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PURPOSE: Previous studies evaluating ticagrelor drug-drug interactions have not differentiated intestinal versus systemic mechanisms, which we do here. METHODS: Using recently published methodologies from our laboratory to differentiate metabolic- from transporter-mediated drug-drug interactions, a critical evaluation of five published ticagrelor drug-drug interactions was carried out to investigate the purported clinical significance of enzymes and transporters in ticagrelor disposition. RESULTS: The suggested CYP3A4 inhibitors, ketoconazole and diltiazem, displayed unchanged mean absorption time (MAT) and time of maximum concentration (T(max)) values as was expected, i.e., the interactions were mainly mediated by metabolic enzymes. The potential CYP3A4/P-gp inhibitor cyclosporine also showed an unchanged MAT value. Further analysis assuming there was no P-gp effect suggested that the increased AUC and unchanged t(1/2) for ticagrelor after cyclosporine administration were attributed to the inhibition of intestinal CYP3A4 rather than P-gp. Rifampin, an inducer of CYP3As after multiple dosing, unexpectedly showed decreased MAT and T(max) values, which cannot be completely explained. In contrast, grapefruit juice, an intestinal CYP3A/P-gp/OATP inhibitor, significantly increased MAT and T(max) values for ticagrelor, which may be due to activation of P-gp or inhibition of OATPs expressed in intestine. CONCLUSIONS: This study provides new insight into the role of transporter pathways in ticagrelor intestinal absorption by examining potential MAT and T(max) changes mediated by drug-drug interactions.

PURPOSE: The involvement of the intestinally expressed xenobiotic transporters P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) have been implicated in apixaban disposition based on in vitro studies. Recommendations against co-administration of apixaban with inhibitors of these efflux transporters can be found throughout the literature as well as in the apixaban FDA label. However, the clinical relevance of such findings is questionable due to the high permeability and high solubility characteristics of apixaban. METHODS: Using recently published methodologies to discern metabolic- from transporter- mediated drug-drug interactions, a critical evaluation of all published apixaban drug-drug interaction studies was conducted to investigate the purported clinical significance of efflux transporters in apixaban disposition. RESULTS: Rational examination of these clinical studies using basic pharmacokinetic theory does not support the clinical significance of intestinal efflux transporters in apixaban disposition. Further, there is little evidence that efflux transporters are clinically significant determinants of systemic clearance. CONCLUSIONS: Inhibition or induction of intestinal CYP3A4 can account for exposure changes of apixaban in all clinically significant drug-drug interactions, and lack of intestinal CYP3A4 inhibition can explain all studies with no exposure changes, regardless of the potential for these perpetrators to inhibit intestinal or systemic efflux transporters.

Purpose: To develop a small-scale set-up to rapidly and accurately determine the intrinsic dissolution rate (IDR) and apparent solubility of poorly water-soluble compounds. Methods: The IDR and apparent solubility (S-app) were measured in fasted state simulated intestinal fluid (FaSSIF) for six model compounds using wet-milled controlled suspensions (1.0% (w/w) PVP and 0.2% (w/w) SDS) and the mu DISS Profiler. Particle size distribution was measured using a Zetasizer and the total surface area was calculated making use of the density of the compound. Powder and disc dissolution were performed and compared to the IDR of the controlled suspensions. Results: The IDR values obtained from the controlled suspensions were in excellent agreement with IDR from disc measurements. The method used low amount of compound (mu g-scale) and the experiments were completed within a few minutes. The IDR values ranged from 0.2-70.6 mu g/min/cm(2) and the IDR/S-app ratio ranged from 0.015 to 0.23. This ratio was used to indicate particle size sensitivity on intestinal concentrations reached for poorly water-soluble compounds. Conclusions: The established method is a new, desirable tool that provides the means for rapid and highly accurate measurements of the IDR and apparent solubility in biorelevant dissolution media. The IDR/S-app is proposed as a measure of particle size sensitivity when significant solubilization may occur.

Purpose To examine the utility of human plasma as an assay medium in Caco-2 permeability studies to overcome poor mass balance and inadequate sink conditions frequently encountered with lipophilic compounds. Methods Caco-2 permeability was assessed for reference compounds with known transport mechanisms using either pH 7.4 buffer or human plasma as the assay medium in both the apical and basolateral chambers. When using plasma, Papp values were corrected for the unbound fraction in the donor chamber. The utility of the approach was assessed by measuring the permeability of selected antimalarial compounds using the two assay media. Results Caco-2 cell monolayer integrity and P-gp transporter function were unaffected by the presence of human plasma in the donor and acceptor chambers. For many of the reference compounds having good mass balance with buffer as the medium, higher Papp values were observed with plasma, likely due to improved acceptor sink conditions. The lipophilic antimalarial compounds exhibited low mass balance with buffer, however the use of plasma markedly improved mass balance allowing the determination of more reliable Papp values. Conclusions The results support the utility of human plasma as an alternate Caco-2 assay medium to improve mass balance and permeability measurements for lipophilic compounds. Electronic supplementary material The online version of this article (10.1007/s11095-018-2493-3) contains supplementary material, which is available to authorized users.

