Microclimate pH Research Articles

Implications of changes in physical state of drugs in poly(lactide-co-glycolide) matrices upon exposure to moisture and release medium

The objective of this study was to investigate the influence of the drug physical state (dissolved or dispersed) in poly(lactide-co-glycolide) (PLGA) on drug release and potential changes in physical state upon contact with moisture or release medium. PLGA matrices containing risperidone, ibuprofen or dexamethasone were prepared as films by solvent casting or as implants by hot melt extrusion. The physical state of the drugs in the polymer was examined by DSC and polarized light microscopy and the in-vitro drug release was compared for different drug physical states.Incubation of drug-PLGA matrices at 40 °C/75% RH (relative humidity) resulted in a decrease in microclimate pH in the PLGA matrices upon moisture uptake, dissolving initially dispersed risperidone because of an increased drug solubility in the matrix. In contrast, initially dissolved ibuprofen precipitated in the matrix because of a decrease in drug solubility at lower pH values and a fast water uptake. Dexamethasone did not show any change of physical state under the same conditions; the drug is barely soluble in the dry matrix and its solubility is not pH dependent. Risperidone was released faster from PLGA matrices with dissolved versus dispersed drug, substantially reducing the lag time. The crystallization process of ibuprofen could be inhibited by the use of a higher molecular weight PLGA grade. The inhibition resulted in a faster release from solid solution than from the corresponding solid dispersion. However, ibuprofen, which was deliberately precipitated on the surface of a PLGA matrix by incubation at 40 °C/75% RH, was able to bridge the lag time and led to a linearization of a typical PLGA release profile.In conclusion, a change in the physical state of the drug in PLGA affects the drug release profile. A change can be induced by incubation at 40 °C/75% RH. Furthermore, moisture contact and storage conditions of PLGA drug delivery systems should be carefully considered during handling to avoid undesired drug release changes.

Mapping in vivo microclimate pH distribution in exenatide-encapsulated PLGA microspheres.

The pH inside the aqueous pores of poly(lactic-co-glycolic acid) (PLGA) microspheres, often termed microclimate pH (μpH), has been widely evaluated in vitro and shown to commonly be deleterious to pH-labile encapsulated drug molecules. However, whether the in vitro μpH is representative of the actual in vivo values has long been remained a largely unresolved issue. Herein we quantitatively mapped, for the first time, the in vivo μpH distribution kinetics inside degrading PLGA microspheres by combining two previously validated techniques, a cage implant system and confocal laser scanning microscopy. PLGA (50/50, Mw=24-38kDa, acid-end capped and ester-capped) microsphere formulations with and without encapsulating exenatide, a pH-labile peptide that is known to be unstable when pH>4.5, were administered to rats subcutaneously via cage implants for up to 6weeks. The results were compared with two different in vitro conditions. Strikingly, the in vivo μpH developed similarly to the low microsphere concentration in vitro condition with 1-μm nylon bags but very different from conventional high microsphere concentration sample-and-separate conditions. Improved maintenance of stable external pH in the release media for the former condition may have been one important factor. Stability of exenatide remaining inside microspheres was evaluated by mass spectrometry and found that it was steadily degraded primarily via pH-dependent acylation with a trend that slightly paralleled the changes in μpH. This methodology may be useful to elucidate pH-triggered instability of PLGA encapsulated drugs in vivo and for improving in vivo-predictive in vitro conditions for assessing general PLGA microsphere performance.

Slc26a3 deletion alters pH-microclimate, mucin biosynthesis, microbiome composition and increases the TNFα expression in murine colon.

