Antiherpes simplex virus type 2 activity of the antimicrobial peptide subtilosin

In the present study we evaluated the antiviral activity of subtilosin, a cyclical peptide isolated from Bacillus amyloliquefaciens, against herpes simplex virus type 2 (HSV-2) in cell cultures and we investigated subtilosin mode of action. We determined, using a virus yield inhibition assay, that non cytotoxic concentrations of subtilosin inhibit HSV-2 replication in Vero cell cultures. Subtilosin strongly inhibited extracellular and total virus production even when it was added at 8 h post-infection indicating that not only virus release but also viral particle formation is impeded by the antiviral peptide. Although viral glycoprotein gD level of expression is not affected by the bacteriocin, an altered pattern of gD intracellular localization was detected by immunofluorescence assay in subtilosin treated culture. On the other hand, at high concentrations subtilosin displays virucidal action.Fil: Quintana, Verónica Mara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Torres, Nicolás. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Wachsman, Mónica B.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Sinko, Patrick J.. State University of New Jersey; Estados UnidosFil: Castilla, Viviana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Chikindas, Michael. State University of New Jersey; Estados Unido

Estimating human drug oral absorption kinetics from Caco-2 permeability using an absorptiondisposition model: model development and evaluation and derivation of analytical solutions for k (a) and F (a).

ABSTRACT Intestinal transcellular permeability (P m ), measured across cell lines such as Caco-2 cells in vitro, is often used for assessing oral drug absorption potential in humans. However, the quantitative link between in vitro permeability and apparent in vivo absorption kinetics, based on drug appearance in plasma, is poorly understood. In the current study, a novel absorptiondisposition kinetic model that links traditional pharmacokinetic and mass transfer models was developed. Analytical solutions of k a and F a were deduced, and using Caco-2 permeability, F a in humans was predicted for 51 structurally diverse compounds. Predicted F a values were similar to and correlated highly with their corresponding experimental values with an average error of 1.88 Ϯ 1.06% (Ϫ17 to 22%) and r 2 ϭ 0.934. Simulated concentration profiles for 17 of 18 drugs corresponded to observed plasma concentration profiles in healthy volunteers. The equilibrium solution for k a (k a,eq ) was found to be a key determinant of F a , whereas under sink conditions, k a is likely to be a determinant of plasma concentration kinetics. The current version of the model offers a quantitative approach for predicting human oral absorption kinetics from in vitro permeability. It also establishes, for the first time, a quantitative link between P m and k a and between k a,eq and F a . This will facilitate better in vitro or in situ-in vivo correlations since it establishes a basis for incorporating permeability coefficients from the various experimental formats based on drug loss or appearance that are commonly used in the laboratory for permeability determination. Oral administration is the most commonly used drug-dosing route. Therefore, the ability to predict the rate and extent of absorption of drug candidates after oral administration is crucial during the preclinical phase of development. Such knowledge complements high throughput drug screening and allows scientists to select the best drug candidates early in the drug development cycle. Drug absorption from the gastrointestinal (GI) tract is affected by many factors. Besides the physiological conditions of the GI tract (e.g., absorptive surface area, local pH, food effects, intestinal transit time, and passive intestinal permeability) and chemical properties of the drug (e.g., solubility, molecular size, and stability), intestinal transporters and enzymes are being increasingly implicated in controlling oral drug absorptio

Characterization of the oral absorption of several aminopenicillins: Determination of intrinsic membrane absorption parameters in the rat intestine in situ

The absorption mechanism of several penicillins was characterized using in situ single-pass intestinal perfusion in the rat. The intrinsic membrane absorption parameters were determined using a modified boundary layer model (fitted value +/- S.E.): J*max = 11.78 +/- 1.88 mM, Km = 15.80 +/- 2.92 mM, P*m = 0, J*c = 0.75 +/- 0.04 for ampicillin; J*max = 0.044 +/- 0.018 mM, Km = 0.058 +/- 0.026 mM, P*m = 0.558 +/- 0.051, P*c = 0.757 +/- 0.088 for amoxicillin; and J*max = 16.30 +/- 3.40 mM, Km = 14.00 +/- 3.30 mM, P*m = 0, P*c = 1.14 +/- 0.05 for cyclacillin. All of the aminopenicillins studied demonstrated saturable absorption kinetics as indicated by their concentration-dependent wall permeabilities. Inhibition studies were performed to confirm the existence of a nonpassive absorption mechanism. The intrinsic wall permeability (P*w) of 0.01 mM ampicillin was significantly lowered by 1 mM amoxicillin and the P*w of 0.01 mM amoxicillin was reduced by 2 mM cephradine consistent with competitive inhibition.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29839/1/0000186.pd

Formulation predictive dissolution (fPD) testing to advance oral drug product development: an introduction to the US FDA funded ‘21st Century BA/BE’ project