PURPOSE: The study evaluates the use of new in vitro primary human cell-based organotypic small intestinal (SMI) microtissues for predicting intestinal drug absorption and drug-drug interaction. METHODS: The SMI microtissues were reconstructed using human intestinal fibroblasts and enterocytes cultured on a permeable support. To evaluate the suitability of the intestinal microtissues to model drug absorption, the permeability coefficients across the microtissues were determined for a panel of 11 benchmark drugs with known human absorption and Caco-2 permeability data. Drug-drug interactions were examined using efflux transporter substrates and inhibitors. RESULTS: The 3D–intestinal microtissues recapitulate the structural features and physiological barrier properties of the human small intestine. The microtissues also expressed drug transporters and metabolizing enzymes found on the intestinal wall. Functionally, the SMI microtissues were able to discriminate between low and high permeability drugs and correlated better with human absorption data (r(2) = 0.91) compared to Caco-2 cells (r(2) = 0.71). Finally, the functionality of efflux transporters was confirmed using efflux substrates and inhibitors which resulted in efflux ratios of >2.0 fold and by a decrease in efflux ratios following the addition of inhibitors. CONCLUSION: The SMI microtissues appear to be a useful preclinical tool for predicting drug bioavailability of orally administered drugs.

Purpose Currently, the FDA allows biowaivers for Class I (high solubility and high permeability) and Class III (high solubility and low permeability) compounds of the Biopharmaceutics Classification System (BCS). Scientific evidence should be provided to support biowaivers for BCS Class I and Class III (high solubility and low permeability) compounds. Methods Data on the effects of excipients on drug permeability are needed to demonstrate that commonly used excipients do not affect the permeability of BCS Class III compounds, which would support the application of biowaivers to Class III compounds. This study was designed to generate such data by assessing the permeability of four BCS Class III compounds and one Class I compound in the presence and absence of five commonly used excipients. Results The permeability of each of the compounds was assessed, at three to five concentrations, with each excipient in two different models: Caco-2 cell monolayers, and in situ rat intestinal perfusion. No substantial increases in the permeability of any of the compounds were observed in the presence of any of the tested excipients in either of the models, with the exception of disruption of Caco-2 cell monolayer integrity by sodium lauryl sulfate at 0.1 mg/ml and higher. Conclusion The results suggest that the absorption of these four BCS Class III compounds would not be greatly affected by the tested excipients. This may have implications in supporting biowaivers for BCS Class III compounds in general.

PurposeCompound solubility serves as a surrogate indicator of oral biopharmaceutical performance. Between infancy and adulthood, marked compositional changes in gastrointestinal (GI) fluids occur. This study serves to assess how developmental changes in GI fluid composition affects compound solubility.MethodsSolubility assessments were conducted in vitro using biorelevant media reflective of age-specific pediatric cohorts (i.e., neonates and infants). Previously published adult media (i.e., FaSSGF, FeSSGF, FaSSIF.v2, and FeSSIF.v2) were employed as references for pediatric media development. Investigations assessing age-specific changes in GI fluid parameters (i.e., pepsin, bile acids, pH, osmolality, etc.) were collected from the literature and served to define the composition of neonatal and infant media. Solubility assessments at 37°C were conducted for seven BCS Class II compounds within the developed pediatric and reference adult media.ResultsFor six of the seven compounds investigated, solubility fell outside an 80–125% range from adult values in at least one of the developed pediatric media. This result indicates a potential for age-related alterations in oral drug performance, especially for compounds whose absorption is delimited by solubility (i.e., BCS Class II).ConclusionDevelopmental changes in GI fluid composition can result in relevant discrepancies in luminal compound solubility between children and adults.

Purpose To identify conditions allowing the use of cell-based models for studies of drug absorption during in vitro lipolysis of lipid-based formulations (LBFs). Methods Caco-2 was selected as the cell-based model system. Monolayer integrity was evaluated by measuring mannitol permeability after incubating Caco-2 cells in the presence of components available during lipolysis. Pure excipients and formulations representing the lipid formulation classification system (LFCS) were evaluated before and after digestion. Porcine mucin was evaluated for its capacity to protect the cell monolayer. Results Most undigested formulations were compatible with the cells (II-LC, IIIB-LC, and IV) although some needed mucin to protect against damaging effects (II-MC, IIIB-MC, I-LC, and IIIA-LC). The pancreatic extract commonly used in digestion studies was incompatible with the cells but the Caco-2 monolayers could withstand immobilized recombinant lipase. Upon digestion, long chain formulations caused more damage to Caco-2 cells than their undigested counterparts whereas medium chain formulations showed better tolerability after digestion. Conclusions Most LBFs and components thereof (undigested and digested) are compatible with Caco-2 cells. Pancreatic enzyme is not tolerated by the cells but immobilized lipase can be used in combination with the cell monolayer. Mucin is beneficial for critical formulations and digestion products. Electronic supplementary material The online version of this article (10.1007/s11095-017-2327-8) contains supplementary material, which is available to authorized users.

We developed simulation and modeling methods to predict the in vivo pharmacokinetic profiles of acyclovir, following escalating oral doses of valacyclovir, in wildtype and Pept1 knockout mice. We also quantitated the contribution of specific intestinal segments in the absorption of valacyclovir in these mice. Simulations were conducted using a mechanistic advanced compartmental absorption and transit (ACAT) model implemented in GastroPlus™. Simulations were performed for 3 h post-dose in wildtype and Pept1 knockout mice following single oral doses of 10, 25, 50 and 100 nmol/g valacyclovir, and compared to experimentally observed plasma concentration-time profiles of acyclovir. Good fits were obtained in wildtype and Pept1 knockout mice. Valacyclovir was primarily absorbed from duodenum (42%) and jejunum (24%) of wildtype mice, with reduced uptake from ileum (3%) and caecum/colon (1%), for a total of 70% absorption. In contrast, the absorption of valacyclovir in Pept1 knockout mice was slow and sustained throughout the entire intestinal tract in which duodenum (4%), jejunum (14%), ileum (10%) and caecum/colon (12%) accounted for a total of 40% absorption. The ACAT model bridged the gap between in situ and in vivo experimental findings, and facilitated our understanding of the complicated intestinal absorption processes of valacyclovir.