SLC26A3 (DRA) mediates the absorption of luminal Cl- in exchange for HCO3- in the distal intestine. Its expression is lost in congenital chloride diarrhoea (CLD) and strongly decreased in the presence of intestinal inflammation. To characterize the consequences of a loss of Slc26a3 beyond disturbed electrolyte transport, colonic mucus synthesis, surface accumulation and composition, pH microclimate, microbiome composition and development of inflammation was studied in slc26a3-/- mice. The epithelial surface pH microclimate and the surface mucus accumulation in vivo was assessed by two photon microscopy in exteriorized mid colon of anaesthetized slc26a3-/- and wt littermates. Mucus synthesis, composition and inflammatory markers were studied by qPCR and immunohistochemistry and microbiome composition by 16S rRNA sequencing. Colonic pH microclimate was significantly more acidic in slc26a3-/- and to a lesser extent in cftr-/- than in wt mice. Goblet cell thecae per crypt were decreased in slc26a3-/- and increased in cftr-/- colon. Mucus accumulation in vivo was reduced, but much less so than in cftr-/- colon, which is possibly related to the different colonic fluid balance. Slc26a3-/- colonic luminal microbiome displayed strong decrease in diversity. These alterations preceded and maybe causally related to increased mucosal TNFα mRNA expression levels and leucocyte infiltration in the mid-distal colon of slc26a3-/- but not of cftr-/- mice. These findings may explain the strong increase in the susceptibility of slc26a3-/- mice to DSS damage, and offer insight into the mechanisms leading to an increased incidence of intestinal inflammation in CLD patients.

Slc26a3 (DRA) Deficient Mice Display an Acidic Colonic pH‐microclimate, Develop a Strongly Altered Microbiome and Colonic Inflammation

BackgroundThe anion transporter Slc26a3 (DRA‐Down Regulated in Adenoma) is localized on the apical membrane of the colonic mucosa and is functionally involved in the absorption of luminal chloride (Cl−) in exchange for bicarbonate ions (HCO3−). Mutations in the dra gene result in congenital chloride diarrhoea (CLD) which is characterized by secretory diarrhoea, loss of Cl− in the stool, dehydration and metabolic alkalosis. These patients also have a higher risk of incidence of acute as well chronic intestinal inflammation (Wedenoja et al. Hum. Mut 2011). We previously reported low colonic HCO3− output rates and an increased susceptibility to Dextran Sodium Sulfate (DSS) damage in slc26a3−/− mice (Xiao, F. et al. Acta Phys. 2015).AimThe present study was undertaken to explore whether the slc26a3−/− mice develop spontaneous intestinal inflammation, and whether an altered microbiome composition may be the underlying molecular mechanism for inflammation.MethodsColonic surface pH was measured in vivo by two photon microscopy, the inflammatory state of the mucosa by Quantitative PCR (qPCR) and immunohistochemistry, the intestinal microbiome by 16S rRNA sequencing, and the effect of the microbiome of the slc26a3−/− colon on the development of inflammation in germ‐free mice by fecal crossing experiments.ResultsThe slc26a3−/− mice developed distal colonic inflammation evidenced by an increase in proinflammatory cytokine expression and the number of inflammatory cells in the colonic mucosa/submucosa at the time of sacrifice. The slc26a3−/− microbiome was significantly different from that of cohoused wild type (WT) littermates, with a strongly decreased diversity, and an increased percentage of several proinflammatory species. Transmission of this dysbiotic microbiome into germ free mice did not lead to a development of inflammation in the recipient mice. Instead, the species richness in the germ free mice gavaged with slc26a3−/− microbiome had increased, and the percentage of proinflammatory species decreased, in comparison to its donor microbiome.ConclusionsSlc26a3−/− mice display a very low colonic pH microclimate and develop spontaneous intestinal inflammation. The microbiome of slc26a3−/− colon is “dysbiotic” with strongly reduced diversity. However, transmission of this microbiome into germ‐free mice does not result in inflammation, but does result in microbiome normalization, suggesting that the reduced pH microclimate and reduced colonic transit time is necessary for the development of intestinal dysbiosis.Support or Funding InformationVolkswagen Foundation VW Vorab, DFG project SE460/17‐1 and 19‐1 and SFB621/C9This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A pro-angiogenic degradable Mg-poly(lactic-co-glycolic acid) implant combined with rhbFGF in a rat limb ischemia model.

Magnesium (Mg)-based implant has been already used in patients with critical limb ischemia. Site-specific controlled release of recombinant human basic fibroblast growth factor (rhbFGF), has become a promising approach to improve peripheral vascular disease. We report here on a novel combination implant composed of spiral magnesium and a coating made using poly(lactic-co-glycolic acid) (PLGA) with encapsulated rhbFGF (Mg-PLGA-rhbFGF). The preparation method does not involve any complex processes and results in a high encapsulation efficiency (approximately 100%). The degradation of metal Mg raise the microclimate pH in the PLGA polymer, which could well preserve the bioactivity of rhbFGF incorporated in the implant. Mg-PLGA-based, sustained local delivery of rhbFGF promotes post-ischemic angiogenesis and blood flow recovery in rat limb ischemic model. This work marks the first report for controlled release of rhbFGF in combination with metal Mg, and suggests potential therapeutic usefulness of Mg-PLGA-rhbFGF for tissue ischemia.

