Linking the Gastrointestinal Behavior of Ibuprofen with the Systemic Exposure between and within Humans-Part 1: Fasted State Conditions.

The goal of this project was to explore and to statistically evaluate the responsible gastrointestinal (GI) factors that are significant factors in explaining the systemic exposure of ibuprofen, between and within human subjects. In a previous study, we determined the solution and total concentrations of ibuprofen as a function of time in aspirated GI fluids, after oral administration of an 800 mg IR tablet (reference standard) of ibuprofen to 20 healthy volunteers in fasted state conditions. In addition, we determined luminal pH and motility pressure recordings that were simultaneously monitored along the GI tract. Blood samples were taken to determine ibuprofen plasma levels. In this work, an in-depth statistical and pharmacokinetic analysis was performed to explain which underlying GI variables are determining the systemic concentrations of ibuprofen between (inter-) and within (intra-) subjects. In addition, the obtained plasma profiles were deconvoluted to link the fraction absorbed with the fraction dissolved. Multiple linear regressions were performed to explain and quantitatively express the impact of underlying GI physiology on systemic exposure of the drug (in terms of plasma Cmax/AUC and plasma Tmax). The exploratory analysis of the correlation between plasma Cmax/AUC and the time to the first phase III contractions postdose (TMMC-III) explains ∼40% of the variability in plasma Cmax for all fasted state subjects. We have experimentally shown that the in vivo intestinal dissolution of ibuprofen is dependent upon physiological variables like, in this case, pH and postdose phase III contractions. For the first time, this work presents a thorough statistical analysis explaining how the GI behavior of an ionized drug can explain the systemic exposure of the drug based on the individual profiles of participating subjects. This creates a scientifically based and rational framework that emphasizes the importance of including pH and motility in a predictive in vivo dissolution methodology to forecast the in vivo performance of a drug product. Moreover, as no extensive first-pass metabolism is considered for ibuprofen, this study demonstrates how intraluminal drug behavior is reflecting the systemic exposure of a drug.

Linking the Gastrointestinal Behavior of Ibuprofen with the Systemic Exposure between and within Humans-Part 2: Fed State.

Exploring the intraluminal behavior of an oral drug product in the human gastrointestinal (GI) tract remains challenging. Many in vivo techniques are available to investigate the impact of GI physiology on oral drug behavior in fasting state conditions. However, little is known about the intraluminal behavior of a drug in postprandial conditions. In a previous report, we described the mean solution and total concentrations of ibuprofen after oral administration of an immediate-release (IR) tablet in fed state conditions. In parallel, blood samples were taken to assess systemic concentrations. The purpose of this work was to statistically evaluate the impact of GI physiology (e.g., pH, contractile events) within and between individuals (intra and intersubject variability) for a total of 17 healthy subjects. In addition, a pharmacokinetic (PK) analysis was performed by noncompartmental analysis, and PK parameters were correlated with underlying physiological factors (pH, time to phase III contractions postdose) and study parameters (e.g., ingested amount of calories, coadministered water). Moreover, individual plasma profiles were deconvoluted to assess the fraction absorbed as a function of time, demonstrating the link between intraluminal and systemic behavior of the drug. The results demonstrated that the in vivo dissolution of ibuprofen depends on the present gastric pH and motility events at the time of administration. Both intraluminal factors were responsible for explaining 63% of plasma Cmax variability among all individuals. For the first time, an in-depth analysis was performed on a large data set derived from an aspiration/motility study, quantifying the impact of physiology on systemic behavior of an orally administered drug product in fed state conditions. The data obtained from this study will help us to develop an in vitro biorelevant dissolution approach and optimize in silico tools in order to predict the in vivo performance of orally administered drug products, especially in fed state conditions.

Utilization of Gastrointestinal Simulator, an in Vivo Predictive Dissolution Methodology, Coupled with Computational Approach To Forecast Oral Absorption of Dipyridamole.