Purpose To examine if pulmonary P-glycoprotein (P-gp) is functional in an intact lung; impeding the pulmonary absorption and increasing lung retention of P-gp substrates administered into the airways. Using calculated physico-chemical properties alone build a predictive Quantitative Structure-Activity Relationship (QSAR) model distinguishing whether a substrate’s pulmonary absorption would be limited by P-gp or not. Methods A panel of 18 P-gp substrates were administered into the airways of an isolated perfused mouse lung (IPML) model derived from Mdr1a/Mdr1b knockout mice. Parallel intestinal absorption studies were performed. Substrate physico-chemical profiling was undertaken. Using multivariate analysis a QSAR model was established. Results A subset of P-gp substrates (10/18) displayed pulmonary kinetics influenced by lung P-gp. These substrates possessed distinct physico-chemical properties to those P-gp substrates unaffected by P-gp (8/18). Differential outcomes were not related to different intrinsic P-gp transporter kinetics. In the lung, in contrast to intestine, a higher degree of non-polar character is required of a P-gp substrate before the net effects of efflux become evident. The QSAR predictive model was applied to 129 substrates including eight marketed inhaled drugs, all these inhaled drugs were predicted to display P-gp dependent pulmonary disposition. Conclusions Lung P-gp can affect the pulmonary kinetics of a subset of P-gp substrates. Physico-chemical relationships determining the significance of P-gp to absorption in the lung are different to those operative in the intestine. Our QSAR framework may assist profiling of inhaled drug discovery candidates that are also P-gp substrates. The potential for P-gp mediated pulmonary disposition exists in the clinic. Electronic supplementary material The online version of this article (doi:10.1007/s11095-017-2220-5) contains supplementary material, which is available to authorized users.

In vitro lipolysis is widely utilized for predicting in vivo performance of oral lipid-based formulations (LBFs). However, evaluation of LBFs in the absence of an absorption sink may have limited in vivo relevance. This study aimed at employing biopharmaceutical modeling to simulate LBF digestion and drug supersaturation in a continuous absorptive environment.Three fenofibrate-loaded LBFs were characterized in vitro (dispersion and lipolysis) and drug precipitation was monitored using in-line Raman spectroscopy. In vitro data were combined with pharmacokinetic data derived from an in vivo study in pigs to simulate intestinal LBF transit. This biopharmaceutical model allowed calculation of lipolysis-triggered drug supersaturation while drug and lipolysis products are absorbed from the intestine.The biopharmaceutical model predicted that, in a continuous absorption environment, fenofibrate supersaturation was considerably lower compared to in vitro lipolysis (non-sink). Hence, the extensive drug precipitation observed in vitro was predicted to be unlikely in vivo. The absorption of lipolysis products increased drug supersaturation, but drug precipitation was unlikely for highly permeable drugs.Biopharmaceutical modeling is a valuable approach for predicting LBFs performance in vivo. In the absence of in vitro tools simulating absorptive conditions, modeling strategies should be further considered.

To determine the effect of PepT1 on the absorption and disposition of cefadroxil, including the potential for saturable intestinal uptake, after escalating oral doses of drug.The absorption and disposition kinetics of [3H]cefadroxil were determined in wild-type and PepT1 knockout mice after 44.5, 89.1, 178, and 356 nmol/g oral doses of drug. The pharmacokinetics of [3H]cefadroxil were also determined in both genotypes after 44.5 nmol/g intravenous bolus doses.PepT1 deletion reduced the area under the plasma concentration-time profile (AUC0-120) of cefadroxil by 10-fold, the maximum plasma concentration (Cmax) by 17.5-fold, and increased the time to reach a maximum plasma concentration (Tmax) by 3-fold. There was no evidence of nonlinear intestinal absorption since AUC0-120 and Cmax values changed in a dose-proportional manner. Moreover, the pharmacokinetics of cefadroxil were not different between genotypes after intravenous bolus doses, indicating that PepT1 did not affect drug disposition. Finally, no differences were observed in the peripheral tissue distribution of cefadroxil (i.e., outside gastrointestinal tract) once these tissues were corrected for differences in perfusing blood concentrations.The findings demonstrate convincingly the critical role of intestinal PepT1 in both the rate and extent of oral administration for cefadroxil and potentially other aminocephalosporin drugs.

Biomagnetic methods have been designed for a wide range of applications. Recently, such methods have been proposed as alternatives to scintigraphy for evaluating of a number of pharmaceutical processes in vitro as well as under the influence of gastrointestinal physiological parameters. In this review, physical characterization as well as the most recent applications of Superconducting Quantum Interference Device (SQUID), Anisotropic Magnetoresistive (AMR) and AC Biosusceptometry (ACB) in the pharmaceutical research will be explored. Moreover, their current status and how these technologies can be employed to improve the knowledge about the impact of gastrointestinal physiology on drug delivery in association with pharmacokinetic outcomes, termed pharmacomagnetography, will be presented.

金沢大学医薬保健研究域薬学系, Purpose. Solute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1. Methods. We first constructed octn1 gene knockout (octn1-/-) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined. Results. The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1-/- mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed, the highest distribution to small intestines and extensive renal reabsorption of [3H]ergothioneine, both of which were much reduced in octn1-/- mice. The octn1 -/- mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn's disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis. Conclusions. These results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases. © 2010 Springer Science+Business Media, LLC.

