The relationship between angiogenesis and the immune response in carcinogenesis and the progression of malignant disease

Recent studies have demonstrated that angiogenesis and suppressed cell- mediated immunity (CMI) play a central role in the pathogenesis of malignant disease facilitating tumour growth, invasion and metastasis. In the majority of tumours, the malignant process is preceded by a pathological condition or exposure to an irritant which itself is associated with the induction of angiogenesis and/or suppressed CMI. These include: cigarette smoking, chronic bronchitis and lung cancer; chronic oesophagitis and oesophageal cancer; chronic viral infections such as human papilloma virus and ano-genital cancers, chronic hepatitis B and C and hepatocellular carcinoma, and Epstein- Barr virus (EBV) and lymphomas; chronic inflammatory conditions such as Crohn's disease and ulcerative colitis and colorectal cancer; asbestos exposure and mesothelioma and excessive sunlight exposure/sunburn and malignant melanoma. Chronic exposure to growth factors (insulin-like growth factor-I in acromegaly), mutations in tumour suppressor genes (TP53 in Li Fraumeni syndrome) and long-term exposure to immunosuppressive agents (cyclosporin A) may also give rise to similar environments and are associated with the development of a range of solid tumours. The increased blood supply would facilitate the development and proliferation of an abnormal clone or clones of cells arising as the result of: (a) an inherited genetic abnormality; and/or (b) acquired somatic mutations, the latter due to local production and/or enhanced delivery of carcinogens and mutagenic growth factors. With progressive detrimental mutations and growth-induced tumour hypoxia, the transformed cell, to a lesser or greater extent, may amplify the angiogenic process and CMI suppression, thereby facilitating further tumour growth and metastasis. There is accumulating evidence that long-term treatment with cyclo-oxygenase inhibitors (aspirin and indomethacin), cytokines such as interferon-α, anti-oestrogens (tamoxifen and raloxifene) and captopril significantly reduces the incidence of solid tumours such as breast and colorectal cancer. These agents are anti-angiogenic and, in the case of aspirin, indomethacin and interferon-α have proven immunomodulatory effects. Collectively these observations indicate that angiogenesis and suppressed CMI play a central role in the development and progression of malignant disease. (C) 2000 Elsevier Science Ltd.

Effect of pregnant mare serum gonadotropin on the induction and degradation of FSH and LH receptors in the granulosa cell of the immature rat

The relative induction of FSH and LH receptors in the granulosa cells of immature rat ovary by pregnant mare serum gonadotropin (PMSG) has been studied. A single injection of PMSG (15 IU) brought about a 3- and 12-fold increase in FSH and LH receptor concentration,respectively, in the granulosa cells. Maximal concentration was reached by 72 h but the receptor levels showed a sharp decline during the next 24–48 h. The kinetic properties of the newly formed FSH receptors were indistinguishable from the pre-existing ones. The induced FSH receptors were functional as demonstrated by an increase in the in vitro responsiveness of the cells to exogenous FSH in terms of progesterone production. Treatment of immature rats with cyanoketone, an inhibitor of Δ5,3β-hydroxysteroid dehydrogenase, prior to PMSG injection effectively reduced the PMSG-stimulated increase in the serum estradiol, uterine weight and LH receptors but had no effect on the FSH receptor induction. The ability of PMSG to induce gonadotropin receptors can be arrested at any given time by injecting its antibody, thereby suggesting a continuous need for the hormonal inducer. Estrogen in the absence of the primary inducer was unable to maintain the induced LH and FSH receptor concentration. Inhibition of prostaglandin synthesis using indomethacin also had no effect on either the induction or degradation of gonadotropin receptors. Administration of PMSG antiserum, 48 h after PMSG injection, brought about a rapid decline in the induced receptors over the next 24 h, with a rate constant and \iota 1/2 of 0.078 h−1 and 8.9 h for FSH receptors and 0.086 h−1 and 8.0 h for the LH receptors, respectively.