Encapsulation of Pancreatic Lipase in Hydrogel Beads with Self-Regulating Internal pH Microenvironments: Retention of Lipase Activity after Exposure to Gastric Conditions.

Oral delivery of lipase is important for individuals with exocrine pancreatic insufficiency; however, lipase loses activity when exposed to the highly acidic gastric environment. In this study, pancreatic lipase was encapsulated in hydrogel beads fabricated from alginate (gel former), calcium chloride (cross-linker), and magnesium hydroxide (buffer). Fluorescence confocal microscopy imaging was used to map the pH microclimate within the hydrogel beads under simulated gastrointestinal tract (GIT) conditions. The pH within buffer-free beads rapidly decreased when they moved from mouth (pH 6.3) to stomach (pH <4), leading to a loss of lipase activity in the small intestine. Conversely, the pH inside buffer-loaded beads remained close to neutral in the mouth (pH 7.33) and stomach (pH 7.39), leading to retention of lipase activity in the small intestine, as shown by pH-stat analysis of lipid digestion. The presence of the encapsulated buffer also reduced bead shrinkage under gastric conditions.

A biomimetic approach to active self-microencapsulation of proteins in PLGA

A biomimetic approach to organic solvent-free microencapsulation of proteins based on the self-healing capacity of poly (dl)-lactic-co-glycolic acid (PLGA) microspheres containing glycosaminoglycan-like biopolymers (BPs), was examined. To screen BPs, aqueous solutions of BP [high molecular weight dextran sulfate (HDS), low molecular weight dextran sulfate (LDS), chondroitin sulfate (CS), heparin (HP), hyaluronic acid (HA), chitosan (CH)] and model protein lysozyme (LYZ) were combined in different molar and mass ratios, at 37°C and pH7. The BP-PLGA microspheres (20–63μm) were prepared by a double water–oil–water emulsion method with a range of BP content, and trehalose and MgCO3 to control microclimate pH and to create percolating pores for protein. Biomimetic active self-encapsulation (ASE) of proteins [LYZ, vascular endothelial growth factor165 (VEGF) and fibroblast growth factor (FgF-20)] was accomplished by incubating blank BP-PLGA microspheres in low concentration protein solutions at ~24°C, for 48h. Pore closure was induced at 42.5°C under mild agitation for 42h. Formulation parameters of BP-PLGA microspheres and loading conditions were studied to optimize protein loading and subsequent release. LDS and HP were found to bind >95% LYZ at BP:LYZ>0.125 w/w, whereas HDS and CS bound >80% LYZ at BP:LYZ of 0.25–1 and <0.33, respectively. HA-PLGA microspheres were found to be not ideal for obtaining high protein loading (>2% w/w of LYZ). Sulfated BP-PLGA microspheres were capable of loading LYZ (~2–7% w/w), VEGF (~4% w/w), and FgF-20 (~2% w/w) with high efficiency. Protein loading was found to be dependent on the loading solution concentration, with higher protein loading obtained at higher loading solution concentration within the range investigated. Loading also increased with content of sulfated BP in microspheres. Release kinetics of proteins was evaluated in-vitro with complete release media replacement. Rate and extent of release were found to depend upon volume of release (with non-sink conditions observed <5ml release volume for ~18mg loaded BP-PLGA microspheres), ionic strength of release media and loading solution concentration. HDS-PLGA formulations were identified as having ideal loading and release characteristics. These optimal microspheres released ~73–80% of the encapsulated LYZ over 60days, with >90% of protein being enzymatically active. Nearly 72% of immunoreactive VEGF was similarly released over 42days, without significant losses in heparin binding affinity in the release medium.