金沢大学医薬保健研究域薬学系 金沢大学医薬保健研究域医学系, Purpose. The oral bioavailability of some therapeutic agents is markedly lower in cynomolgus monkeys than in humans. We investigated small-intestinal absorption of the P-glycoprotein (P-gp) substrates etoposide and digoxin in monkeys to clarify the influence of efflux transport on their intestinal permeability. Methods. The pharmacokinetics of etoposide and digoxin was examined in monkeys and rats after oral and intravenous administration. Intestinal permeability and segmental differences in permeability were investigated with an Ussing-type chamber. Results. The bioavailability of etoposide was 12.9 and 13.9% in monkeys and rats, respectively. Total body clearance of etoposide in monkeys was much less than hepatic blood flow, suggesting that the bioavailability would be limited at intestinal absorption. Marked vectorial transport of etoposide in the secretory direction was observed in rats, especially in the lower small intestine, and segmental differences were consistent with the distribution of P-gp expression. Vectorial transport was minimal in monkey small intestine. Our kinetic analysis indicated that P-gp contributes little to the intestinal permeability of etoposide and digoxin in monkeys, and apical uptake is rate-limiting. Conclusion. Low bioavailability of etoposide in monkeys is due to poor intestinal uptake resulting from low influx from the apical side, rather than secretion via P-gp. © 2008 Springer Science+Business Media, LLC.

To quantitate and predict colon-specific 9-aminocamptothecin (9-AC) release from the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-9-AC conjugate and its absorption behavior after oral administration in rats.Drug distribution in the gastrointestinal (GI) tract and the plasma concentration-time profile of 9-AC released from the HPMA copolymer conjugate were predicted using the degradation, transit, and absorption rate constants in cecum. The fate of 9-AC in cecum and liver was measured by in-situ cecum absorption and liver perfusion.Following oral administration of the conjugate, 9-AC was released rapidly in cecum. Based on the pharmacokinetic model, up to 60% of the dose was in the cecum at ~6 h, and 7% of the dose still remained there at 24 h. The predicted plasma concentration curve for released 9-AC after an oral dose of 3 mg/kg of 9-AC equivalent increased gradually and reached a peak of 98 nM at 7 h, then started decreasing slowly to 16 nM at 24 h. The bioavailability value was estimated as 0.31 after the first-pass elimination.A pharmacokinetic model delineated the impact of GI transit, drug absorption rate, and first-pass metabolism on drug disposition following oral administration of HPMA copolymer-9-AC conjugate in rats.

Purpose To evaluate the pharmacological activity of insulin-loaded alginate/chitosan nanoparticles following oral dosage in diabetic rats. Methods Nanoparticles were prepared by ionotropic pre-gelation of an alginate core followed by chitosan polyelectrolyte complexation. In vivo activity was evaluated by measuring the decrease in blood glucose concentrations in streptozotocin induced, diabetic rats after oral administration and flourescein (FITC)-labelled insulin tracked by confocal microscopy. Results Nanoparticles were negatively charged and had a mean size of 750 nm, suitable for uptake within the gastrointestinal tract due to their nanosize range and mucoadhesive properties. The insulin association efficiency was over 70% and insulin was released in a pH-dependent manner under simulated gastrointestinal conditions. Orally delivered nanoparticles lowered basal serum glucose levels by more than 40% with 50 and 100 IU/kg doses sustaining hypoglycemia for over 18 h. Pharmacological availability was 6.8 and 3.4% for the 50 and 100 IU/kg doses respectively, a significant increase over 1.6%, determined for oral insulin alone in solution and over other related studies at the same dose levels. Confocal microscopic examinations of FITC-labelled insulin nanoparticles showed clear adhesion to rat intestinal epithelium, and internalization of insulin within the intestinal mucosa. Conclusion The results indicate that the encapsulation of insulin into mucoadhesive nanoparticles was a key factor in the improvement of its oral absorption and oral bioactivity.

There is significant interest in the application of nanoparticles to deliver immunostimulatory signals to cells. We hypothesized that curdlan (immune stimulating polymer) could be conjugated to PLGA and nanoparticles from this copolymer would possess immunostimulatory activity, be non-cytotoxic and function as an effective sustained drug release system.Carbodiimide chemistry was employed to conjugate curdlan to PLGA. The conjugate (C-PLGA) was characterized using (1)H and (13)C NMR, FTIR, DSC and TGA. Nanoparticles were synthesized using an emulsion-solvent evaporation technique. Immunostimulatory activity was characterized in THP-1 derived macrophages. MTT assay and real-time impedance measurements were used to characterize polymer and nanoparticle toxicity and uptake in macrophages. Drug delivery capability was assessed across Caco-2 cells using rifampicin as a model drug.Spectral characterization confirmed successful synthesis of C-PLGA. C-PLGA nanoparticles enhanced phosphorylated ERK production in macrophages indicating cell stimulation. Nanoparticles provided slow release of rifampicin across Caco-2 cells. Polymers but not nanoparticles altered the adhesion profiles of the macrophages. Impedance measurements suggested Ca(2+) dependent uptake of nanoparticles by the macrophages.PLGA nanoparticles with macrophage stimulating and sustained drug delivery capabilities have been prepared. These nanoparticles can be used to stimulate macrophages and concurrently deliver drug in infectious disease therapy.