Mesoporous silica- and silicon-based materials as carriers for poorly water soluble drugs

New chemical entities with unfavorable water solubility properties are continuously emerging in drug discovery. Without pharmaceutical manipulations inefficient concentrations of these drugs in the systemic circulation are probable. Typically, in order to be absorbed from the gastrointestinal tract, the drug has to be dissolved. Several methods have been developed to improve the dissolution of poorly soluble drugs. In this study, the applicability of different types of mesoporous (pore diameters between 2 and 50 nm) silicon- and silica-based materials as pharmaceutical carriers for poorly water soluble drugs was evaluated. Thermally oxidized and carbonized mesoporous silicon materials, ordered mesoporous silicas MCM-41 and SBA-15, and non-treated mesoporous silicon and silica gel were assessed in the experiments. The characteristic properties of these materials are the narrow pore diameters and the large surface areas up to over 900 m²/g. Loading of poorly water soluble drugs into these pores restricts their crystallization, and thus, improves drug dissolution from the materials as compared to the bulk drug molecules. In addition, the wide surface area provides possibilities for interactions between the loaded substance and the carrier particle, allowing the stabilization of the system. Ibuprofen, indomethacin and furosemide were selected as poorly soluble model drugs in this study. Their solubilities are strongly pH-dependent and the poorest (< 100 µg/ml) at low pH values. The pharmaceutical performance of the studied materials was evaluated by several methods. In this work, drug loading was performed successfully using rotavapor and fluid bed equipment in a larger scale and in a more efficient manner than with the commonly used immersion methods. It was shown that several carrier particle properties, in particular the pore diameter, affect the loading efficiency (typically ~25-40 w-%) and the release rate of the drug from the mesoporous carriers. A wide pore diameter provided easier loading and faster release of the drug. The ordering and length of the pores also affected the efficiency of the drug diffusion. However, these properties can also compensate the effects of each other. The surface treatment of porous silicon was important in stabilizing the system, as the non-treated mesoporous silicon was easily oxidized at room temperature. Different surface chemical treatments changed the hydrophilicity of the porous silicon materials and also the potential interactions between the loaded drug and the particle, which further affected the drug release properties. In all of the studies, it was demonstrated that loading into mesoporous silicon and silica materials improved the dissolution of the poorly soluble drugs as compared to the corresponding bulk compounds (e.g. after 30 min ~2-7 times more drug was dissolved depending on the materials). The release profile of the loaded substances remained similar also after 3 months of storage at 30°C/56% RH. The thermally carbonized mesoporous silicon did not compromise the Caco-2 monolayer integrity in the permeation studies and improved drug permeability was observed. The loaded mesoporous silica materials were also successfully compressed into tablets without compromising their characteristic structural and drug releasing properties. The results of this research indicated that mesoporous silicon/silica-based materials are promising materials to improve the dissolution of poorly water soluble drugs. Their feasibility in pharmaceutical laboratory scale processes was also confirmed in this thesis.

Molecular dynamics simulations of PPI dendrimer-drug complexes

Dendrimeric nanoparticles are potential drug delivery devices which can enhance the solubility of hydrophobic drugs, thus increasing their bioavailability and sustained release action. A quantitative understanding of the dendrimer-drug interactions can give valuable insight into the solubility and release profile of hydrophobic drug molecules in various solvent conditions. Fully atomistic molecular dynamics (MD) simulations have been performed to study the interactions of G5 PPIEDA (G5 ethylenediamine cored poly(propylene imine)) dendrimer and two well known drugs (Famotidine and Indomethacin) at different pH conditions. The study suggested that at low pH the dendrimer-drug complexes are thermodynamically unstable as compared to neutral and high pH conditions. Calculated Potential of Mean Force (PMF) by umbrella sampling showed that the release of drugs from the dendrimer at low pH is spontaneous, median release at neutral pH and slow release at high pH. In addition, Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) binding free energy calculations were also performed at each umbrella sampling window to identify the various energy contributions. To understand the effect of dendrimer chemistry and topology on the solubility and release profile of drugs, this study is extended to explore the solubility and release profile of phenylbutazone drug complexed with G3 poly(amidoamine) and G4 diaminobutane cored PPI dendrimers. The results indicate that the pH-induced conformational changes in dendrimer, ionization states, dendrimer type and pK(a) of the guest molecules influence the free energy barrier and stability of complexation, and thus regulate drug loading, solubility and release.