Weakly basic drugs exhibit a pH-dependent dissolution profile in the gastrointestinal (GI) tract, which makes it difficult to predict their oral absorption profile. The aim of this study was to investigate the utility of the gastrointestinal simulator (GIS), a novel in vivo predictive dissolution (iPD) methodology, in predicting the in vivo behavior of the weakly basic drug dipyridamole when coupled with in silico analysis. The GIS is a multicompartmental dissolution apparatus, which represents physiological gastric emptying in the fasted state. Kinetic parameters for drug dissolution and precipitation were optimized by fitting a curve to the dissolved drug amount-time profiles in the United States Pharmacopeia apparatus II and GIS. Optimized parameters were incorporated into mathematical equations to describe the mass transport kinetics of dipyridamole in the GI tract. By using this in silico model, intraluminal drug concentration-time profile was simulated. The predicted profile of dipyridamole in the duodenal compartment adequately captured observed data. In addition, the plasma concentration-time profile was also predicted using pharmacokinetic parameters following intravenous administration. On the basis of the comparison with observed data, the in silico approach coupled with the GIS successfully predicted in vivo pharmacokinetic profiles. Although further investigations are still required to generalize, these results indicated that incorporating GIS data into mathematical equations improves the predictability of in vivo behavior of weakly basic drugs like dipyridamole.

Unpredictable Performance of pH-Dependent Coatings Accentuates the Need for Improved Predictive in Vitro Test Systems.

First introduced in the second half of the 19th century, enteric coatings are commonly used to protect acid-labile drugs, reduce the risk of gastric side effects due to irritating drugs, or for local drug delivery to the lower gastrointestinal (GI) tract. The currently available enteric-coatings are based on pH-sensitive weakly acidic polymers. Despite the long history of their use, the causes behind their performance often being unpredictable have not been properly investigated with most of the attention being focused only on the gastric emptying. However, little attention has been given to the postgastric emptying disintegration and dissolution of these dosage forms. This lack of attention has contributed to the difficulty in predicting the in vivo behavior of these dosage forms and to cases of bioavailability problems with some enteric-coated products. Therefore, increased attention needs to be given to this issue.

In Vivo Dissolution and Systemic Absorption of Immediate Release Ibuprofen in Human Gastrointestinal Tract under Fed and Fasted Conditions.

In vivo drug dissolution in the gastrointestinal (GI) tract is largely unmeasured. The purpose of this clinical study was to evaluate the in vivo drug dissolution and systemic absorption of the BCS class IIa drug ibuprofen under fed and fasted conditions by direct sampling of stomach and small intestinal luminal content. Expanding current knowledge of drug dissolution in vivo will help to establish physiologically relevant in vitro models predictive of drug dissolution. A multilumen GI catheter was orally inserted into the GI tract of healthy human subjects. Subjects received a single oral dose of ibuprofen (800 mg tablet) with 250 mL of water under fasting and fed conditions. The GI catheter facilitated collection of GI fluid from the stomach, duodenum, and jejunum. Ibuprofen concentration in GI fluid supernatant and plasma was determined by LC-MS/MS. A total of 23 subjects completed the study, with 11 subjects returning for an additional study visit (a total of 34 completed study visits). The subjects were primarily white (61%) and male (65%) with an average age of 30 years. The subjects had a median [min, max] weight of 79 [52, 123] kg and body mass index of 25.7 [19.4, 37.7] kg/m2. Ibuprofen plasma levels were higher under fasted conditions and remained detectable for 28 h under both conditions. The AUC0-24 and Cmax were lower in fed subjects vs fasted subjects, and Tmax was delayed in fed subjects vs fasted subjects. Ibuprofen was detected immediately after ingestion in the stomach under fasting and fed conditions until 7 h after dosing. Higher levels of ibuprofen were detected in the small intestine soon after dosing in fasted subjects compared to fed. In contrast to plasma drug concentration, overall gastric concentrations remained higher under fed conditions due to increased gastric pH vs fasting condition. The gastric pH increased to near neutrality after feedingbefore decreasing to acidic levels after 7 h. Induction of the fed state reduced systemic levels but increased gastric levels of ibuprofen, which suggest that slow gastric emptying and transit dominate the effect for plasma drug concentration. The finding of high levels of ibuprofen in stomach and small intestine 7 h post dosing was unexpected. Future work is needed to better understand the role of various GI parameters, such as motility and gastric emptying, on systemic ibuprofen levels in order to improve in vitro predictive models.

