (BNZ) is the only alternative for Chagas disease treatment in Brazil. This drug has low solubility, which restricts its dissolution rate. Thus, the present work aimed to study the BNZ interactions in binary systems with beta cyclodextrin (β-CD) and hydroxypropyl-beta cyclodextrin (HP-β-CD), in order to increase the apparent aqueous solubility of drug. The influence of seven hydrophilic polymers, triethanolamine (TEA) and 1-methyl-2- pyrrolidone (NMP) in benznidazole apparent aqueous solubility, as well as the formation of inclusion complexes was also investigated. The interactions in solution were predicted and investigated using phase solubility diagram methodology, nuclear magnetic resonance of protons (RMN) and molecular modeling. Complexes were obtained in solid phase by spray drying and physicochemical characterization included the UV-Vis spectrophotometric spectroscopy in the infrared region, scanning electron microscopy, X-ray diffraction and dissolution drug test from the different systems. The increment on apparent aqueous solubility of drug was achieved with a linear type (AL) in presence of both cyclodextrins at different pH values. The hydrophilic polymers and 1-methyl-2-pyrrolidone contributes to the formation of inclusion complexes, while the triethanolamine decreased the complex stability constant (Kc). The log-linear model applied for solubility diagrams revealed that both triethanolamine and 1-methyl-2-pyrrolidone showed an action cosolvent (both solvents) and complexing (1-methyl-2-pyrrolidone). The best results were obtained with complexes involving 1-methyl-2-pyrrolidone and hydroxypropylbeta- cyclodextrin, with an increased of benznidazole solubility in 27.9 and 9.4 times, respectively. The complexes effectiveness was proven by dissolution tests, in which the ternary complexes and physical mixtures involving 1-methyl- 2-pyrrolidone and both cyclodextrins investigated showed better results, showing the potential use as novel pharmaceutical ingredient, that leads to increased benznidazole bioavailability

Estudo de compatibilidade e estabilidade térmica do ácido retinóico e hidroquinona por termogravimetria

Retinoic acid (RA) and hydroquinone (HQ) assets are widely used in pharmaceutical and cosmetic formulations, for having depigmenting properties and are largely produced in drugstores. To assist in the development of formulations containing the active RA and HQ National Forms of Brazilian Pharmacopoeia (2005 and 2012 ) proposes formulations with different excipients such as cetyl alcohol (AC), cetostearyl alcohol (ACT), methylparaben (MTP), propyl paraben ( PPB), glycerin (GLY), dipropylene glycol (DPG), imidazolidinil urea ( IMD ), cyclomethicone (CCM ), butylated hydroxytoluene (BHT), octyl stearate (ETO), EDTA, decil oleate (ODC) and hydroxipropymethyl celullose (HPMC). One of the difficulties found in most cosmetic formulations is the large number of incompatibilities between the components of the formulations, so the aim this study was to evaluate thermal stability and interactions between these active pharmaceutical ingredients and excipients. The depigmenting agents were analyzed by DSC and TG and excipients were analyzed by TG. The dynamic thermogravimetric curves were obtained on a SHIMADZU thermobalance, model DTG-60, using an alumina crucible, at the heating rate of 10ºC min-1, in the temperature range of 25-900 ºC, under an atmosphere of nitrogen at 50 mL min-1. The DSC curves were obtained using Shimadzu calorimeter, model DSC-60, using aluminum crucible, at the heating rate of 10ºC min-1, in the temperature range of 25-400ºC. The thermogravimetric and calorimetric curves were analyzed using TASYS software SHIMADZU. In this study no were found interactions between AR and the following excipients: MTP, PPB, IMD, ODC, EDTA, CCM, ETO, HPMC. However, were found interactions with the following excipients: AC, ACT, BHT, GLI and DPG. For HQ were found interactions with IMD and DPG. Interactions remained even changing proportions of the mixtures and the ternary. Thus, the studies conducted with excipients of National Formulary from 2005 and 2012 showed that these new excipients do not interact by thermogravimetry with the active pharmaceutical ingredients of this study