Potencial antiinflamatório do extrato aquoso de Echinodorus macrophyllus e de suas frações em modelo de inflamação aguda

A Echinodorus macrophyllus (Alismataceae), conhecida como chapéu de couro no Brasil, é usada popularmente para tratar doenças reumáticas e inflamatórias. Neste trabalho, foram avaliados os efeitos antiinflamatórios do extrato aquoso de E. macrophyllus (EAEm) e suas frações etanólicas no modelo murino de air pouch. Para a obtenção das frações, 7 g do EAEm foram aplicadas em uma coluna cromatográfica aberta de sílica gel eluída com diferentes concentrações de etanol. Os cromatogramas do EAEm/frações foram obtidos usando um sistema de HPLC. Foram obtidas quatro frações, duas delas com maior rendimento. Resumidamente, a bolha de ar foi induzida pela injeção de 5 mL de ar estéril (s.c) no dorso de camundongos SW machos (25-35 g). Após 3 dias, mas 3 mL de ar estéril foram injetados para manter a bolha. No sexto dia, cada grupo (n = 4) foi tratado intraperitoneal (ip) ou oralmente (v.o) com EAEm (25 ou 250 mg/kg), Fr20 ou Fr40 (2,5, 25, 50 ou 100 mg/kg) e os controles com indometacina (10 mg/kg, v.o.) ou veículo (salina). Uma hora depois, 1 mL de salina ou de carragenina 1% estéril foi injetada dentro da bolha. Após 4 h, a cavidade foi lavada com NaCl 0,9%, EDTA 2 mM (1 mL), para a determinação do número de leucócitos, volume do exsudato e concentração de proteínas. Células do exsudato foram preparadas em citocentrífuga e coradas pelo método do Panótico para a contagem diferencial dos leucócitos. Cortes histológicos coletados dos diferentes grupos foram fixados com formol tamponado 10% (pH 7,4) por 7 dias, corados com HE e analisados em MO. A análise da expressão da iNOS e da COX-2 foi realizada em células do exsudato por RT-PCR. O acúmulo de nitrito (NO2−) no sobrenadante do cultivo de células RAW 264.7 foi determinado usando um ensaio colorimétrico baseado na reação de Griess. Os resultados foram expressos como média EP e comparados usando ANOVA seguido de teste de Dunnet. Os experimentos foram realizados em triplicata. No modelo air pouch, a injeção de carragenina 1% aumentou tanto a migração celular quanto a concentração de proteína no exsudato. Contudo, enquanto o pré-tratamento com a Fr40 aumentou a resposta inflamatória, o pré-tratamento com o EAEm e a Fr20, sobretudo por via i.p., inibiu esta resposta quando comparado ao grupo controle tratado apenas com o veículo. Assim, foram observadas as seguintes razões de inibição da migração de células: EAEm, i.p. a 25 mg/kg (66,44%) e a 250 mg/kg (87,27%) e Fr20 a 2,5 mg/kg (26,89%), 25 mg/kg (60,06%), 50 mg/kg (63,13%) e a 100 mg/kg (77,47%). Em relação à contagem diferencial, o EAEm e a Fr20 afetaram principalmente o número de neutrófilos, inibindo sua migração no exsudato. O EAEm e a Fr20 também reduziram a concentração total de proteínas no exsudato principalmente no tratamento i.p.; EAEm a 25 e 250 mg/kg mostrou 3,33 0,55 e 2,05 0,51 mg/mL, respectivamente, quando comparado aos grupos controles (Indometacina 2.88 0.64 mg/mL; Veículo 5.48 0.88 mg/mL). A Fr20 a 2,5, 25, 50 e 100 mg/kg mostrou 4,788 0,444, 1,417 0,519, 2,474 0,529 e 2,215 0, 361 mg/mL. A análise histológica mostrou infiltrado celular, principalmente composto de leucócitos polimorfonucleares ao longo da derme inflamada de animais tratados com veículo. O tratamento com o EAEm ou Fr20 reduziu a infiltração de leucócitos no tecido inflamado. Além disso, o tratamento com o EAEm e a Fr20 mostrou atividade supressora sobre a expressão de iNOS e COX-2, e mostrou efeitos inibitórios na produção de NO induzida por LPS. Concluindo, todos estes resultados confirmam o potencial antiinflamatório sugerido para esta planta e fornecem uma base para a compreensão de seus mecanismos moleculares de ação. Contudo, outros estudos devem ser realizados para melhor elucidar as vias pelas quais o EAEm e a Fr20 exercem seus efeitos antiinflamatórios. Além disso, estudos fitoquímicos devem ser realizados para identificar os compostos ativos no EAEm e na Fr20.

