Metabolomics Insights of the Immunomodulatory Activities of Phlorizin and Phloretin on Human THP-1 Macrophages.

Dihydrochalcones, phlorizin (PZ) and its aglycone phloretin (PT), have evidenced immunomodulatory effects through several mechanisms. However, the differential metabolic signatures that lead to these properties are largely unknown. Since macrophages play an important role in the immune response, our study aimed to characterise human THP-1 macrophages under PZ and PT exposure. A multiplatform-based untargeted metabolomics approach was used to reveal metabolites associated with the anti-inflammatory mechanisms triggered by the dihydrochalcones in LPS-stimulated macrophages, for the first time. Results showed differential phenotypic response in macrophages for all treatments. Dihydrochalcone treatment in LPS-stimulated macrophages mimics the response under normal conditions, suggesting inhibition of LPS response. Antagonistic effects of dihydrochalcones against LPS was mainly observed in glycerophospholipid and sphingolipid metabolism besides promoting amino acid biosynthesis. Moreover, PT showed greater metabolic activity than PZ. Overall, the findings of this study yielded knowledge about the mechanisms of action PZ and PT at metabolic level in modulating inflammatory response in human cells.

Comparative oral and intravenous pharmacokinetics of phlorizin in rats having type 2 diabetes and in normal rats based on phase II metabolism.

Phlorizin (PHZ), a type of dihydrochalcone widely found in Rosaceae such as apples, is the first compound discovered as a sodium-glucose cotransporter (SGLT) inhibitor. It has been confirmed to improve the symptoms of diabetes and diabetic complications effectively. Like other flavonoids, the bioavailability challenge of PHZ is the wide phase I and II metabolism in the digestive tract. In this study, we investigated the pharmacokinetics and contribution of phase II metabolism after the oral and intravenous administrations of PHZ in rats having type 2 diabetes (T2D) and in normal rats. The phase II metabolism characteristics of PHZ were investigated by treating plasma samples with ß-glucuronidase/sulfatase. The contribution ratio of phase II metabolism of PHZ ranged from 41.9% to 69.0% after intravenous injection with three doses of PHZ in normal rats. Compared with the observations for normal rats, AUC0-t and Cmax of PHZ significantly increased and T1/2 of PHZ significantly decreased in T2D rats. PHZ was converted into phloretin (PHT) through an enzyme-catalyzed hydrolysis reaction, and PHT was further transformed into conjugates with glycose after both oral and intravenous administrations. Moreover, it was found that the bioavailability of PHZ was about 5% in T2D rats, which was significantly higher than that in normal rats (0%). In conclusion, compared with the observations for normal rats, the pharmacokinetic characteristics of PHZ significantly changed in T2D rats through oral and intravenous administrations. The bioavailability of PHZ significantly increased in T2D rats. Besides, the phase II metabolites of PHT were the major existing forms in blood after oral and intravenous administrations. Our results indicated that the phase II metabolism characteristics of PHZ should be considered when PHZ is applied for the treatment of diabetes as a drug or functional food.

Selective SGLT2 inhibition by tofogliflozin reduces renal glucose reabsorption under hyperglycemic but not under hypo- or euglycemic conditions in rats.

To understand the risk of hypoglycemia associated with urinary glucose excretion (UGE) induced by sodium-glucose cotransporter (SGLT) inhibitors, it is necessary to know the relationship between the ratio of contribution of SGLT2 vs. SGLT1 to renal glucose reabsorption (RGR) and the glycemic levels in vivo. To examine the contributions of SGLT2 and SGLT1 in normal rats, we compared the RGR inhibition by tofogliflozin, a highly specific SGLT2 inhibitor, and phlorizin, an SGLT1 and SGLT2 (SGLT1/2) inhibitor, at plasma concentrations sufficient to completely inhibit rat SGLT2 (rSGLT2) while inhibiting rSGLT1 to different degrees. Under hyperglycemic conditions by glucose titration, tofogliflozin and phlorizin achieved ≥50% inhibition of RGR. Under hypoglycemic conditions by hyperinsulinemic clamp, RGR was reduced by 20-50% with phlorizin and by 1-5% with tofogliflozin, suggesting the smaller contribution of rSGLT2 to RGR under hypoglycemic conditions than under hyperglycemic conditions. Next, to evaluate the hypoglycemic potentials of SGLT1/2 inhibition, we measured the plasma glucose (PG) and endogenous glucose production (EGP) simultaneously after UGE induction by SGLT inhibitors. Tofogliflozin (400 ng/ml) induced UGE of about 2 mg·kg⁻¹·min⁻¹ and increased EGP by 1-2 mg·kg⁻¹·min⁻¹, resulting in PG in the normal range. Phlorizin (1,333 ng/ml) induced UGE of about 6 mg·kg⁻¹·min⁻¹ and increased EGP by about 4 mg·kg⁻¹·min⁻¹; this was more than with tofogliflozin, but the minimum PG was lower. These results suggest that the contribution of SGLT1 to RGR is greater under lower glycemic conditions than under hyperglycemic conditions and that SGLT2-selective inhibitors pose a lower risk of hypoglycemia than SGLT1/2 inhibitors.