Based on adherence to intestinal mucosa, intralumenally administered liposomal formulations of 5-aminosalicylate (5-ASA) and 6-mercaptopurine (6-MP) were studied for their potential to enhance local drug delivery to intestinal tissue for the treatment of inflammatory bowel disease. 5-ASA was encapsulated in standard phospholipid liposomes while 6-MP required encapsulation in nonphospholipid liposomes to obtain equivalent drug loading. Encapsulation efficiency was measured by size-exclusion chromatography/high-performance liquid chromatogtaphy (HPLC). Liposomal formulations or solution of the drugs were injected into unligated jejunum to compare pharmacokinetics and into ligated loops of rat ileum and colon to evaluate local delivery. Dextran sulfate and acetic acid induced colitis were used as models of lower intestinal inflammation. Plasma, tissue and luminal drug and metabolite levels were measured by liquid scintillation counting or HPLC. Encapsulation efficiency of 6-MP was dependent on lipid content and composition. While liposomal encapsulation significantly reduced systemic absorption of 5-ASA this was not the case for 6-MP. Liposomal adherence to intestinal tissue resulted in increased tissue levels for 5-ASA; however, 6-MP local tissue levels were not improved compared to solution drug. Nonphospholipid liposomes optimize encapsulation of 6-MP. While liposomal formulations show potential for local drug delivery to diseased bowel, drug physicochemical properties, absorption, and metabolic profiles dictate tissue-targeting potential. Liposomes reduce systemic availability from paracellular absorption of hydrophilic 5-ASA, but fail to improve local tissue delivery of 6-MP, a molecule absorbed by passive membrane permeation that undergoes extensive first- pass metabolism.

Purpose. Isolating the relative contributions of parallel transcellular and paracellular transport to the intestinal absorption of small hydrophilic molecules has proven experimentally challenging. In this report, lumenal appearance of drug metabolite is utilized as a tool to assess the contribution of paracellular transport to the absorption of cimetidine and 5-aminosalicylate (5ASA) in rat small intestine.

Purpose. The aim of this research was to determine the mechanism by which a co-administered meal decreases the oral absorption of bidisomide and does not influence the oral absorption of the chemically-related antiarrhythmic agent, disopyramide.

Purpose. To study the uptake of biodegradable microparticles in Caco-2 cells. Methods. Biodegradable microparticles of polylactic polyglycolic acid co-polymer (PLGA 50:50) of mean diameters 0.1 μm, 1 μm, and 10 μm containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker were formulated by a multiple emulsion technique. The Caco-2 cell monolayers were incubated with each diameter microparticles (100 μg/ml) for two hours. The microparticle uptake in Caco-2 cells was studied by confocal microscopy and also by quantitating the 6-coumarin content of the microparticles taken up by the cells. The effects of microparticle concentration, and incubation time and temperature on microparticle cell uptake were also studied. Results. The study demonstrated that the Caco-2 cell microparticle uptake significantly depends upon the microparticle diameter. The 0.1 μm diameter microparticles had 2.5 fold greater uptake on the weight basis than the 1 μm and 6 fold greater than the 10 μm diameter microparticles. Similarly in terms of number the uptake of 0.1 μm diameter microparticles was 2.7 × 103 fold greater than the 1 μm and 6.7 × 106 greater than the 10 μm diameter microparticles. The efficiency of uptake of 0.1 μm diameter microparticles at 100 μg/ml concentration was 41% compared to 15% and 6% for the 1 μm and the 10 μm diameter microparticles, respectively. The Caco-2 cell microparticle (0.1 μm) uptake increased with concentration in the range of 100 μg/ml to 500 μg/ml which then reached a plateau at higher concentration. The uptake of microparticles increased with incubation time, reaching a steady state at two hours. The uptake was greater at an incubation temperature of 37°C compared to at 4°C. Conclusions. The Caco-2 cell microparticle uptake was microparticle diameter, concentration, and incubation time and temperature dependent. The small diameter microparticles (0.1 μm) had significantly greater uptake compared to larger diameter microparticles. The results thus suggest that the mechanism of uptake of microparticles in Caco-2 cell is particle diameter dependent. Caco-2 cells are used as an in vitro model for gastrointestinal uptake, and therefore the results obtained in these studies could be of significant importance in optimizing the microparticle-based oral drug delivery systems.

The bioavailability of dipyridamole, a poorly soluble weak base, was evaluated in 11 healthy, older subjects (> or = 65 years), 6 with a low fasting gastric pH (control) and 5 with a fasting gastric pH > 5 (achlorhydric), in a randomized, crossover design. Subjects received 50 mg dipyridamole as a single oral dose both with and without pretreatment with 40 mg famotidine (control subjects) or 1360 mg glutamic acid HCl (achlorhydric subjects). Gastric pH was monitored by Heidelberg radiotelemetric capsule. Gastric emptying of 99mTc-radiolabeled orange juice was measured. Gastric pH appeared to be a primary determinant in dipyridamole absorption in the elderly. Elevated gastric pH resulted in compromised dipyridamole absorption compared to low-gastric pH conditions in all cases. The administration of glutamic acid hydrochloride to achlorhydric subjects prior to the dose of dipyridamole corrected for the decreased Cmax and AUC(0-36) exhibited in achlorhydric subjects without pretreatment. Tmax and ka were slower in achlorhydrics, although pretreatment with glutamic acid HCl tended to normalize these parameters. Based on these results, it would be beneficial for achlorhydrics to take glutamic acid hydrochloride prior to taking dipyridamole and other medications which need a low gastric pH for complete absorption. The administration of 40 mg famotidine was successful in elevating the gastric pH to > 5 in all subjects and maintained it at > 5 for at least 3 hr in all subjects tested.(ABSTRACT TRUNCATED AT 250 WORDS)

To clarify sotalol's classification in the BCS versus BDDCS systems through cellular, rat everted sac and PAMPA permeability studies.Studies were carried out in Madin Darby canine kidney (MDCK) and MDR1-transfected MDCK (MDCK-MDR1) cell lines, rat everted gut sacs and the Parallel Artificial Membrane Permeability Assay (PAMPA) system. Three-hour transport studies were conducted in MDCK cell lines (with apical pH changes) and MDCK-MDR1 cells (with and without the P-glycoprotein inhibitor GG918); male Sprague-Dawley rats (300~350 g) were used to prepare everted sacs. In the PAMPA studies, drug solutions at different pH's were dosed in each well and incubated for 5 h. Samples were measured by LC-MS/MS, or liquid scintillation counting and apparent permeability (P(app)) was calculated.Sotalol showed low permeability in all of the cultured-cell lines, everted sacs and PAMPA systems. It might be a border line P-glycoprotein substrate. The PAMPA study showed that sotalol's permeability increased with a higher apical pH, while much less change was found in MDCK cells.The low permeability rate for sotalol correlates with its Class 3 BDDCS assignment and lack of in vivo metabolism.