Fracionamento biomonitorado de fração isolada do extrato hexânico de Pterodon polygalaeoflorus

O gênero Pterodon compreende algumas espécies largamente distribuídas sobre a região central do Brasil. Seus frutos são comercialmente disponíveis no mercado da flora medicinal sendo amplamente utilizados pelas suas propriedades farmacológicas como antirreumático, anti-inflamatório e analgésicas. O objetivo deste trabalho foi biomonitorar o fracionamento do extrato hexânico de Pterodon polygalaeflorus Benth. (ExPpg), utilizando modelos de inflamação aguda (edema de pata e bolha de ar). O fracionamento/sub-fracionamento foi realizado por cromatografia em coluna de sílica e as amostras analisadas por cromatografia em camada fina e cromatografia gasosa acoplada a espectrômetro de massas. O edema de pata foi induzido em camundongos SW por injeção (s.c.) de carragenina. Uma hora antes da inoculação de carragenina os animais foram tratados v.o. com veículo (EtOH 15%, 1,25% Tween-20), indometacina (10 mg/kg p.c.) ou ExHPpg/frações/sub-frações de Ppg. No modelo bolha de ar, a cavidade foi desenvolvida em camundongos SW através da injeção de ar estéril (s.c.) no dorso e a inflamação induzida por carragenina. Uma hora antes de inocular a carragenina os animais foram tratados (v.o) com o veículo, indometacina (10 mg/kg) ou ExHPpg/Fr2Ppg/sub-frações. Após 4 h o exsudato da bolha foi coletado para contagem total e diferencial de leucócitos (Panótico rápido) e dosagem de proteínas (biureto), e a pele referente à bolha foi removida para análises macroscópica e histológica (HE). Também foi realizado estudo de migração de neutrófilos pelo ensaio do transwell. Após demonstração do efeito antiedematogênico do ExHPpg, este foi fracionado em quatro frações. A fração Fr2Ppg, mais ativa no modelo de edema de pata, também inibiu os diferentes parâmetros inflamatórios avaliados no modelo de bolha de ar, com a menor dose testada, e foi sub-fracionada em cinco sub-frações. Destas, as SF2.1 e SF2.2 foram as que mostraram melhor efeito anti-inflamatório pelo modelo da bolha de ar. Em relação ao grupo controle com carragenina, o ExHppg, Fr2Ppg, SF 2.1 e SF 2.2, na dose 0,02 mg/kg, exerceram inibições de 70,6%, 62,8%, 54,7% e 79% no número total de células no exsudato, reduções de 76,8%, 76,9%, 71,1% e 73,3% na concentração de proteína, respectivamente. Com a dose de 0,2 mg/kg, foram observadas inibições apenas para ExHPpg e SF 2.1, com intensidades menores, na leucometria total (62,9% e 48,62%, respectivamente), e na concentração de proteína para SF 2.1 e SF 2.2 (reduções de 68,2% e 30,4%, respectivamente). As análises macroscópica e histológica mostraram redução importante da vasodilatação e do infiltrado inflamatório pelo tratamento com o ExHppg, Fr2Ppg, SF 2.1 e SF 2.2. No ensaio de transwell a Fr2Ppg exibiu 31,4% de inibição na migração de neutrófilos. As sub-frações SF2.1 e SF2.2 foram avaliadas por GC-MS, identificando-se de diversos compostos majoritários. Em resumo, este trabalho confirma o potencial anti-inflamatório da espécie Pterodon polygalaeflorus e mostra um fracionamento efetivo do ExHPpg quanto à composição e ação anti-inflamatória.

