Novel Thermal Analytical Techniques to Characterize Drugs and Drug Delivery

This thesis encompasses three significant projects. The study includes the characterization and evaluation of the properties of a commercial contraceptive transdermal patch, Ortho Evra® by Dielectric Analysis and Differential Scanning Calorimetry (DSC). This study helps in monitoring the mobility of the drug and transport properties by Isothermal and Scanning Dielectric Analysis as a function of temperature and frequency. The drugs in this product are norelgestromin and ethinyl estradiol. DSC was used to detect any crystalline character of the drugs by their fusion properties. Having no melting endotherm and detecting a glass transition temperature suggested that the drugs in the patches were amorphous. The amorphous form of the drug has more bioavailability. The isothermal DEA a plot of Log frequency vs. reciprocal temperature (K) revealed two critical modulating frequencies at body temperature 37°C for the two API drugs with DEA peak frequencies at 460 and 560 Hz. The main project includes studying the polarization of macro and micro molecular liquid drugs by Dielectric Analysis, B-Cell (a customized isothermal dielectric device) and Differential Scanning Calorimetry. This study demonstrated in vitro transport of selected non-ionic high (e.g. insulin) and low (e.g. Diphenhydramine) molecular weight drugs through excised biological tissue membranes using alternating current (AC) electrokinetics. This new technique of the drug delivery system enhances benefits over systemic oral therapies, in which clinically sufficient quantities of the active ingredient do not reach the intended target organ and/or use of the drugs result in serious side effects. An optimally-tuned low-voltage applied AC electrical field has been found capable of inducing polarization and delivering micro and macromolecules through a biological membrane. The relationships between factors such as delivery time, AC voltage amplitude and frequency, and transported drug concentration were investigated. A factorial design was used to estab

Novel Thermal Analytical Techniques to Characterize Drugs and Drug Delivery

This thesis encompasses three significant projects. The study includes the characterization and evaluation of the properties of a commercial contraceptive transdermal patch, Ortho Evra® by Dielectric Analysis and Differential Scanning Calorimetry (DSC). This study helps in monitoring the mobility of the drug and transport properties by Isothermal and Scanning Dielectric Analysis as a function of temperature and frequency. The drugs in this product are norelgestromin and ethinyl estradiol. DSC was used to detect any crystalline character of the drugs by their fusion properties. Having no melting endotherm and detecting a glass transition temperature suggested that the drugs in the patches were amorphous. The amorphous form of the drug has more bioavailability. The isothermal DEA a plot of Log frequency vs. reciprocal temperature (K) revealed two critical modulating frequencies at body temperature 37°C for the two API drugs with DEA peak frequencies at 460 and 560 Hz. The main project includes studying the polarization of macro and micro molecular liquid drugs by Dielectric Analysis, B-Cell (a customized isothermal dielectric device) and Differential Scanning Calorimetry. This study demonstrated in vitro transport of selected non-ionic high (e.g. insulin) and low (e.g. Diphenhydramine) molecular weight drugs through excised biological tissue membranes using alternating current (AC) electrokinetics. This new technique of the drug delivery system enhances benefits over systemic oral therapies, in which clinically sufficient quantities of the active ingredient do not reach the intended target organ and/or use of the drugs result in serious side effects. An optimally-tuned low-voltage applied AC electrical field has been found capable of inducing polarization and delivering micro and macromolecules through a biological membrane. The relationships between factors such as delivery time, AC voltage amplitude and frequency, and transported drug concentration were investigated. A factorial design was used to estab

Analysis of proteins with the 'hot dog' fold: Prediction of function and identification of catalytic residues of hypothetical proteins

<p>Abstract</p> <p>Background</p> <p>The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites.</p> <p>Results</p> <p>We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins.</p> <p>Conclusion</p> <p>The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.</p

A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome

Structural Basis for the Variation in Triclosan Affinity to Enoyl Reductases

Bacteria synthesize fatty acids in a dissociated type pathway different from that in humans. Enoyl acyl carrier protein reductase, which catalyzes the final step of fatty acid elongation, has been validated as a potential antimicrobial drug target. Triclosan is known to inhibit this enzyme effectively. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. With this in view, interactions between triclosan, the cofactor $NADH/NAD^+$ and the enzyme from five different species, one plant and four of microbial origin, have been examined in the available crystal structures. A comparison of these structures shows major structural differences at the substrate/inhibitor/cofactor-binding loop. The analysis reveals that the conformation of this flexible loop and the binding affinities of triclosan to each of these enzymes are strongly correlated

Crystal structure of dimeric FabZ of Plasmodium falciparum reveals conformational switching to active hexamers by peptide flips