Over the past decade, formulation predictive dissolution (fPD) testing has gained increasing attention. Another mindset is pushed forward where scientists in our field are more confident to explore the in vivo behavior of an oral drug product by performing predictive in vitro dissolution studies. Similarly, there is an increasing interest in the application of modern computational fluid dynamics (CFD) frameworks and high-performance computing platforms to study the local processes underlying absorption within the gastrointestinal (GI) tract. In that way, CFD and computing platforms both can inform future PBPK-based in silico frameworks and determine the GI-motility-driven hydrodynamic impacts that should be incorporated into in vitro dissolution methods for in vivo relevance. Current compendial dissolution methods are not always reliable to predict the in vivo behavior, especially not for biopharmaceutics classification system (BCS) class 2/4 compounds suffering from a low aqueous solubility. Developing a predictive dissolution test will be more reliable, cost-effective and less time-consuming as long as the predictive power of the test is sufficiently strong. There is a need to develop a biorelevant, predictive dissolution method that can be applied by pharmaceutical drug companies to facilitate marketing access for generic and novel drug products. In 2014, Prof. Gordon L. Amidon and his team initiated a far-ranging research program designed to integrate (1) in vivo studies in humans in order to further improve the understanding of the intraluminal processing of oral dosage forms and dissolved drug along the gastrointestinal (GI) tract, (2) advancement of in vitro methodologies that incorporates higher levels of in vivo relevance and (3) computational experiments to study the local processes underlying dissolution, transport and absorption within the intestines performed with a new unique CFD based framework. Of particular importance is revealing the physiological variables determining the variability in in vivo dissolution and GI absorption from person to person in order to address (potential) in vivo BE failures. This paper provides an introduction to this multidisciplinary project, informs the reader about current achievements and outlines future directions

Development and Characterization of a Biomimetic Passive Diffusion Membrane and in Vivo Relevant Dissolution Methodology

Preclinical evaluation of modern solid oral dosage forms requires more advanced in vitro devices due to the trend of formulating low solubility, high permeability compounds for commercial therapeutics. Current compendial dissolution methodologies may not be suitable for such compounds due to high fluid shear rates, heterogeneity of shear rates, suboptimal fluid flow, unrealistic fluid volumes, and ultimately the lack of an experimental component that represents the absorption process. Here a dissolution apparatus is introduced that overcomes some limitations of the compendial apparatus using poly(dimethyl siloxane) (PDMS) as an in vitro biomimetic analogue that simulates the passive drug absorption process. PDMS is biomimetic because of similarities in small molecule transport such as mechanism, ionization selectivity, and lipophilicity. Nine molecular probes were used to evaluate the transport pathways and properties of PDMS to simulate human oral absorption rates. The transport pathways through PDMS are analogous to transcellular (TCDT) and paracellular (PCDT) drug transport pathways. PDMS PCDT was assessed using positronium annihilation life-time spectroscopy (PALS) and partition experiments; TCDT was assessed using diffusion and partition experiments. PALS determined that PDMS pores were uniform (diameter~0.85nm), isolated, and void volume was unaffected by drug accumulation. A strong linear correlation exists between predicted octanol-water partition coefficients and PDMS partition coefficients (LogPPDMS=0.736 x LogPO-W–0.971, R2=0.981). The characteristics of an ultra-thin large area PDMS membrane (UTLAM) dissolution/absorption system with respect to pH, partition coefficient (K), aqueous boundary layer (ABL), drug particle size, and administered dose were measured. A pH dependent solution diffusion model and a particle size-dose dependent particle dissolution-absorption model were derived. A 1.5” hydrofoil design was implemented to reduce particle coning, promote particle re-suspension, and control bulk fluid shear. PDMS membranes were successfully fabricated to thicknesses of 11.2 ± 0.5µm to 12.5± 0.2µm, and a UTLAM active surface area of 21.8-25.2cm2 (44.4cm2 total area) was achieved. Experiments between pH 1.9-12.5 were investigated using ibuprofen as a model weak acid drug. Partition coefficient (K) is the dominant factor determining absorption and the ABL played a significant role in absorption when the drug is mostly non-ionized. In aqueous and membrane limited diffusion absorption, there was a significant shift towards higher pH in the half of maximum absorption rate caused by K and ABL. Number and mass particle size distributions (PSD) measured and dissolution was studied. Particle dissolution was determined to occur in the ABL adjacent to the PDMS membrane and has a significant effect on flux of drug across the PDMS membrane. Suspension experiments conducted with ibuprofen particle size radii of 3.7µm, 18µm, 117µm at doses of 0.2, 1, 10, and 40mg/mL. Significant enhancements in flux were observed with increasing dose and decreasing particle size in accordance with theoretical predictions of particle dissolution within the ABL. In all PSD’s, an unaccounted increase in flux was observed at high doses and was attributed to particle settling on the horizontal PDMS membrane. In conclusion, the UTLAM PDMS diffusion cell exhibits improved hydrodynamics in the donor phase and the PDMS membrane mimics in vivo relevant passive absorption kinetics through PDMS membranes.PHDPharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/151729/1/sinkopd_1.pdfDescription of sinkopd_1.pdf : Restricted to UM users only