Measurement of in vivo Gastrointestinal Release and Dissolution of Three Locally Acting Mesalamine Formulations in Regions of the Human Gastrointestinal Tract.

As an orally administered, locally acting gastrointestinal drug, mesalamine products are designed to achieve high local drug concentration in the gastrointestinal (GI) tract for the treatment of ulcerative colitis. The aim of this study was to directly measure and compare drug dissolution of three mesalamine formulations in human GI tract and to correlate their GI concentration with drug concentration in plasma. Healthy human subjects were orally administered Pentasa, Apriso, or Lialda. GI fluids were aspirated from stomach, duodenum, proximal jejunum, mid jejunum, and distal jejunum regions. Mesalamine (5-ASA) and its primary metabolite acetyl-5-mesalamine (Ac-5-ASA) were measured using LC-MS/MS. GI tract pH was measured from each GI fluid sample, which averaged 1.82, 4.97, 5.67, 6.17, and 6.62 in the stomach, duodenum, proximal jejunum, middle jejunum, and distal jejunum, respectively. For Pentasa, high levels of 5-ASA in solution were observed in the stomach, duodenum, proximal jejunum, mid jejunum, and distal jejunum from 1 to 7 h. Apriso had minimal 5-ASA levels in stomach, low to medium levels of 5-ASA in duodenum and proximal jejunum from 4 to 7 h, and high levels of 5-ASA in distal jejunum from 3 to 7 h. In contrast, Lialda had minimal 5-ASA levels from stomach and early small intestine. A composite appearance rate (CAR) was calculated from the deconvolution of individual plasma concentration to reflect drug release, dissolution, transit, and absorption in the GI tract. Individuals dosed with Pentasa had high levels of CAR from 1 to 10 h; individuals dosed with Apriso had low levels of CAR from 1 to 4 h and high levels of CAR from 5 to 10 h; Lialda showed minimal levels of CAR from 0 to 5 h, then increased to medium levels from 5 to 12 h, and then decreased to further lower levels after 12 h. In the colon region, Pentasa and Apriso showed similar levels of accumulated 5-ASA excreted in the feces, while Lialda showed slightly higher 5-ASA accumulation in feces. However, all three formulations showed similar levels of metabolite Ac-5-ASA in the feces. These results provide direct measurement of drug dissolution in the GI tract, which can serve as a basis for investigation of bioequivalence for locally acting drug products.

Low Buffer Capacity and Alternating Motility along the Human Gastrointestinal Tract: Implications for in Vivo Dissolution and Absorption of Ionizable Drugs.

In this study, we determined the pH and buffer capacity of human gastrointestinal (GI) fluids (aspirated from the stomach, duodenum, proximal jejunum, and mid/distal jejunum) as a function of time, from 37 healthy subjects after oral administration of an 800 mg immediate-release tablet of ibuprofen (reference listed drug; RLD) under typical prescribed bioequivalence (BE) study protocol conditions in both fasted and fed states (simulated by ingestion of a liquid meal). Simultaneously, motility was continuously monitored using water-perfused manometry. The time to appearance of phase III contractions (i.e., housekeeper wave) was monitored following administration of the ibuprofen tablet. Our results clearly demonstrated the dynamic change in pH as a function of time and, most significantly, the extremely low buffer capacity along the GI tract. The buffer capacity on average was 2.26 µmol/mL/ΔpH in fasted state (range: 0.26 and 6.32 µmol/mL/ΔpH) and 2.66 µmol/mL/ΔpH in fed state (range: 0.78 and 5.98 µmol/mL/ΔpH) throughout the entire upper GI tract (stomach, duodenum, and proximal and mid/distal jejunum). The implication of this very low buffer capacity of the human GI tract is profound for the oral delivery of both acidic and basic active pharmaceutical ingredients (APIs). An in vivo predictive dissolution method would require not only a bicarbonate buffer but also, more significantly, a low buffer capacity of dissolution media to reflect in vivo dissolution conditions.