Translocation of transfected GLUT2 to the apical membrane in rat intestinal IEC-6 cells.

AIM: In this study, we transfected the full length cDNA of glucose transporter 2 (GLUT2) into IEC-6 cells (which lack GLUT2 expression) to investigate GLUT2 translocation in enterocytes. The purpose of this study was to investigate cellular mechanisms of GLUT2 translocation and its signaling pathway. METHODS: Rat GLUT2 cDNA was transfected into IEC-6 cells. Glucose uptake was measured by incubating cell monolayers with glucose (0.5-50 mM), containing (14)C-D-glucose and (3)H-L-glucose, to measure stereospecific, carrier-mediated and passive uptake. We imaged GLUT2 immunoreactivity by confocal fluorescence microscopy. We evaluated the GLUT2 inhibitor (1 mM phloretin), SGLT1 inhibitor (0.5 mM phlorizin), disrupting microtubular integrity (2 µM nocodazole and 0.5 µM cytochalasin B), protein kinase C (PKC) inhibitors (50 nM calphostin C and 10 µM chelerythrine), and PKC activator (50 nM phorbol 12-myristate 13-acetate: PMA). RESULTS: In GLUT2-IEC cells, the K(m) (54.5 mM) increased compared with non-transfected IEC-6 cells (7.8 mM); phloretin (GLUT2 inhibitor) inhibited glucose uptake to that of non-transfected IEC-6 cells (P < 0.05). Nocodazole and cytochalasin B (microtubule disrupters) inhibited uptake by 43-58% only at glucose concentrations ≥25 and 50 mM and the 10-min incubations. Calphostin C (PKC inhibitor) reproduced the inhibition of nocodazole; PMA (a PKC activator) enhanced glucose uptake by 69%. Exposure to glucose increased the GFP signal at the apical membrane of GLUT-1EC cells. CONCLUSION: IEC-6 cells lacking GLUT2 translocate GLUT2 apically when transfected to express GLUT2. Translocation of GLUT2 occurs through glucose stimulation via a PKC-dependent signaling pathway and requires integrity of the microtubular skeletal structure.

Pharmacological profile of ipragliflozin (ASP1941), a novel selective SGLT2 inhibitor, in vitro and in vivo.

The pharmacological profile of ipragliflozin (ASP1941; (1S)-1,5-anhydro-1-C-{3-[(1-benzothiophen-2-yl)methyl]-4-fluorophenyl}-D: -glucitol compound with L: -proline (1:1)), a novel SGLT2 selective inhibitor, was investigated. In vitro, the potency of ipragliflozin to inhibit SGLT2 and SGLT1 and stability were assessed. In vivo, the pharmacokinetic and pharmacologic profiles of ipragliflozin were investigated in normal mice, streptozotocin-induced type 1 diabetic rats, and KK-A(y) type 2 diabetic mice. Ipragliflozin potently and selectively inhibited human, rat, and mouse SGLT2 at nanomolar ranges and exhibited stability against intestinal glucosidases. Ipragliflozin showed good pharmacokinetic properties following oral dosing, and dose-dependently increased urinary glucose excretion, which lasted for over 12 h in normal mice. Single administration of ipragliflozin resulted in dose-dependent and sustained antihyperglycemic effects in both diabetic models. In addition, once-daily ipragliflozin treatment over 4 weeks improved hyperglycemia with a concomitant increase in urinary glucose excretion in both diabetic models. In contrast, ipragliflozin at pharmacological doses did not affect normoglycemia, as was the case with glibenclamide, and did not influence intestinal glucose absorption and electrolyte balance. These results suggest that ipragliflozin is an orally active SGLT2 selective inhibitor that induces sustained increases in urinary glucose excretion by inhibiting renal glucose reabsorption, with subsequent antihyperglycemic effect and a low risk of hypoglycemia. Ipragliflozin has, therefore, the therapeutic potential to treat hyperglycemia in diabetes by increasing glucose excretion into urine.