Loss of carbonic anhydrase IX expression impairs gastric mucosal defence against luminal acid (1109.13)

Background: The exoenzyme carbonic anhydrase IX is strongly expressed in tumours. In normal tissues, CA IX is expressed during embryogenesis but downregulated postnatally, except in the stomach. Aim and Methods: We studied the cellular differentiation pattern and acid secretory capacity in gastric mucosa from CAIX KO and WT mice at age 8 days to 1,3,6,9,12 months. Surface pH and mucus layer thickness, as well as the ability of the surface epithelial cells to withstand a luminal acid load was assessed in young adult CAIX KO and WT mice by in vivo two photon microscopy. Results: CAIX KO gastric mucosa developed a unique phenotype with a gradual expansion of the surface cell zone, and regression of the parietal cell zone to the lower gland area. Concomitantly, the acid secretory capacity, which was normal at young age, declined. HCO3‐ output into the lumen and mucus layer thickness were not significantly different between CAIX KO and WT. However, the ability of the surface cells to withstand a luminal acid load, as well as to maintain a more alkaline surface pH than the luminal pH, was significantly impaired in CAIX KO compared to WT stomach. Conclusion: We speculate that the CAIX at the basolateral membrane of the gastric surface cells rapidly converts extruded protons with blood‐borne HCO3‐ to CO2 and thus augments interstitial buffer capacity, surface cell pHi regulation, and maintenance of the pH microclimate in the mucus layer.Grant Funding Source: Supported by DFG Se 13‐4 and 9‐6 to Ursula Seidler

Non-invasive in vivo characterization of microclimate pH inside in situ forming PLGA implants using multispectral fluorescence imaging

The pH inside drug delivery systems influences directly the physical and chemical behavior of its ingredients specifically their solubility and stability. These properties significantly affect the release performance of the formulations as well as the pharmacological effect. Therefore, the determination of the microclimate pH (μpH) inside the drug delivery systems is of great importance and interest.Implants are considered to be attractive parenteral drug delivery systems used for the long-term application of drugs and of peptides. Poly(lactide-co-glycolide) (PLGA) is the most frequently used and extensively researched polymer for implant preparation. However it is known that the microclimate pH (μpH) within the PLGA implants can also drop dramatically. This pH drop can cause peptide or protein instabilities as well as drug insolubilities and further decomposition. Although the internal pH behavior of PLGA implants and microparticles has been studied in vitro, no data about the μpH behavior in in situ forming implants has yet been described. This is due to the fact, that there is no reliable non-invasive method available to measure directly and continuously the pH in vivo. Therefore, the question if in vitro measurement results are potentially assignable remains unclear.In this study, the μpH of in situ forming PLGA implants were mapped in vitro, in vivo, and ex vivo. A non-invasive in vivo pH measurement method using the multispectral Maestro fluorescence imaging system was developed. The in vivo experiments performed, not only enabled the authors of this article to make certain assumptions about μpH behavior but also emphasized certain expectations regarding the solvent replacement in the core area of the implant as well as the release profile of hydrophilic substances.The experiments emphasized the broad application range of the fluorescence imaging technique for non-invasive monitoring of μpH values in drug delivery systems in vivo.

Comparative Studies on the Influences of Primary Emulsion Preparation on Properties of Uniform-Sized Exenatide-Loaded PLGA Microspheres

It is well known that primary emulsion (W1/O) preparation process (by ultrasonication or homogenization) plays an important role in the properties of drug-loaded microspheres, such as encapsulation efficiency, release behavior and pharmacodynamics. However, its involved mechanism has not been intensively and systematically studied, partly because that broad size distribution of the resultant particles prepared by conventional preparation can greatly disturb the analysis and reliability of the results. Here, we focused on the relevant studies. In order to eliminate the disturbance caused by broad size distribution, uniform-sized exenatide-loaded poly(DL-lactic-co-glycolic acid) (PLGA) microspheres were prepared by Shirasu Porous Glass (SPG) premix membrane emulsification. The properties of microspheres whose W1/O was formed by ultrasonication (UMS) and homogenization (HMS) were compared including in vitro release, pharmacology and so forth. HMS exhibited fast release rate and hyperglycemic efficacy within first 14 days, but declined afterwards. Comparatively, UMS showed slower polymer degradation, more acidic microclimate pH (μpH) in vitro, and stable drug release with sustained efficacy during 1 month in vivo. HMS was desirable for the 2-week-sustained release in vivo, while UMS was more appropriate for the longer time release (about 1 month). These comparative researches can provide guidance for emulsion-microsphere preparation routs in pharmaceutics.