A theoretical analysis for estimating the extent of intestinal peptide and peptide analogue absorption was developed on the basis of a mass balance approach that incorporates convection, permeability, and reaction. The macroscopic mass balance analysis (MMBA) was extended to include chemical and enzymatic degradation. A microscopic mass balance analysis, a numerical approach, was also developed and the results compared to the MMBA. The mass balance equations for the fraction of a drug absorbed and reacted in the tube were derived from the general steady state mass balance in a tube: [formula: see text] where M is mass, z is the length of the tube, R is the tube radius, Pw is the intestinal wall permeability, kr is the reaction rate constant, C is the concentration of drug in the volume element over which the mass balance is taken, VL is the volume of the tube, and vz is the axial velocity of drug. The theory was first applied to the oral absorption of two tripeptide analogues, cefaclor (CCL) and cefatrizine (CZN), which degrade and dimerize in the intestine. Simulations using the mass balance equations, the experimental absorption parameters, and the literature stability rate constants yielded a mean estimated extent of CCL (250-mg dose) and CZN (1000-mg dose) absorption of 89 and 51%, respectively, which was similar to the mean extent of absorption reported in humans (90 and 50%). It was proposed previously that 15% of the CCL dose spontaneously degraded systematically; however, our simulations suggest that significant CCL degradation occurs (8 to 17%) presystemically in the intestinal lumen.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro permeability of a series of both hydrophilic and lipophilic compounds, as defined by the octanol/water partition coefficient, was measured in four segments of rabbit, monkey, and dog intestine using a side-by-side diffusion cell. A linear relationship was established for tissue resistance to hydrophilic compound diffusion in jejunum and colon among rabbit, monkey, and dog. The results suggest that rabbit jejunum is twice as permeable as monkey and dog jejunum. The colonic tissues of monkey, rabbit, and dog demonstrate similar permeabilities. Measuring the permeabilities of different tissues with compounds of similar physicochemical properties allows comparison of tissue restriction to transport. Thus, in vitro permeability measurements may be used to investigate physiological differences of various intestinal tissue segments that influence tissue permeability. Investigating the permeability of different intestinal segments from various species could allow the identification of an appropriate in vitro intestinal permeability model that will lead to the prediction of intestinal absorption in humans, eliminating the need for extensive and often misleading in vivo animal testing.

In vitro bile salt binding equilibria and kinetic studies were performed with cholestyramine to determine how these factors influence bile sequestrant efficacy in vivo. Chloride ion at physiologic concentrations caused more than a twofold reduction in glycocholate (GCH) binding, compared to binding in the absence of salt, over a range of GCH concentrations and was also observed to displace bound GCH. In addition, chloride ion displaced from cholestyramine as a result of bile salt binding was measured using a chloride selective electrode, and the results show that bile salt binding is due to ion exchange. Comparison of the results of the equilibrium binding experiments to human data shows that the effect of anion binding competition alone cannot account for the lack of efficacy of cholestyramine. Consideration of other effects, such as additional binding competition or poor availability for binding, based on data from the literature, shows that adequate bile salt binding potential exists and that these interferences are not major factors influencing resin efficacy. In kinetic studies, both binding uptake of GCH and displacement of GCH from cholestyramine by chloride ion were relatively rapid, indicating that cholestyramine should equilibrate rapidly with bile salts in the GI tract. Based on these findings, it is suggested that the low efficacy of cholestyramine is a result mainly of its relatively poor ability to prevent bile salt reabsorption in the ileum.

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 x 10(-5) +/- 0.53 x 10(-5) and 1.56 x 10(-5) +/- 0.65 x 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The single-pass intestinal perfusion technique has been used extensively to estimate the wall permeability in rats. The unbiased membrane parameters can be obtained only when the aqueous resistance is properly accounted for. This aqueous resistance was calculated numerically from a convective diffusive mass transfer model, including both passive and carrier-mediated transport at the intestinal wall. The aqueous diffusion layer resistance was shown to be best described by a function of the form, [formula: see text] where G zeta, P*m, P*c, Km, and Co are, respectively, Graetz number, passive permeability, carrier-mediated permeability, Michaelis constant, and the drug concentration entering the tube. Asterisked are dimensionless quantities obtained by multiplying the permeability constants with R/D, where R and D being radius and drug diffusivity, respectively. A, B, C, D and E were obtained by a least-squares nonlinear regression method, giving values of 1.05, 1.74, 1.27, 0.0659, and 0.377, respectively, over the range of 0.001 less than or equal to G zeta less than or equal to 0.5, 0.01 less than or equal to P*m less than or equal to 10, 0.01 less than or equal to P*c less than or equal to 10, and 0.01 less than or equal to Km/Co less than or equal to 100. This aqueous resistance was found to converge to those calculated from Levich's boundary layer solution in low Graetz range, indicating the correct theoretical limit. Using an iteration method, the equation was shown to be useful in extracting the intrinsic membrane permeability from the experimental data.