Pharmacokinetic and pharmacodynamic modeling of the effect of an sodium-glucose cotransporter inhibitor, phlorizin, on renal glucose transport in rats.

A pharmacokinetic and pharmacodynamic (PK-PD) model for the inhibitory effect of sodium-glucose cotransporter (SGLT) inhibitors on renal glucose reabsorption was developed to predict in vivo efficacy. First, using the relationship between renal glucose clearance and plasma glucose level in rats and both the glucose affinity and transport capacity obtained from in vitro vesicle experiments, a pharmacodynamic model analysis was performed based on a nonlinear parallel tube model to express the renal glucose transport mediated by SGLT1 and SGLT2. This model suitably expressed the relationship between plasma glucose level and renal glucose excretion. A PK-PD model was developed next to analyze the inhibitory effect of phlorizin on renal glucose reabsorption. The PK-PD model analysis was performed using averaged concentrations of both the drug and glucose in plasma and the corresponding renal glucose clearance. The model suitably expressed the concentration-dependent inhibitory effect of phlorizin on renal glucose reabsorption. The in vivo inhibition constants of phlorizin for SGLT in rats were estimated to be 67 nM for SGLT1 and 252 nM for SGLT2, which are similar to the in vitro data reported previously. This suggests that the in vivo efficacy of SGLT inhibitors could be predicted from an in vitro study based on the present PK-PD model. The present model is based on physiological and biochemical parameters and, therefore, would be helpful in understanding individual differences in the efficacy of an SGLT inhibitor.

Inhibidores del cotransportador sodio-glucosa tipo 2(SGLT2): de la glucosuria renal familiar al tratamiento de la diabetes mellitus tipo 2 / Sodium-glucose cotransporter type 2 inhibitors (SGLT2): from familial renal glycosuria to the treatment of type 2 diabetes mellitus

Durante siglos, el riñón se ha considerado principalmente un órgano de eliminación y un regulador de la sal y del equilibrio iónico. A pesar de que una vez se pensó que era la causa estructural de la diabetes, y que en los últimos años ha sido ignorado como regulador de la homeostasis de la glucosa, actualmente es reconocido como un actor importante en el ámbito de la regulación del metabolismo glucídico. Durante el ayuno, el 55% de la glucosa proviene de la gluconeogénesis. Sólo 2 órganos tienen esta capacidad: el hígado y el riñón. Este último es responsable del 20% de la producción total deglucosa y del 40% de la producida por la gluconeogénesis. Hoy en día tenemos una mejor comprensión de la fisiología del transporte de glucosa renal a través de transportadores específicos, como el cotransportador sodio-glucosa tipo 2(SGLT2 por sus siglas en inglés: Sodium Glucose Cotransporter). Un compuesto natural, floricina, se aisló a principios de1800 y durante décadas desempeñó un papel importante enla diabetes y la investigación de la fisiología renal. Finalmente, en el nexo de estos descubrimientos antes mencionados, se reconoció el efecto de compuestos floricina-like en los transportadores de glucosa renal, lo que ha ofrecido un nuevo mecanismo para el tratamiento de la hiperglucemia. Esto ha llevado al desarrollo de varias modalidades terapéuticas potencialmente eficaces para el tratamiento de la diabetes (AU)

For centuries, the kidney has been considered primarily an organ of elimination and a regulator of salt and ion balance. Although once thought that the kidney was the structural cause of diabetes, which in recent years has been ignored as a regulator of glucose homeostasis, is now recognized as a major player in the field of metabolic regulation carbohydrate. During fasting, 55% of the glucose comes from gluconeogenesis. Only 2 organs have this capability: the liver and kidney. The latter is responsible for 20% of total glucose production and 40%of that produced by gluconeogenesis. Today we have a better understanding of the physiology of renal glucose transport via specific transporters, such as type 2 sodiumglucose cotransporter (SGLT2). A natural compound, phlorizin, was isolated in early 1800 and for decades played an important role in diabetes and renal physiology research. Finally, at the nexus of these findings mentioned above, recognized the effect of phlorizin-like compounds in the renal glucose transporter, which has offered a new mechanism to treat hyperglycemia. This has led to the development of several potentially effective treatment modalities for the treatment of diabetes (AU)

[Sodium-glucose cotransporter type 2 inhibitors (SGLT2): from familial renal glucosuria to the treatment of type 2 diabetes mellitus]. / Inhibidores del cotransportador sodio-glucosa tipo 2 (SGLT2): de la glucosuria renal familiar al tratamiento de la diabetes mellitus tipo 2.