Participação dos fatores derivados do endotélio no efeito vasodilatador da clonidina

Os mecanismos envolvidos na ação vasodilatadora da clonidina ainda não foram completamente elucidados. Investigamos os mecanismos potencialmente envolvidos utilizando o leito arterial mesentérico (LAM) isolado de rato. No LAM pré-contraído, clonidina (10-300 pmol) induz relaxamento dose-dependente, que foi significativamente inibido pela remoção do endotélio (ácido deoxicólico) e pelos inibidores do receptor α2-adrenérgico, ioimbina (1-3 μM) e rauwolscina (1 μM). A vasodilatação endotélio-dependente induzida pela clonidina foi reduzida pelo inibidor da oxido nítrico sintase (NOS), L-NAME (0.3 mM) e pelo inibidor da guanilato ciclase, ODQ (10 μM), mas não foi alterada pela indometacina (3-10 μM). Na presença do L-NAME, o efeito vasodilatador da clonidina foi adicionalmente reduzido pela solução de potássio elevado (45 mM). Os inibidores dos canais de K⁺ dependentes de cálcio (K_ca), caribdotoxina (ChTx; 0.1 μM) e apamina (0.1 μM) também reduziram a vasodilatação induzida pela clonidina, contudo, esta resposta não foi adicionalmente inibida na presença de L-NAME, como foi observado para acetilcolina (10 pmol). Na presença do bloqueador dos canais de K⁺ dependentes de ATP, glibenclamida (10 μM), o efeito inibitório da associação ChTx, apamina e L-NAME foi aumentado. Em contraste, a vasodilatação induzida pela clonidina não foi afetada pelo inibidor dos canais de potássio dependentes de voltagem (K_v), 4-aminopiridina (4-AP, 1 mM). Concluindo, nossos resultados demonstram que clonidina ativa receptores α2-adrenérgicos no LAM e que a vasodilatação dependente de endotélio é mediada pela ativação da via NO-GMPc e também envolve a ativação de canais de KC_{a e KATP.} Um fator hiperpolarizante derivado do endotélio (EDHF) também parece participar do efeito vasodilatador da clonidina.

Gastroprotective potential of Buddleja scordioides Kunth: Effects into the modulation of antioxidant enzymes and inflammation markers in an in vivo model

Ethnopharmacological relevance: A common plant used to treat several gastric disorders is Buddleja scordioides Kunth,commonly known as salvilla. Aim of thes tudy: To detect inflammatory markers,in order to evaluate the gastroprotective potential of salvilla infusions,as this could have beneficial impact on the population exposed to gastric ulcers and colitis. Materials and methods: The present work attempted infusions were prepared with B. scordioides (1% w/w) lyophilized and stored.Total phenolic content and GC–MS analysis were performed. Wistar rats were divided into five groups a negative vehicle control,an indomethacin group,and three experimental groups,named preventive,curative,and suppressive. All rats were sacrificed under deep ether anesthesia(6h)after the last oral administration of indomethacin/infusion.The rat stomachs were promptly excised,weighed,and chilled in ice-cold and 0.9%NaCl.Histological analysis,nitrites quantification and immunodetection assays were done. Results: B.scordioides infusions markedly reduced the visible hemorrhagic lesions induced byindomethacin in rat stomachs,also showed down-regulation of COX2, IL-8 and TNFα and up-regulation of COX-1with a moderate down-regulation of NFkB and lower amount of nitrites.However,this behavior was dependent on the treatment,showing most down-regulation of COX-2,TNFα and IL-8 in the curative treatment;more down-regulation of NF-kB in the preventive treatment;and more up-regulation of COX-1 for the suppressor and preventive treatments. Conclusion: The anti-inflammatory potential of B. scordioides infusions could be related with the presence of polyphenols as quercetin in the infusion and how this one is consumed.

Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation

Objective: Enhanced oxidative stress is involved in mediating the endothelial dysfunction associated with hypertension. The aim of this study was to investigate the relative contributions of pro-oxidant and anti-oxidant enzymes to the pathogenesis of endothelial dysfunction in genetic hypertension. Methods: Dilator responses to endothelium-dependent and endothelium-independent agents such as acetylcholine (ACh) and sodium nitroprusside were measured in the thoracic aortas of 28-week-old spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar Kyoto rats (WKY). The activity and expression (mRNA and protein levels) of endothelial nitric oxide synthase (eNOS), p22-phox, a membrane-bound component of NAD(P)H oxidase, and antioxidant enzymes, namely, superoxide dismutases (CuZn- and Mn-SOD), catalase and glutathione peroxidase (GPx), were also investigated in aortic rings. Results: Relaxant responses to ACh were attenuated in phenylephrine-precontracted SHR aortic rings, despite a 2-fold increase in eNOS expression and activity. Although the activity and/or expression of SODs, NAD(P)H oxidase (p22-phox) and GPx were elevated in SHR aorta, catalase activity and expression remained unchanged compared to WKY. Pretreatment of SHR aortic rings with the inhibitor of xanthine oxidase, allopurinol, and the inhibitor of cyclooxygenase, indomethacin, significantly potentiated ACh-induced relaxation. Pretreatment of SHR rings with catalase and Tiron, a superoxide anion (O) scavenger, increased the relaxant responses to the levels observed in WKY rings whereas pyrogallol, a O -generator, abolished relaxant responses to ACh. Conclusion: These data demonstrate that dysregulation of several enzymes, resulting in oxidative stress, contributes to the pathogenesis of endothelial dysfunction in SHR and indicate that the antioxidant enzyme catalase is of particular importance in the reversal of this defect. © 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Novel Supercritical Carbon Dioxide Impregnation Technique for the Production of Amorphous Solid Drug Dispersions: A Comparison to Hot Melt Extrusion

The formulation of BCS Class II drugs as amorphous solid dispersions has been shown to provide advantages with respect to improving the aqueous solubility of these compounds. While hot melt extrusion (HME) and spray drying (SD) are among the most common methods for the production of amorphous solid dispersions (ASDs), the high temperatures often required for HME can restrict the processing of thermally labile drugs, while the use of toxic organic solvents during SD can impact on end-product toxicity. In this study, we investigated the potential of supercritical fluid impregnation (SFI) using carbon dioxide as an alternative process for ASD production of a model poorly water-soluble drug, indomethacin (INM). In doing so, we produced ASDs without the use of organic solvents and at temperatures considerably lower than those required for HME. Previous studies have concentrated on the characterization of ASDs produced using HME or SFI but have not considered both processes together. Dispersions were manufactured using two different polymers, Soluplus and polyvinylpyrrolidone K15 using both SFI and HME and characterized for drug morphology, homogeneity, presence of drug-polymer interactions, glass transition temperature, amorphous stability of the drug within the formulation, and nonsink drug release to measure the ability of each formulation to create a supersaturated drug solution. Fully amorphous dispersions were successfully produced at 50% w/w drug loading using HME and 30% w/w drug loading using SFI. For both polymers, formulations containing 50% w/w INM, manufactured via SFI, contained the drug in the γ-crystalline form. Interestingly, there were lower levels of crystallinity in PVP dispersions relative to SOL. FTIR was used to probe for the presence of drug-polymer interactions within both polymer systems. For PVP systems, the nature of these interactions depended upon processing method; however, for Soluplus formulations this was not the case. The area under the dissolution curve (AUC) was used as a measure of the time during which a supersaturated concentration could be maintained, and for all systems, SFI formulations performed better than similar HME formulations.

Comparative Study of Different Methods for the Prediction of Drug− Polymer Solubility

In this study, a comparison of different methods to predict drug−polymer solubility was carried out on binary systems consisting of five model drugs (paracetamol, chloramphenicol, celecoxib, indomethacin, and felodipine) and polyvinylpyrrolidone/vinyl acetate copolymers (PVP/VA) of different monomer weight ratios. The drug−polymer solubility at 25 °C was predicted using the Flory−Huggins model, from data obtained at elevated temperature using thermal analysis methods based on the recrystallization of a supersaturated amorphous solid dispersion and two variations of the melting point depression method. These predictions were compared with the solubility in the low molecular weight liquid analogues of the PVP/VA copolymer (N-vinylpyrrolidone and vinyl acetate). The predicted solubilities at 25 °C varied considerably depending on the method used. However, the three thermal analysis methods ranked the predicted solubilities in the same order, except for the felodipine−PVP system. Furthermore, the magnitude of the predicted solubilities from the recrystallization method and melting point depression method correlated well with the estimates based on the solubility in the liquid analogues, which suggests that this method can be used as an initial screening tool if a liquid analogue is available. The learnings of this important comparative study provided general guidance for the selection of the most suitable method(s) for the screening of drug−polymer solubility.