Potential Development of Tumor-Targeted Oral Anti-Cancer Prodrugs: Amino Acid and Dipeptide Monoester Prodrugs of Gemcitabine.

One of the main obstacles for cancer therapies is to deliver medicines effectively to target sites. Since stroma cells are developed around tumors, chemotherapeutic agents have to go through stroma cells in order to reach tumors. As a method to improve drug delivery to the tumor site, a prodrug approach for gemcitabine was adopted. Amino acid and dipeptide monoester prodrugs of gemcitabine were synthesized and their chemical stability in buffers, resistance to thymidine phosphorylase and cytidine deaminase, antiproliferative activity, and uptake/permeability in HFF cells as a surrogate to stroma cells were determined and compared to their parent drug, gemcitabine. The activation of all gemcitabine prodrugs was faster in pancreatic cell homogenates than their hydrolysis in buffer, suggesting enzymatic action. All prodrugs exhibited great stability in HFF cell homogenate, enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase, and deamination by cytidine deaminase compared to their parent drug. All gemcitabine prodrugs exhibited higher uptake in HFF cells and better permeability across HFF monolayers than gemcitabine, suggesting a better delivery to tumor sites. Cell antiproliferative assays in Panc-1 and Capan-2 pancreatic ductal cell lines indicated that the gemcitabine prodrugs were more potent than their parent drug gemcitabine. The transport and enzymatic profiles of gemcitabine prodrugs suggest their potential for delayed enzymatic bioconversion and enhanced resistance to metabolic enzymes, as well as for enhanced drug delivery to tumor sites, and cytotoxic activity in cancer cells. These attributes would facilitate the prolonged systemic circulation and improved therapeutic efficacy of gemcitabine prodrugs.

In Vitro Dissolution of Fluconazole and Dipyridamole in Gastrointestinal Simulator (GIS), Predicting in Vivo Dissolution and Drug-Drug Interaction Caused by Acid-Reducing Agents.

Weakly basic drugs typically exhibit pH-dependent solubility in the physiological pH range, displaying supersaturation or precipitation along the gastrointestinal tract. Additionally, their oral bioavailabilities may be affected by coadministration of acid-reducing agents that elevate gastric pH. The purpose of this study was to assess the feasibility of a multicompartmental in vitro dissolution apparatus, Gastrointestinal Simulator (GIS), in predicting in vivo dissolution of certain oral medications. In vitro dissolution studies of fluconazole, a BCS class I, and dipyridamole, a BCS class II weak bases (class IIb), were performed in the GIS as well as United States Pharmacopeia (USP) apparatus II and compared with the results of clinical drug-drug interaction (DDI) studies. In both USP apparatus II and GIS, fluconazole completely dissolved within 60 min regardless of pH, reflecting no DDI between fluconazole and acid-reducing agents in a clinical study. On the other hand, seven-fold and 15-fold higher concentrations of dipyridamole than saturation solubility were observed in the intestinal compartments in GIS with gastric pH 2.0. Precipitation of dipyridamole was also observed in the GIS, and the percentage of dipyridamole in solution was 45.2 ± 7.0%. In GIS with gastric pH 6.0, mimicking the coadministration of acid-reducing agents, the concentration of dipyridamole was equal to its saturation solubility, and the percentage of drug in solution was 9.3 ± 2.7%. These results are consistent with the clinical DDI study of dipyridamole with famotidine, which significantly reduced the Cmax and area under the curve. An In situ mouse infusion study combined with GIS revealed that high concentration of dipyridamole in the GIS enhanced oral drug absorption, which confirmed the supersaturation of dipyridamole. In conclusion, GIS was shown to be a useful apparatus to predict in vivo dissolution for BCS class IIb drugs.

Intestinal permeability study of minoxidil: assessment of minoxidil as a high permeability reference drug for biopharmaceutics classification.