Intestinal Absorption of Peptides and Peptide Analogues: Implications of Fasting Pancreatic Serine Protease Levels and pH on the Extent of Oral Absorption in Dogs and Humans

In order to describe and predict the impact of intestinal metabolism on peptide absorption, intestinal chymotrypsin activity, flow rate, and pH were characterized in fasted, duodenally fistulated dogs as a function of gastrointestinal (GI) motility phase. GI motility was classified as either active or quiescent. Cumulative volume, F(t) , and volumetric flow rate, Q(t) , curves were constructed and the data were sorted according to motility phase. The mean ± SE active phase pH was 6.4 ± 0.3, whereas the quiescent phase pH was 7.3 ± 0.3. The difference between the mean active and the mean quiescent phase pH values was significant. The active and quiescent phase flow rates (ml/min) were also significantly different, at values of 1.2 ± 0.2 and 0.28 ± 0.07, respectively. The active phase flow rates were consistent among the dogs studied; however, the quiescent phase flow rates were highly variable among the dogs. The variability of the quiescent phase flow rates was expected since phase II of the GI motility cycle is characterized by intermediate, irregular spike activity. The mean active and quiescent phase chymotrypsin activities were 1.87 × 10 -5 ± 0.53 × 10 -5 and 1.56 × 10 -5 ± 0.65 × 10 -5 M , respectively. The active phase values were not statistically different among dogs, however, the quiescent phase values were found to be highly variable among dogs. The difference between the active and the quiescent phase chymotrypsin mean levels, however, was not statistically significant. The chymotrypsin levels determined in dogs were found to be approximately 10 times greater than those reported in humans. The significance of fasted-state chymotrypsin levels is discussed with respect to the impact of GI metabolism on peptide and peptide-like drug absorption in dogs. Further, given the intestinal metabolic differences between dogs and humans, the suitability of using the dog model for predicting the oral absorption of peptides in humans is discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41569/1/11095_2004_Article_305100.pd

Predicting oral drug absorption in man for compounds absorbed by carrier mediated and passive absorption processes.

Predicting drug absorption and drug absorption variation can considerably aid the selection of c and idates in the drug discovery process as well as identify ways to optimize oral drug delivery in patients. An approach to estimating the fraction dose absorbed in humans is developed based on a macroscopic mass balance analysis. The analysis utilizes membrane absorption parameters, calculated from intestinal perfusion experiments in rats, for drugs absorbed by both carrier mediated and passive adsorption mechanisms. The analysis suggests that the absorption number, An, and the dose to solubility ratio, 1/S$\\sp*$, are key parameters for predicting drug absorption where An = P$\\sp*\\sb{\\rm e}$Gz, P$\\sp*\\sb{\\rm e}$ is the dimensionless effective permeability, and Gz is the Graetz number. Three equations predicting fraction absorbed are developed for the following cases of drug in solution: drug concentration (1) below the solubility, (2) initially exceeding the solubility, and (3) always greater than the solubility. Model compounds used for the correlations included those absorbed by carrier mediated and /or passive absorption processes. Literature values for intestinal wall permeability are used for the passively absorbed compounds whereas the absorption parameters for the carrier mediated compounds were determined from intestinal perfusion experiments in rats. Human fraction dose absorbed (F) data and An showed an excellent correlation. The theoretical analysis, confirmed by experimental results, demonstrates that two of the fundamental parameters controlling drug absorption are the absorption number and the dose to solubility ratio. The $\\beta$-lactam antibiotic intestinal absorption mechanism is characterized using single pass perfusion technique in rats. The membrane absorption parameters, J$\\sp*\\sb{\\rm max}$(maximal flux), K$\\sb{\\rm m}$(Michaelis Constant), and P$\\sp*\\sb{\\rm c}$(carrier permeability) for amoxicillin, cephalexin, cephradine, cefatrizine, cefaclor, and cefadroxil were determined. Analysis of the data using a modified boundary layer method revealed nonpassive membrane transport. Competitive absorption studies performed with $\\beta$-lactam antibiotics, amino acids, and several small peptides suggest that absorption interactions between carrier mediated compounds, including other drugs, peptides or amino acids, may be clinically significant and may account for a second possible mechanism along with delayed gastric emptying for the delay in antibiotic plasma levels.Ph.D.Pharmaceutical sciencesUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/161975/1/8821654.pd