The Use of High Surface Area Silica and Supercritical Carbon Dioxide to Enhance Solubility of Poorly Water-Soluble Drugs

Purpose Poor water-solubility of BCS class II drugs can limit their commercialization because of reduced oral bioavailability. It has been reported that loading of drug by adsorption onto porous silica would enhance drug solubility due to the increased surface area available for solvent diffusion. In this work, solid dispersions are formed using supercritical carbon dioxide (scCO2). The aim of this research was to characterise the solid-state properties of scCO2 dispersion and to investigate the impact of altering scCO2 processing conditions on final amorphous product performance that could lead to enhancement of drug dissolution rate for BCS class II drugs. Methods Indomethacin (IND) was purchased from Sigma-Aldrich (Dorset, UK) and was used as a model drug with two grades of high surface area silica (average particle sizes 3&[micro] and 7&[micro]), which were obtained directly from Grace-Davison (Germany). Material crystallinity was evaluated using powder X-ray diffraction (PXRD, Rigaku™, miniflex II, Japan) and high-speed differential scanning calorimetry (Hyper-DSC 8000, Perkin Elmer, USA). Materials were placed in a high-pressure vessel consisting of a CO2 cylinder, a Thar™ Technologies P50 high-pressure pump and a 750 ml high-pressure vessel (Thar, USA). Physical mixtures were exposed to CO2 gas above its critical conditions. SEM imaging and elemental analysis were conducted using a Jeol 6500 FEGSEM (Advanced MicroBeam Inc., Austria). Drug release was examined using USP type II dissolution tester (Caleva™, UK). Results The two grades of silica were found to be amorphous using PXRD and Hyper-DSC. Using PXRD, it was shown that an increase in incubation time and pressure resulted in a decrease in the crystalline content. Drug release profiles from the two different silica formulations prepared under the same conditions are shown in Figure 1. It was found that there was a significant enhancement in drug release, which was influenced, by silica type and other experiment conditions such as temperature, pressure and exposure time. SEM imaging and elemental analysis showed drug deposited inside silica pores as well as on the outer surface. Conclusion This project has shown that silica carrier platforms may be used as an alternative approach to generating polymeric solid dispersions of amorphous drugs exhibiting enhanced solubility.

Analgesic exposure in pregnant rats affects fetal germ cell development with inter-generational reproductive consequences

Analgesics which affect prostaglandin (PG) pathways are used by most pregnant women. As germ cells (GC) undergo developmental and epigenetic changes in fetal life and are PG targets, we investigated if exposure of pregnant rats to analgesics (indomethacin or acetaminophen) affected GC development and reproductive function in resulting offspring (F1) or in the F2 generation. Exposure to either analgesic reduced F1 fetal GC number in both sexes and altered the tempo of fetal GC development sex-dependently, with delayed meiotic entry in oogonia but accelerated GC differentiation in males. These effects persisted in adult F1 females as reduced ovarian and litter size, whereas F1 males recovered normal GC numbers and fertility by adulthood. F2 offspring deriving from an analgesic-exposed F1 parent also exhibited sex-specific changes. F2 males exhibited normal reproductive development whereas F2 females had smaller ovaries and reduced follicle numbers during puberty/adulthood; as similar changes were found for F2 offspring of analgesic-exposed F1 fathers or mothers, we interpret this as potentially indicating an analgesic-induced change to GC in F1. Assuming our results are translatable to humans, they raise concerns that analgesic use in pregnancy could potentially affect fertility of resulting daughters and grand-daughters.

Motorização de fármacos na água bruta e na água para consumo humano por cromatolografia líquida de ultra eficiência associada à espectrometria de massa tandem

Tese de doutoramento, Farmácia (Bromatologia), Universidade de Lisboa, Faculdade de Farmácia, 2014

Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity.

AIMS: Aldosterone plays a crucial role in cardiovascular disease. 'Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the 'endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. METHODS AND RESULTS: C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high 'endogenous' aldosterone) and in 'exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. CONCLUSION: Obesity-induced endothelial dysfunction depends on the 'endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.