The purpose of this study was to evaluate minoxidil as a high permeability reference drug for Biopharmaceutics Classification System (BCS). The permeability of minoxidil was determined in in situ intestinal perfusion studies in rodents and permeability studies across Caco-2 cell monolayers. The permeability of minoxidil was compared with that of metoprolol, an FDA reference drug for BCS classification. In rat perfusion studies, the permeability of minoxidil was somewhat higher than that of metoprolol in the jejunum, while minoxidil showed lower permeability than metoprolol in the ileum. The permeability of minoxidil was independent of intestinal segment, while the permeability of metoprolol was region-dependent. Similarly, in mouse perfusion study, the jejunal permeability of minoxidil was 2.5-fold higher than that of metoprolol. Minoxidil and metoprolol showed similar permeability in Caco-2 study at apical pH of 6.5 and basolateral pH of 7.4. The permeability of minoxidil was independent of pH, while metoprolol showed pH-dependent transport in Caco-2 study. Minoxidil exhibited similar permeability in the absorptive direction (AP-BL) in comparison with secretory direction (BL-AP), while metoprolol had higher efflux ratio (ER > 2) at apical pH of 6.5 and basolateral pH of 7.4. No concentration-dependent transport was observed for either minoxidil or metoprolol transport in Caco-2 study. Verapamil did not alter the transport of either compounds across Caco-2 cell monolayers. The permeability of minoxidil was independent of both pH and intestinal segment in intestinal perfusion studies and Caco-2 studies. Caco-2 studies also showed no involvement of carrier mediated transport in the absorption process of minoxidil. These results suggest that minoxidil may be an acceptable reference drug for BCS high permeability classification. However, minoxidil exhibited higher jejunal permeability than metoprolol and thus to use minoxidil as a reference drug would raise the permeability criteria for BCS high permeability classification.

Comparison of the permeability of metoprolol and labetalol in rat, mouse, and Caco-2 cells: use as a reference standard for BCS classification.

The purpose of this study was to investigate labetalol as a potential high permeability reference standard for the application of Biopharmaceutics Classification Systems (BCS). Permeabilities of labetalol and metoprolol were investigated in animal intestinal perfusion models and Caco-2 cell monolayers. After isolating specific intestinal segments, in situ single-pass intestinal perfusions (SPIP) were performed in rats and mice. The effective permeabilities (Peff) of labetalol and metoprolol, an FDA standard for the low/high Peff class boundary, were investigated in two different segments of rat intestine (proximal jejunum and distal ileum) and in the proximal jejunum of mouse. No significant difference was found between Peff of metoprolol and labetalol in the jejunum and ileum of rat (0.33 ± 0.11 × 10(-4) vs 0.38 ± 0.06 × 10(-4) and 0.57 ± 0.17 × 10(-4) vs 0.64 ± 0.30 × 10(-4) cm/s, respectively) and in the jejunum of mouse (0.55 ± 0.05 × 10(-4) vs 0.59 ± 0.13 × 10(-4) cm/s). However, Peff of metoprolol and labetalol were 1.7 and 1.6 times higher in the jejunum of mouse, compared to the jejunum of rat, respectively. Metoprolol and labetalol showed segmental-dependent permeability through the rat intestine, with increased Peff in the distal ileum in comparison to the proximal jejunum. Most significantly, Peff of labetalol was found to be concentration-dependent. Decreasing concentrations of labetalol in the perfusate resulted in decreased Peff compared to Peff of metoprolol. The intestinal epithelial permeability of labetalol was lower than that of metoprolol in Caco-2 cells at both apical pH 6.5 and 7.5 (5.96 ± 1.96 × 10(-6) vs 9.44 ± 3.44 × 10(-6) and 15.9 ± 2.2 × 10(-6) vs 23.2 ± 7.1 × 10(-6) cm/s, respectively). Labetalol exhibited higher permeability in basolateral to apical (BL-AP) compared to AP-BL direction in Caco-2 cells at 0.1 times the highest dose strength (HDS) (46.7 ± 6.5 × 10(-6) vs 14.2 ± 1.5 × 10(-6) cm/s). The P-gp inhibitor, verapamil, significantly increased AP-BL and decreased BL-AP direction transport of labetalol. Overall, labetalol showed high Peff in rat and mouse intestinal perfusion models similar to metoprolol at a concentration based on HDS. However, the concentration-dependent permeability of labetalol in mice due to P-gp and the inhibition study with verapamil in Caco-2 cells indicated that labetalol is not an ideal reference standard for BCS classification.