For centuries, the kidney has been considered primarily an organ of elimination and a regulator of salt and ion balance. Although once thought that the kidney was the structural cause of diabetes, which in recent years has been ignored as a regulator of glucose homeostasis, is now recognized as a major player in the field of metabolic regulation carbohydrate. During fasting, 55% of the glucose comes from gluconeogenesis. Only 2 organs have this capability: the liver and kidney. The latter is responsible for 20% of total glucose production and 40% of that produced by gluconeogenesis. Today we have a better understanding of the physiology of renal glucose transport via specific transporters, such as type 2 sodium-glucose cotransporter  (SGLT2). A natural compound, phlorizin, was isolated in early 1800 and for decades played an important role in diabetes and renal physiology research. Finally, at the nexus of these findings mentioned above, recognized the effect of phlorizin-like compounds in the renal glucose transporter, which has offered a new mechanism to treat hyperglycemia. This has led to the development of several potentially effective treatment modalities for the treatment of diabetes.

Estrogenic and antiestrogenic activities of phloridzin.

Phloridzin, a phloretin 2'-beta-D-glucoside, belongs to dihydrochalcones and mainly exists in the fruits of Malus pumila Mill., Lithocarpus polystachyus REHD and the root skins, stems, tender leaves and fruits of Malus hupehensis. It has many pharmacological activities, such as regulating blood sugar level and blood pressure, protecting heart, scavenging of oxygen free radicals and antioxidant injuries. Thus, market demand of products containing phloridzin is increasing year by year. Our research results demonstrated that phloridzin is provided with a double directional adjusting function of estrogenic and antiestrogenic activities. It showed significant effects on the proliferation of estrogen sensitive estrogen receptor (ER) (+)MCF-7 cells in the absence of estrogen. When added with 17beta-estradiol, phloridzin showed antagonism on estradiol-induced MCF-7 cell proliferation, but it did not significantly affect proliferation of estrogen insensitive ER (-)MDA-MB-231 cells. Phloridzin induced beta-galactosidase activity in a yeast two-hybrid assay. Light increase of the uterine weight and serum estradiol content of mouse was observed when the glucoside was administered orally for 7 d. After oral administration, phloridzin was found mainly in the blood and a small part was metabolized to phloretin. Our investigation proved that phloridzin was distributed at the target organ and played the role of phytoestrogen.

Stabilization of enzyme-susceptible glucoside bonds of phloridzin through conjugation with poly(gamma-glutamic acid).

We designed and prepared poly(gamma-glutamic acid)s (gamma-PGA) bearing phloridzin, which is an inhibitor of Na(+)/glucose cotransporter 1 (SGLT1), via a non-biodegradable omega-amino triethylene glycol linker. Properties of gamma-PGA-phloridzin conjugates (PGA-PRZ) were examined because our previous research revealed that PGA-PRZ with a 15% phloridzin content suppressed an increase in the blood glucose level after oral administration of D-glucose in rats, even though intact phloridzin scarcely affected the glucose-induced hyperglycemic effect. In uptake experiments using rat small intestinal brush-border membrane vesicles (BBMVs), the conjugation resulted in a 10-fold increase in the inhibitor concentration giving half-maximum inhibition of SGLT1-mediated D-glucose uptake, indicating that the inhibitory effect on the uptake was considerably reduced. On the other hand, beta-glucosidase-susceptible glucoside bonds of phloridzin were stabilized through conjugation with gamma-PGA. D-glucose, which is essential for the inhibition of SGLT1, was not released from PGA-PRZ with a phloridzin content of greater than 15% incubated with BBMVs, despite the immediate release of D-glucose from intact phloridzin. It was strongly indicated that the improved stability resulted in the difference in pharmacological activities between the conjugate and phloridzin. We also concluded that the toxic phloretin was not released from the conjugates. These results suggest that gamma-PGA-phloridzin conjugates have potential as oral anti-diabetic drugs with high safety.

Polymer-phloridzin conjugates as an anti-diabetic drug that inhibits glucose absorption through the Na+/glucose cotransporter (SGLT1) in the small intestine.