Protein-loaded comb-shape copolymer-based pH-responsive nanoparticles to improve the stability of proteins

Extensive studies have shown that poly(lactide-co-glycolide) (PLGA) and its derivatives are excellent biomaterials for drug delivery systems. However, their use in protein delivery is limited due to their harsh internal pH environment. In this study, we designed an amphiphilic comb-shaped copolymer, lactobionic acid-poly(ethylene oxide)-ε-polylysine-g-PLGA (LPEP), for protein delivery. Depending on the ratio of PLGA contents, the internal pH of LPEP nanoparticles (NPs) was adjustable, which was verified by a quantitative ratiometric method. The protein delivery system was able to meet the needs of the appropriate pH microclimate for the integrity of proteins. Circular dichroism spectroscopy revealed that the protein released from LPEP NPs was more stable than that released from PLGA NPs. In addition, cytotoxicity studies involving HL-7702 cells and acute toxicity experiments in Kunming mice showed that the NPs had good biocompatibility. In vivo experiments showed the circulation time and tissue distribution of LPEP NPs. Taken together, our results suggest that the LPEP NP system is a promising candidate for protein delivery.

The microclimate pH in poly(d,l-lactide-co-hydroxymethyl glycolide) microspheres during biodegradation

The microclimate pH (μpH) in biodegradable polymers, such as poly(d,l-lactic-co-glycolic acid) (PLGA) 50/50, commonly falls to deleterious acidic levels during biodegradation, resulting in instability of encapsulated acid-labile molecules. The μpH distribution in microspheres of a more hydrophilic polyester, poly(d,l-lactide-co-hydroxymethyl glycolide) (PLHMGA), was measured and compared to that in PLGA 50/50 of similar molecular weight and degradation time scales. pH mapping in the polymers was performed after incubation under physiological conditions by using a previously validated ratiometric method employing confocal laser scanning microscopy (CLSM). Confocal μpH maps revealed that PLHMGA microspheres, regardless of copolymer composition, developed a far less acidic μpH during 4 weeks of incubation compared with microspheres from PLGA. A pH-independent fluorescent probe marker of polymer matrix diffusion of μpH-controlling water-soluble acid degradation products, bodipy, was observed by CLSM to diffuse ∼3–7 fold more rapidly in PLHMGA compared to PLGA microspheres, consistent with much more rapid release of acids observed from the hydrophilic polymer during bioerosion. Hence, PLHMGA microspheres are less susceptible to acidification during degradation as compared to similar PLGA formulations, and therefore, PLHMGA may be more suitable to deliver acid labile molecules such as proteins.

Mapping Microclimate pH Distribution inside Protein-Encapsulated PLGA Microspheres Using Confocal Laser Scanning Microscopy

The pH in the aqueous pores of poly(lactide-co-glycolide) (PLGA) matrix, also referred to as microclimate pH (μpH), is often uncontrolled, ranging from highly acidic to neutral pH range. The μpH distribution inside protein-encapsulated PLGA microspheres was quantitatively evaluated using confocal laser scanning microscopy. The fluorescent response of Lysosensor yellow/blue dextran used to map μpH in PLGA was influenced by the presence of encapsulated protein. The nonprotonated form of pyridyl group on the fluorescence probe at neutral pH was responsible for the interference, which was dependent on the type and concentration of protein. A method for correction of this interference based on estimating protein concentration inside the microspheres was established and validated. After correction of the influence, the μpH distribution kinetics inside microspheres was evaluated for different PLGA 50/50 microsphere formulations under physiological conditions for 4 weeks. Generally, the μpH acidity increased with the progression of incubation time. The coincorporation of poorly soluble base, magnesium carbonate, in the microspheres prolonged the appearance of detectable acidity for up to 3 weeks. Co-addition of an acetate buffer was able to control the μpH over a slightly acidic range (around pH 4.7) after two week incubation. Microspheres prepared from a lower polymer concentration exhibited a higher μpH, likely owing to reduced diffusional resistance to acidic degradation products. The stability of protein was enhanced by addition of MgCO(3), acetate buffer, or by reduced polymer concentration in the preparation, as evidenced by more soluble protein recovered after incubation. Hence, the μpH imaging technique developed can be employed in the future for optimization of formulation strategies for controlling μpH and stabilizing encapsulated proteins.

Concentration-dependent exsorption of quinidine in the rat intestine.