Leu-enkephalin (YGGFL) and several analogues were chosen as model peptides for the study of peptide absorption and hydrolysis in the rat jejunum. An HPLC assay was adapted to detect YGGFL or the analogues and metabolites. Peptide hydrolysis was studied in the rat jejunum using a single-pass perfusion method. Extensive hydrolysis of YGGFL was observed in the rat jejunum and approaches to reduce its metabolism were studied. The brush border enzymes are a major site of enkephalin hydrolysis. Lumenal peptidases were secondary to the brush border enzymes in hydrolyzing the enkephalins in this system. In the in situ perfusion system, YGGFL is hydrolyzed primarily to Tyr and GGFL by the brush border aminopeptidase and to YGG and FL by brush border endopeptidase. Lowering the jejunal pH below 5.0 significantly reduces aminopeptidase activity and, to a lesser extent, endopeptidase activity. An aminopeptidase inhibitor, amastatin, produced more pronounced inhibitory effects at higher pH and the endopeptidase inhibitors, tripeptides YGG and GGF, are effective even below pH 5.0. Coperfusion of YGGFL with a combination of aminopeptidase and endopeptidase inhibitors, e.g., amastatin and YGG, is more effective in inhibiting hydrolysis since both metabolic pathways are inhibited. Leu-D(Ala)2-enkephalin, while showing enhanced stability against aminopeptidase hydrolysis, is hydrolyzed at the Gly-Phe bond by the endopeptidase. Its hydrolysis is not affected by pH changes or amastatin but is decreased by YGG. The YGGFL wall permeability was estimated and is not a limiting factor for oral absorption.

Polyethylene glycols (PEGs) 600, 1000, and 2000 were used to study the molecular weight permeability dependence in the rat nasal and gastrointestinal mucosa. Absorption of the PEGs was measured by following their urinary excretion over a 6-hr collection period. HPLC methods were used to separate and quantitate the individual oligomeric species present in the PEG samples. The permeabilities of both the gastrointestinal and the nasal mucosae exhibited similar molecular weight dependencies. The steepest absorption dependence for both mucosae occurs with the oligomers of PEG 600, where the extent of absorption decreases from approximately 60% to near 30% over a molecular weight range of less than 300 daltons. Differences in the absorption characteristics between the two sites appear in the molecular weight range spanned by PEG 1000. For these oligomers, the mean absorption from the nasal cavity is approximately 14%, while that from the gastrointestinal tract is only 9%. For PEG 2000, mean absorption decreases to 4% following intranasal application and below 2% following gastrointestinal administration. Within the PEG 1000 and 2000 samples, however, very little molecular weight dependency is seen among the oligomers. In the range studied, a distinct molecular weight cutoff was not apparent at either site.

The intestinal uptake mechanism of the purine analogue, acyclovir, was investigated in rat jejunum using in vitro and in situ methods. The pyrimidine, uracil, was used as a reference compound for carrier-mediated transport, while the purine analogue, caffeine, served as the reference compound for passive diffusion. With the in vitro intestinal ring method, acyclovir uptake was linear in the concentration range 0.01–5 mM. No significant competition for uptake was observed with uracil, 6-mercaptopurine, hypoxanthine, caffeine, or adenine. In addition, use of 2,4-dinitrophenol (DNP), ouabain, or K+ substituted buffer did not reduce the rate of acyclovir uptake. The in situ single-pass perfusion method yielded a wall permeability of ∼0.2, which did not vary consistently with increasing concentration. Coperfusion of acyclovir with DNP did not decrease the wall permeability. None of the data provided evidence of a carrier-mediated transport system, and it was concluded that the uptake mechanism of acyclovir in the rat jejunum is predominantly via passive diffusion.

We recommend that regulatory agencies add the extent of drug metabolism (i.e., >or=90% metabolized) as an alternate method in defining Class 1 marketed drugs suitable for a waiver of in vivo studies of bioequivalence. That is, >or=90% metabolized is an additional methodology that may be substituted for >or=90% absorbed. We propose that the following criteria be used to define>or=90% metabolized for marketed drugs: Following a single oral dose to humans, administered at the highest dose strength, mass balance of the Phase 1 oxidative and Phase 2 conjugative drug metabolites in the urine and feces, measured either as unlabeled, radioactive labeled or nonradioactive labeled substances, account for >or=90% of the drug dosed. This is the strictest definition for a waiver based on metabolism. For an orally administered drug to be >or=90% metabolized by Phase 1 oxidative and Phase 2 conjugative processes, it is obvious that the drug must be absorbed. This proposal, which strictly conforms to the present>or=90% criteria, is a suggested modification to facilitate a number of marketed drugs being appropriately assigned to Class 1.

PURPOSE: The aims of the study are to develop and evaluate an in vitro rat intestine segmental perfusion model for the prediction of the oral fraction absorbed of compounds and to assess the ability of the model to study intestinal metabolism. METHODS: The system consisted of a perfusion cell with a rat intestinal segment and three perfusion circulations (donor, receiver, and rinsing circulation). Lucifer yellow (LY) was applied as internal standard together with test compounds in the donor circulation. To validate the model, the permeability of eight noncongeneric passively absorbed drugs was determined. Intestinal N-demethylation of verapamil into norverapamil was followed in the donor and receiver circulations by high-performance liquid chromatography analysis. RESULTS: The in vitro model allowed ranking of the tested compounds according to their in vivo absorption potential. The Spearman's correlation coefficient between the oral fraction absorbed in humans and the ratio of permeation coefficient of test compound to the permeation coefficient of LY within the same experiment was 0.98 (P < 0.01). Moreover, intestinal N-demethylation of verapamil, its permeation, and the permeation of its metabolite norverapamil could be assessed in parallel. CONCLUSIONS: Up to six permeation kinetics can be obtained per rat, and the method has shown to be a valuable tool to estimate human oral absorption.