The crystal structure of beta-hydroxyacyl acyl carrier protein dehydratase of Plasmodium falciparum (PfFabZ) has been determined at a resolution of 2.4 angstrom. PfFabZ has been found to exist as a homodimer (d-PfFabZ) in the crystals of the present study in contrast to the reported hexameric form (h-PfFabZ) which is a trimer of dimers crystallized in a different condition. The catalytic sites of this enzyme are located in deep narrow tunnel-shaped pockets formed at the dimer interface. A histidine residue from one subunit of the dimer and a glutamate residue from the other subunit lining the tunnel form the catalytic dyad in the reported crystal structures. While the position of glutamate remains unaltered in the crystal structure of d-PffabZ compared to that in b-PfFabZ, the histidine residue takes up an entirely different conformation and moves away from the tunnel leading to a His-Phe cis-trans peptide flip at the histidine residue. In addition, a loop in the vicinity has been observed to undergo a similar flip at a Tyr-Pro peptide bond. These alterations not only prevent the formation of a hexamer but also distort the active site geometry resulting in a dimeric form of FabZ that is incapable of substrate binding. The dimeric state and an altered catalytic site architecture make d-PfFabZ distinctly different from the FabZ structures described so far. Dynamic light scattering and size exclusion chromatographic studies clearly indicate a pH-related switching of the dimers to active hexamers. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserv

Thermally-induced Dielectric Relaxation Spectra in Three Aldohexose Monosaccharides

Three aldohexose monosaccharides, d-glucose, d-mannose, and d-galactose, were examined by scanning temperature dielectric analysis (DEA) from ambient temperatures through their melts. Phase transitions, including glass transition (T g) and melting temperature (T m), were evaluated by differential scanning calorimetry (DSC). The monosaccharides were found to exhibit thermally-induced dielectric loss spectra in their amorphous-solid phase before melting. Activation energies for electrical charging of each of the monosaccharides were calculated from an Arrhenius plot of the tan delta (e″/e′, dielectric loss factor/relative permittivity) peak frequency versus reciprocal temperature in Kelvin. The DEA profiles were also correlated with the DSC phase diagrams, showing the changes in electrical behavior associated with solid–solid and solid–liquid transitions

Solid-state Mechanical Properties of Crystalline Drugs and Excipients

Thermal mechanical analysis (TMA) of crystalline drugs and excipients in their pre-melt temperature range performed in this study corroborate their newly found linear dielectric conductivity properties with temperature. TMA of crystalline active pharmacy ingredients (APIs) or excipients shows softening at 30–100 °C below the calorimetric melting phase transition, which is also observed by dielectric analysis (DEA). Acetophenetidin melts at 135 °C as measured calorimetrically by DSC, but softens under a low mechanical stress at 95 °C. At this pre-melting temperature, the crystals collapse under the applied load, and the TMA probe shows rapid displacement. The mechanical properties yield a softening structure and cause a dimensionally slow disintegration resulting in a sharp dimensional change at the melting point. In order to incorporate these findings into a structure–property relationship, several United States Pharmacopeia (USP) melting-point standard drugs were evaluated by TMA, DSC, and DEA, and compared to the USP standard melt temperatures. The USP standard melt temperature for vanillin (80 °C) [1], acetophenetidin (135 °C) [2], and caffeine (235 °C) [3] are easily verified calorimetrically via DSC. The combined thermal analysis techniques allow for a wide variety of the newly discovered physical properties of drugs and excipients

Impact of body mass index on progression of primary immunoglobulin a nephropathy

The role of obesity in the progression of primary glomerular diseases is controversial. A few studies report overweight/obesity as a risk factor for disease progression in immunoglobulin A nephropathy (IgAN), and the real impact of it still remains unclear. The aim of this study was to elucidate the effect of body mass index (BMI) on disease progression and proteinuria in patients with IgAN in Indian population. A cohort of biopsy-proven primary IgAN patients diagnosed between March 2010 and February 2015 who had a follow-up for a minimum of 12 months were included in the study. We defined two groups of patients according to the BMI value at diagnosis: non-obese group (Group N) with BMI 23 Kg/m2 as per Asia-Pacific task force criteria. Baseline characteristics were compared between the groups. The estimated glomerular filtration rate (eGFR) and urine protein-creatinine ratio (UPCR) were followed up at entry time, 6 months, 12 months, and at the end of follow-up. Outcomes studied were change in eGFR, proteinuria, and progression to end-stage renal disease. Statistical analysis was done using the Statistical Package for the Social Sciences version 15.0. Of 51 patients, 25 (49%) had BMI 23 kg/m2 (Group O) (P = 0.01). The baseline clinical, histopathological, and treatment characteristics of both the groups were comparable. The BMI at the time of diagnosis did not have any significant effect on eGFR (P = 0.41) or proteinuria (P = 0.99) at presentation. At the end of follow-up, both the groups had a similar reduction of proteinuria (UPCR) (P = 0.46) and eGFR (P = 0.20). Two patients in each group have reached chronic kidney disease Stage 5. In the present study, BMI at presentation did not have any impact on eGFR or proteinuria, either at diagnosis or at follow-up. It needs further large multicenter randomized control studies to see the effect of BMI on progression of IgAN