During intravenous infusion, the luminal concentration of quinidine was higher than the plasma concentration. The intestinal clearance (CLi) of the drug was measured by dividing the rate of appearance of the drug in the intestinal luminal perfusate by the plasma concentration. The CLi of quinidine was therefore much higher than the rate of luminal perfusion. Over the infusion dose range of 0.1-2 mg h-1, the CLi of quinidine decreased with increasing plasma concentration of quinidine. Adding quinidine into the luminal perfusate had little effect on the CLi of quinidine. Co-administration of quinidine with other agents intravenously did not alter the CLi of salicylic acid and urea, while the same treatment decreased the CLi of theophylline and S-disopyramide. In-vitro experiments on brush-border membrane vesicles showed that quinidine decreased the rate of Na+ uptake and H+ efflux. The inhibition was significant at quinidine concentrations above 20 microM. Quinidine was a more potent inhibitor than amiloride. At quinidine infusion rates less than 2 mg h-1, quinidine concentration in plasma or in the luminal perfusate was at the lower limit of the inhibitory concentration. Microclimate pH at the intestinal surface was also measured. At mid-jejunum, the microclimate pH increased 0.3 pH units by infusing 2 mg h-1 of quinidine, while the microclimate pH at most other measuring sites was not significantly altered by quinidine infusion. It was concluded that quinidine is exsorbed from blood into the intestinal lumen by a carrier-mediated pathway in addition to the passive diffusion. At high plasma concentration, quinidine exsorption becomes saturated.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of theophylline on the intestinal clearance of drugs in rats.

Intestinal exsorption of salicylic acid, urea and quinidine was measured during the perfusion of the rat intestinal lumen with Tyrode solution. The intestinal clearance (CLi) of the three compounds was measured by dividing the rate of appearance in the intestinal luminal perfusate by the plasma concentration of the compound. Co-administration of theophylline (0.2 mg h-1) with the test agents increased the CLi of salicylic acid, did not alter the CLi of urea, but decreased the CLi of quinidine. The effect of theophylline on the CLi of quinidine was enhanced with increasing dose. Theophylline was found to increase microclimate-pH at the intestinal surface, but the magnitude of delta pH alone could not explain the effect of theophylline on the CLi of quinidine. The data, together with previous observations, suggest that the intestinal exsorption of drugs was affected by the microclimate pH and by the unstirred water layer. Theophylline affects CLi of salicylic acid and quinidine partly by increasing the microclimate pH of the intestine. Theophylline may also affect quinidine CLi by inhibiting the carrier-mediated pathway.

Modulation of urea transport across sheep rumen epithelium in vitro by SCFA and CO2

Urea transport across the gastrointestinal tract involves transporters of the urea transporter-B group, the regulation of which is poorly understood. The classical stimulatory effect of CO(2) and the effect of short-chain fatty acids (SCFA) on the ruminal recycling of urea were investigated by using Ussing chamber and microelectrode techniques with isolated ruminal epithelium of sheep. The flux of urea was found to be phloretin sensitive and passive. At a luminal pH of 6.4, but not at 7.4, the addition of SCFA (40 mmol/l) or CO(2)/HCO3- (10% and 25 mmol/l) led to a fourfold increase in urea flux. The stepwise reduction of luminal pH in the presence of SCFA from 7.4 to 5.4 led to a bell-shaped modification of urea transport, with a maximum at pH 6.2. Lowering the pH in the absence of SCFA or CO(2) had no effect. Inhibition of Na(+)/H(+) exchange increased urea flux at pH 7.4, with a decrease being seen at pH 6.4. In experiments with double-barreled, pH-sensitive microelectrodes, we confirmed the presence of an apical pH microclimate and demonstrated the acidifying effects of SCFA on the underlying epithelium. We confirm that the permeability of the ruminal epithelium to urea involves a phloretin-sensitive pathway. We present clear evidence for the regulation of urea transport by strategies that alter intracellular pH, with permeability being highest after a moderate decrease. The well-known postprandial stimulation of urea transport to the rumen in vivo may involve acute pH-dependent effects of intraruminal SCFA and CO(2) on the function of existing urea transporters.