Increasing oral absorption of polar neuraminidase inhibitors: a prodrug transporter approach applied to oseltamivir analogue.

Poor oral absorption is one of the limiting factors in utilizing the full potential of polar antiviral agents. The neuraminidase target site requires a polar chemical structure for high affinity binding, thus limiting oral efficacy of many high affinity ligands. The aim of this study was to overcome this poor oral absorption barrier, utilizing prodrug to target the apical brush border peptide transporter 1 (PEPT1). Guanidine oseltamivir carboxylate (GOCarb) is a highly active polar antiviral agent with insufficient oral bioavailability (4%) to be an effective therapeutic agent. In this report we utilize a carrier-mediated targeted prodrug approach to improve the oral absorption of GOCarb. Acyloxy(alkyl) ester based amino acid linked prodrugs were synthesized and evaluated as potential substrates of mucosal transporters, e.g., PEPT1. Prodrugs were also evaluated for their chemical and enzymatic stability. PEPT1 transport studies included [(3)H]Gly-Sar uptake inhibition in Caco-2 cells and cellular uptake experiments using HeLa cells overexpressing PEPT1. The intestinal membrane permeabilities of the selected prodrugs and the parent drug were then evaluated for epithelial cell transport across Caco-2 monolayers, and in the in situ rat intestinal jejunal perfusion model. Prodrugs exhibited a pH dependent stability with higher stability at acidic pHs. Significant inhibition of uptake (IC(50) <1 mM) was observed for l-valyl and l-isoleucyl amino acid prodrugs in competition experiments with [(3)H]Gly-Sar, indicating a 3-6 times higher affinity for PEPT1 compared to valacyclovir, a well-known PEPT1 substrate and >30-fold increase in affinity compared to GOCarb. The l-valyl prodrug exhibited significant enhancement of uptake in PEPT1/HeLa cells and compared favorably with the well-absorbed valacyclovir. Transepithelial permeability across Caco-2 monolayers showed that these amino acid prodrugs have a 2-5-fold increase in permeability as compared to the parent drug and showed that the l-valyl prodrug (P(app) = 1.7 × 10(-6) cm/s) has the potential to be rapidly transported across the epithelial cell apical membrane. Significantly, only the parent drug (GOCarb) appeared in the basolateral compartment, indicating complete activation (hydrolysis) during transport. Intestinal rat jejunal permeability studies showed that l-valyl and l-isoleucyl prodrugs are highly permeable compared to the orally well absorbed metoprolol, while the parent drug had essentially zero permeability in the jejunum, consistent with its known poor low absorption. Prodrugs were rapidly converted to parent in cell homogenates, suggesting their ability to be activated endogenously in the epithelial cell, consistent with the transport studies. Additionally, l-valyl prodrug was found to be a substrate for valacyclovirase (K(m) = 2.37 mM), suggesting a potential cell activation mechanism. Finally we determined the oral bioavailability of our most promising candidate, GOC-l-Val, in mice to be 23% under fed conditions and 48% under fasted conditions. In conclusion, GOC-l-Val prodrug was found to be a very promising antiviral agent for oral delivery. These findings indicate that the carrier-mediated prodrug approach is an excellent strategy for improving oral absorption of polar neuraminidase inhibitors. These promising results demonstrate that the oral peptide transporter-mediated prodrug strategy has enormous promise for improving the oral mucosal cell membrane permeability of polar, poorly absorbed antiviral agents and treating influenza via the oral route of administration.

The extracellular microenvironment explains variations in passive drug transport across different airway epithelial cell types.

PURPOSE: We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types. METHODS: Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types. RESULTS: Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in the extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells. CONCLUSION: Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types.

Novel inhibitors of bacterial virulence: development of 5,6-dihydrobenzo[h]quinazolin-4(3H)-ones for the inhibition of group A streptococcal streptokinase expression.