Based on a simple tube model for drug absorption, the key parameters controlling drug absorption are shown to be the dimensionless effective permeability, P eff *, and the Graetz number, Gz, when metabolism or solubility/dissolution is not rate controlling. Estimating the Graetz number in humans and assuming that P aq * is not rate controlling gives the following equation for fraction dose absorbed: F = 1− e −2 P*w. The correlation between fraction dose absorbed in humans and P w * determined from steady-state perfused rat intestinal segments gives an excellent correlation. It is of particular significance that the correlation includes drugs that are absorbed by passive and carrier-mediated processes. This indicates that P w * is one of the key variables controlling oral drug absorption and that the correlation may be useful for estimating oral drug absorption in humans regardless of the mechanism of absorption.

Intestinal permeabilities of five dipeptidyl derivatives of L-alpha-methyldopa (I) were studied by an in situ intestinal perfusion method. The dipeptides displayed a significant increase in their permeabilities compared to L-alpha-methyldopa. The increases ranged from 4 to 20 times. These results suggest that the peptide transport system is less structurally specific than the amino acid transport systems and can be used as an absorption pathway for peptide analogues. The kinetic advantage demonstrated by the dipeptide, L-alpha-methyldopa-L-phenylalanine, over the amino acid analogue, L-alpha-methyldopa, suggests that the peptide carrier would be a possible route for improving the intestinal absorption of pharmacologically active amino acid analogues. Furthermore, the preliminary results of in vitro hydrolysis studies of selected dipeptidyl derivatives indicate that the peptide carrier system could be used as a base for a prodrug strategy.

The intestinal absorption mechanism of two ACE inhibitor prodrugs, enalapril and fosinopril, was investigated in rats using a single-pass perfusion method. A modified boundary layer solution was applied to determine the apparent intestinal wall permeability. The prodrug enalapril is well absorbed from rat jejunum, whereas the parent drug, enalaprilat, is poorly absorbed. The permeability of enalapril is concentration dependent and is decreased by the dipeptide Tyr-Gly and by cephradine but not by the amino acids L-leucine or L-phenylalanine, indicating a nonpassive absorption mechanism via the small peptide carrier-mediated transport system. In contrast, fosinopril is readily absorbed by a concentration-independent mechanism without the involvement of the peptide carrier.

The intestinal uptake of phenytoin was studied as a function of concentration, intestinal region, coperfused glucose, and calcium chloride in rat intestinal perfusions and everted intestinal rings. Steady-state intestinal membrane permeabilities were obtained in an in situ perfusion system and initial rates of intestinal tissue uptake were obtained in an in vitro everted ring system as rate of absorption parameters. Steady-state membrane permeabilities were independent of phenytoin perfusion concentration and decreased from duodenum to ileum. Coperfusion of glucose increased, and high calcium chloride concentrations decreased phenytoin permeabilities. While phenytoin uptake in the in vitro ring system was also concentration-independent and depressed by high calcium concentrations, regional variations and glucose enhancement were not observed. Thus, drug–nutrient interactions involved in intestinal absorption from phenytoin solutions are a function of the isolation procedure.

Purpose A versatile methodology is demonstrated for improving dissolution kinetics, gastrointestinal (GI) absorption, and bioavailability of protein kinase inhibitors (PKIs). Methods The approach is based on nanoparticle precipitation by sub- or supercritical CO2 together with a matrix-forming polymer, incorporating surfactants either during or after nanoparticle formation. Notably, striking synergistic effects between hybrid PKI/polymer nanoparticles and surfactant added after particle formation is investigated. Results The hybrid nanoparticles, consisting of amorphous PKI embedded in a polymer matrix (also after 12 months), display dramatically increased release rate of nilotinib in both simulated gastric fluid and simulated intestinal fluid, particularly when surfactants are present on the hybrid nanoparticle surface. Similar results indicated flexibility of the approach regarding polymer identity, drug load, and choice of surfactant. The translation of the increased dissolution rate found in vitro into improved GI absorption and bioavalilability in vivo was demonstrated for male beagle dogs, where a 730% increase in the AUC0–24h was observed compared to the benchmark formulation. Finally, the generality of the formulation approach taken was demonstrated for a range of PKIs. Conclusions Hybrid nanoparticles combined with surfactant represent a promising approach for improving PKI dissolution rate, providing increased GI absorption and bioavailability following oral administration.

Purpose Medium chain fatty acid salts promote absorption by increasing paracellular permeability of the intestinal epithelium. Novel oily suspension (OS) formulation disperses a powder containing sodium caprylate and macromolecules such as octreotide or fluorescent dextran (FD). Formulation safety, macromolecule absorption and pharmacokinetic (PK)/pharmacodynamic (PD) were evaluated. Methods Octreotide/OS toxicity was evaluated in monkeys following 9 months of daily oral enteric-coated capsule administration. The OS permeation effect was also assessed in rats, using FD/OS and octreotide/OS preparations. Octreotide/OS effects on circulating growth hormone (GH) levels were also measured. Results Safety assessment of octreotide/OS in monkeys after 9 months showed minor drug-related findings, comparable to the injectable octreotide. Octreotide exposure levels were similar across the treatment periods. In rats, OS facilitated FD permeation up to 70 kDa in a reversible, spatial and dose-dependent manner, independent of the intestinal dosing site. Following OS administration, the staining pattern of the tight-junction protein, ZO-1, changed transiently, and a paracellular penetration marker, LC-biotin, permeated between adjacent epithelial cells. Enteral octreotide/OS absorption was dose-dependent and suppressed rat GH levels. Conclusions Oral octreotide/OS dosing was shown to be safe in monkeys. OS enhances intestinal absorption of active octreotide, likely by transient alteration of the tight junction protein complex.