Membrane Permeability in the Gastrointestinal Tract: The Interplay between Microclimate pH and Transporters

Some examples of pH- and transporter-dependent permeability, determined in side-by-side diffusion cells, are summarized. We investigated the polarized transport in the mucosal-to-serosal direction of monocarboxylic acid-type drugs through the excised rat jejunal tissue and an artificial membrane. We established that, in vitro, these substances are most probably not transported by monocarboxylate transporter 1, but by passive pH-dependent transport. We also studied various influences on the permeability of fluorescein, a low permeability marker, through isolated rat intestinal segments, Caco-2 cell monolayers, and an artificial membrane. Polarized transport of fluorescein in the serosal-to-mucosal direction through the rat jejunum by multidrug resistance-associated protein was triggered by the addition of D-glucose to the mucosal side, while the pH-dependent increase of fluorescein influx is presumably the consequence of a monocarboxylate transporter and a member of the organic-anion transporting polypeptide family. With permeability experiments through the excised segments of rat small intestine, we ascertained that ciprofloxacin is a low-permeability drug and has higher and pH-dependent transport in the mucosal-to-serosal direction than in the opposite direction. We also established that neither the permeability of fluoroquinolones nor their solubility in different buffers was influenced by the interactions with metal cations.

Duodenal epithelial cells provide a stimulus package for the recovery from an acid load

It is widely appreciated that some of the harshest physiological conditions to which epithelial cells are exposed are found in the lumen of the upper gastrointestinal tract. The lumen-facing apical plasma membrane of these cells is regularly bathed in the ‘gemisch’ of chyme, which is comprised of HCl secreted by gastric parietal cells, hydrolytic enzymes secreted by gastric chief cells and pancreatic acinar cells, and foodstuffs. For digestion and absorption to continue, the luminal HCl must be largely neutralized to a pH that will enable the hydrolytic enzymes from the pancreas and solute transporters of intestinal epithelial cells to function at their optimum. This neutralization occurs via bicarbonate secreted into the mixture by epithelial cells of the duodenum and pancreas. Duodenal epithelial cells are also potentially directly exposed to this acidic chyme arriving from the stomach. Thus, bicarbonate secretion by these cells is critical for their own protection. While it has been recognized that part of the mechanism that protects the duodenum is comprised of an alkaline layer of fluid that is adjacent to the apical brush border membrane of duodenal epithelial cells and is in disequilibrium with the bulk luminal pH (Flemstrom & Kivilaakso, 1983), the mechanisms by which this alkaline zone is generated and regulated were largely unknown. In this issue of The Journal of Physiology, Mizumori and colleagues (Mizumori et al. 2009), using an in vivo duodenal perfusion loop, identify the key players at the apical membrane in rat duodenal epithelial cells that are involved in a feedback loop that regulates bicarbonate secretion in the duodenal response to an acid load. The work is based upon the hypothesis that an ecto-purinergic signalling system regulates this bicarbonate secretion, and the intriguing nature of this hypothesis appears to have been developed both from observations from the literature and from the authors’ unusual familiarity with duodenal physiology. Part of the novelty of their work is found in the collection of apical brush border membrane proteins that they hypothesize cooperate in this feedback loop. In addition to the more recently characterized proteins with defined physiological functions, such as apical membrane purinergic P2Y receptors and the cystic fibrosis transmembrane regulator (CFTR), some of the other proteins are ones that have been known for decades, such as intestinal alkaline phosphatase (IAP) and ecto-ATPase (Hietanen, 1973; Humphreys & Chou, 1979), which have well-characterized luminally oriented biochemical activities, but until now, no apparent physiological function. Using this duodenal perfusion loop, the authors were elegantly able to test directly this hypothesis to arrive at their model for regulated bicarbonate secretion by duodenal epithelial cells. They also benefitted significantly from the ability to use pharmacological probes for their proteins of interest. Their data have led them to propose that when bulk pH is closer to neutral, IAP activity is high. IAP degrades ATP released from duodenal epithelial cells, which in turn decreases signalling by P2Y receptors. Decreased signalling by P2Y receptors then results in decreased bicarbonate secretion. Decreased bicarbonate secretion thus results in a lower microclimate pH, thereby inhibiting IAP and completing the negative feedback loop. In the case of acid exposure, IAP activity is reduced and luminal ATP is increased. Signalling through P2Y receptors is increased and therefore bicarbonate secretion consequently increased. The acid load is neutralized, and the microclimate pH is increased, which then stimulates IAP activity. Again, the feedback loop is completed. So what about CFTR in this process? The authors speculate that CFTR may regulate bicarbonate secretion through regulating ATP release from epithelial cells because they observed that inhibition of CFTR inhibited ATP output by the duodenum. This observation is consistent with their earlier observation that the dysfunction of CFTR reduces bicarbonate secretion; however, interestingly, the duodenum is protected from ulcers in this case (Akiba et al. 2005). In this and previous work (Akiba et al. 2007), the authors have clearly begun to characterize at the molecular level the long-standing question of the regulation of bicarbonate secretion by the duodenum. Of course, as the authors acknowledge, there is more work to be done. One major question is the source of the ATP released from the duodenal epithelial cells: is it released via a membrane transporter or via exocytosis, and what is the precise relationship between CFTR and bicarbonate secretion in duodenal cells as well as in other epithelial cells? Despite these questions, at least this stimulus package is one that has led to recovery from toxic conditions.