Resistance to antibiotics is an increasingly dire threat to human health that warrants the development of new modes of treating infection. We recently identified 1 (CCG-2979) as an inhibitor of the expression of streptokinase, a critical virulence factor in Group A Streptococcus that endows blood-borne bacteria with fibrinolytic capabilities. In this report, we describe the synthesis and biological evaluation of a series of novel 5,6-dihydrobenzo[h]quinazolin-4(3H)-one analogs of 1 undertaken with the goal of improving the modest potency of the lead. In addition to achieving an over 35-fold increase in potency, we identified structural modifications that improve the solubility and metabolic stability of the scaffold. The efficacy of two new compounds 12c (CCG-203592) and 12k (CCG-205363) against biofilm formation in Staphylococcus aureus represents a promising additional mode of action for this novel class of compounds.

Evaluation and prediction of oral drug absorption and bioequivalence with food-druginteraction.

This article reviews the impacts on the in vivo prediction of oral bioavailability (BA) and bioequivalence (BE) based on Biopharmaceutical classification systems (BCS) by the food-drug interaction (food effect) and the gastrointestinal (GI) environmental change. Various in vitro and in silico predictive methodologies have been used to expect the BA and BE of the test oral formulation. Food intake changes the GI physiology and environment, which affect oral drug absorption and its BE evaluation. Even though the pHs and bile acids in the GI tract would have significant influence on drug dissolution and, hence, oral drug absorption, those impacts largely depend on the physicochemical properties of oral medicine, active pharmaceutical ingredients (APIs). BCS class I and III drugs are high soluble drugs in the physiological pH range, food-drug interaction may not affect their BA. On the other hand, BCS class II and IV drugs have pH-dependent solubility, and the more bile acid secretion and the pH changes by food intake might affect their BA. In this report, the GI physiological changes between the fasted and fed states are described and the prediction on the oral drug absorption by food-drug interaction have been introduced.

Harmonizing Biopredictive Methodologies Through the Product Quality Research Institute (PQRI) Part I: Biopredictive Dissolution of Ibuprofen and Dipyridamole Tablets.

Assessing in vivo performance to inform formulation selection and development decisions is an important aspect of drug development. Biopredictive dissolution methodologies for oral dosage forms have been developed to understand in vivo performance, assist in formulation development/optimization, and forecast the outcome of bioequivalence studies by combining them with simulation tools to predict plasma profiles in humans. However, unlike compendial dissolution methodologies, the various biopredictive methodologies have not yet been harmonized or standardized. This manuscript presents the initial phases of an effort to develop best practices and move toward standardization of the biopredictive methodologies through the Product Quality Research Institute (PQRI, https://pqri.org ) entitled "The standardization of in vitro predictive dissolution methodologies and in silico bioequivalence study Working Group." This Working Group (WG) is comprised of participants from 10 pharmaceutical companies and academic institutes. The project will be accomplished in a total of five phases including assessing the performance of dissolution protocols designed by the individual WG members, and then building "best practice" protocols based on the initial dissolution profiles. After refining the "best practice" protocols to produce equivalent dissolution profiles, those will be combined with physiologically based biopharmaceutics models (PBBM) to predict plasma profiles. In this manuscript, the first two of the five phases are reported, namely generating biopredictive dissolution profiles for ibuprofen and dipyridamole and using those dissolution profiles with PBBM to match the clinical plasma profiles. Key experimental parameters are identified, and this knowledge will be applied to build the "best practice" protocol in the next phase.

Selection of suitable prodrug candidates for in vivo studies via in vitro studies; the correlation of prodrug stability in between cell culture homogenates and human tissue homogenates.