Intestinal alkaline phosphatase regulates protective surface microclimate pH in rat duodenum

Regulation of localized extracellular pH (pH(o)) maintains normal organ function. An alkaline microclimate overlying the duodenal enterocyte brush border protects the mucosa from luminal acid. We hypothesized that intestinal alkaline phosphatase (IAP) regulates pH(o) due to pH-sensitive ATP hydrolysis as part of an ecto-purinergic pH regulatory system, comprised of cell-surface P2Y receptors and ATP-stimulated duodenal bicarbonate secretion (DBS). To test this hypothesis, we measured DBS in a perfused rat duodenal loop, examining the effect of the competitive alkaline phosphatase inhibitor glycerol phosphate (GP), the ecto-nucleoside triphosphate diphosphohydrolase inhibitor ARL67156, and exogenous nucleotides or P2 receptor agonists on DBS. Furthermore, we measured perfusate ATP concentration with a luciferin-luciferase bioassay. IAP inhibition increased DBS and luminal ATP output. Increased luminal ATP output was partially CFTR dependent, but was not due to cellular injury. Immunofluorescence localized the P2Y(1) receptor to the brush border membrane of duodenal villi. The P2Y(1) agonist 2-methylthio-ADP increased DBS, whereas the P2Y(1) antagonist MRS2179 reduced ATP- or GP-induced DBS. Acid perfusion augmented DBS and ATP release, further enhanced by the IAP inhibitor l-cysteine, and reduced by the exogenous ATPase apyrase. Furthermore, MRS2179 or the highly selective P2Y(1) antagonist MRS2500 co-perfused with acid induced epithelial injury, suggesting that IAP/ATP/P2Y signalling protects the mucosa from acid injury. Increased DBS augments IAP activity presumably by raising pH(o), increasing the rate of ATP degradation, decreasing ATP-mediated DBS, forming a negative feedback loop. The duodenal epithelial brush border IAP-P2Y-HCO(3-) surface microclimate pH regulatory system effectively protects the mucosa from acid injury.

Acidic microclimate pH distribution in PLGA microspheres monitored by confocal laser scanning microscopy.

The acidic microclimate pH (micropH) distribution inside poly(lactic-co-glycolic acid) (PLGA) microspheres was monitored quantitatively as a function of several formulation variables. A ratiometric method by confocal laser scanning microscopy with Lysosensor yellow/blue dextran was adapted from those previously reported, and micropH distribution kinetics inside microspheres was examined during incubation under physiologic conditions for 4 weeks. Effects of PLGA molecular weight (MW) and lactic/glycolic acid ratio, microspheres size and preparation method, and polymer blending with poly(ethylene glycol) (PEG) were evaluated. micropH kinetics was accurately sensed over a broadly acidic range (2.8 < micropH < 5.8) and was more acidic and variable inside PLGA with lower MW and lactic/glycolic acid ratio. Lower micropH was found in larger microspheres of lower MW polymers, but size effects for lactic-rich polymers were insignificant during 4 weeks. Microspheres prepared by the oil-in-oil emulsion method were less acidic than those prepared by double emulsion, and blending PLGA 50/50 with 20% PEG increased micropH significantly (micropH > 5 throughout incubation). Coupling this method with that previously developed (SNARF-1 dextran for micropH 5.8-8.0) should provide microclimate pH mapping over the entire useful pH range (2.8-8.0) for optimization of PLGA delivery of pH-sensitive bioactive substances.