PURPOSE: To determine the correlations/discrepancies of drug stabilities between in the homogenates of human culture cells and of human tissues. METHODS: Amino acid/dipeptide monoester prodrugs of floxuridine were chosen as the model drugs. The stabilities (half-lives) of floxuridine prodrugs in human tissues (pancreas, liver, and small intestine) homogenates were obtained and compared with ones in cell culture homogenates (AcPC-1, Capan-2, and Caco-2 cells) as well as human liver microsomes. The correlations of prodrug stability in human small bowel tissue homogenate vs. Caco-2 cell homogenate, human liver tissue homogenate vs. human liver microsomes, and human pancreatic tissue homogenate vs. pancreatic cell, AsPC-1 and Capan-2, homogenates were examined. RESULTS: The stabilities of floxuridine prodrugs in human small bowel homogenate exhibited the great correlation to ones in Caco-2 cell homogenate (slope = 1.0-1.3, r2 = 0.79-0.98). The stability of those prodrugs in human pancreas tissue homogenate also exhibited the good correlations to ones in AsPC-1 and Capan-2 cells homogenates (slope = 0.5-0.8, r2 = 0.58-0.79). However, the correlations of prodrug stabilities between in human liver tissue homogenates and in human liver microsomes were weaker than others (slope = 1.3-1.9, r2 = 0.07-0.24). CONCLUSIONS: The correlations of drug stabilities in cultured cell homogenates and in human tissue homogenates were compared. Those results exhibited wide range of correlations between in cell homogenate and in human tissue homogenate (r2 = 0.07 - 0.98). Those in vitro studies in cell homogenates would be good tools to predict drug stabilities in vivo and to select drug candidates for further developments. In the series of experiments, 5'-O-D-valyl-floxuridine and 5'-O-L-phenylalanyl-L-tyrosyl-floxuridine would be selected as candidates of oral drug targeting delivery for cancer chemotherapy due to their relatively good stabilities compared to other tested prodrugs.

In silico prediction of drug dissolution and absorption with variation in intestinal pH for BCS class II weak acid drugs: ibuprofen and ketoprofen.

The FDA Biopharmaceutical Classification System guidance allows waivers for in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage forms only for BCS class I. Extensions of the in vivo biowaiver for a number of drugs in BCS class III and BCS class II have been proposed, in particular, BCS class II weak acids. However, a discrepancy between the in vivo BE results and in vitro dissolution results for BCS class II acids was recently observed. The objectives of this study were to determine the oral absorption of BCS class II weak acids via simulation software and to determine if the in vitro dissolution test with various dissolution media could be sufficient for in vitro bioequivalence studies of ibuprofen and ketoprofen as models of carboxylic acid drugs. The oral absorption of these BCS class II acids from the gastrointestinal tract was predicted by GastroPlus™. Ibuprofen did not satisfy the bioequivalence criteria at lower settings of intestinal pH of 6.0. Further the experimental dissolution of ibuprofen tablets in a low concentration phosphate buffer at pH 6.0 (the average buffer capacity 2.2 mmol l (-1) /pH) was dramatically reduced compared with the dissolution in SIF (the average buffer capacity 12.6 mmol l (-1) /pH). Thus these predictions for the oral absorption of BCS class II acids indicate that the absorption patterns depend largely on the intestinal pH and buffer strength and must be considered carefully for a bioequivalence test. Simulation software may be a very useful tool to aid the selection of dissolution media that may be useful in setting an in vitro bioequivalence dissolution standard.

The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme.

The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

Dissolution Challenges Associated with the Surface pH of Drug Particles: Integration into Mechanistic Oral Absorption Modeling.

The present work aimed to differentiate between in vitro dissolution profiles of ibuprofen as input for GastroPlus™ and to see the impact on systemic exposure. In vitro dissolution profiles of ibuprofen obtained under low- and high-buffered dissolution media were used as input using the z-factor approach. In a second step, a customized surface pH calculator was applied to predict the surface pH of ibuprofen under these low- and high-buffered dissolution conditions. These surface pH values were adopted in GastroPlus™ and simulations were performed to predict the systemic outcome. Simulated data were compared with systemic data of ibuprofen obtained under fasted state conditions in healthy subjects. The slower dissolution rate observed when working under low-buffered conditions nicely matched with the slower dissolution rate as observed during the clinical aspiration study and was in line with the systemic exposure of the drug. Finally, a population simulation was performed to explore the impact of z-factor towards bioequivalence (BE) criteria (so-called safe space). Concerning future perspectives, the customized calculator should be developed in such a way to make it possible to predict the dissolution rate (being informed by the particle size distribution) which, in its turn, can be used as a surrogate to predict the USP2 dissolution curve. Subsequently, validation can be done by using this profile as input for PBPK platforms.