Poly(gamma-glutamic acid)s (gamma-PGA) modified with phloridzin, which is an inhibitor of the Na(+)/glucose cotransporter (SGLT1), via a omega-amino triethylene glycol linker were synthesized. The potential of gamma-PGA-phloridzin conjugates (PGA-PRZs) obtained as a novel oral anti-diabetic drug was examined by in vitro and in vivo experiments. A PGA-PRZ with a 15% phloridzin content inhibited glucose transport from mucosal to serosal sides of the everted rat's small intestine, and its inhibitory effect was as strong as that of intact phloridzin. When the PGA-PRZ was given orally to rats before glucose administration, the glucose-induced hyperglycemic effect was significantly suppressed. On the other hand, reduction of an increase in the blood glucose concentration was scarcely observed when the PGA-PRZ was substituted with a double amount of intact phloridzin. This difference in the biological activity between PGA-PRZ and intact phloridzin might have resulted from the improved stability of a glucoside bond of phloridzin through the conjugation with gamma-PGA. These results suggest that the gamma-PGA-phloridzin conjugates have potential as oral anti-diabetic drugs.

Bioavailability of phloretin and phloridzin in rats.

Phloretin is a flavonoid found exclusively in apples and in apple-derived products where it is present as the glucosidic form, namely, phloridzin (phloretin 2'-O-glucose). In the present study, we compared the changes in plasma and urine concentrations of these two compounds in rats fed a single meal containing 0.25% phloridzin or 0.157% phloretin (corresponding to the ingestion of 22 mg of phloretin equivalents). In plasma, phloretin was recovered mainly as the conjugated forms (glucuronided and/or sulfated) but some unconjugated phloretin was also detected. By contrast, no trace of intact phloridzin was detected in plasma of rats fed a phloridzin meal. These compounds presented different kinetics of absorption; phloretin appeared more rapidly in plasma when rats were fed the aglycone than when fed the glucoside. However, whatever compound was administered, no significant difference in the plasma concentrations of total phloretin were observed 10 h after food intake. At 24 h after the beginning of the meal, the plasma concentrations of phloretin were almost back to the baseline, indicating that this compound was excreted rapidly in urine. The total urinary excretion rate of phloretin was not affected by the forms administered, and was estimated to be 8.5 micromol/24 h in rats fed phloretin or phloridzin. Thus, 10.4% of the ingested dose was recovered in urine after 24 h.

Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl-/GABA (hGAT1) cotransporters.

1. Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na(+)/glucose (hSGLT1) and Na(+)/Cl(-)/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate-dependent current and inhibitor-induced changes in cotransporter conformation. 2. The competitive, non-transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5-orders of magnitude. 3. Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7x12A. Important hydrogen bond interactions occur at five positions bordering this surface. 4. In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site approximately 8A from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non-competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1). 5. Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non-radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors.

Comparison of the intestinal absorption of quercetin, phloretin and their glucosides in rats.

Absorption and metabolism of quercetin and isoquercitrin (quercetin 3-O-glucose) were investigated in rats after in situ perfusion of jejunum plus ileum (15 nmol/min) for 30 min and compared with those of phloretin and phloridzin (phloretin 2'-O-glucose). After perfusion of the glucosides, the corresponding aglycone forms and conjugated derivatives appeared in the lumen. The conjugated metabolites were similar to those recovered after intestinal perfusion of the aglycone forms. Regardless of the aglycone or glucoside perfused, only conjugated forms were present in the mesenteric vein blood draining the perfused segment showing the importance of intestinal conjugation. The hydrolysis of glucosides was a prerequisite step before their conjugation by intestinal enzymes and their transport towards the mucosal and serosal sides. In contrast to phloridzin, lactase phloridzin hydrolase activity did not seem to be an essential pathway for isoquercitrin hydrolysis. The 3-O-glucosylation of quercetin improved the net absorption of the aglycone (P < 0.05), whereas phloretin absorption decreased when present as 2'-O-glucoside (P < 0.05). Whatever the perfused compound, the efficiency of the absorption seemed to be linked to the intestinal conjugation process and to the luminal secretion of metabolites.

A Na+-dependent D-mannose transporter in the apical membrane of chicken small intestine epithelial cells.

The presence of a Na+/D-mannose cotransporter in brush-border membrane vesicles (BBMV) isolated from chicken small intestine was examined. In the presence of an electrochemical gradient for Na+, but not in its absence, D-mannose was accumulated transiently by the BBMV. D-Mannose uptake into the BBMV was energized by both the membrane potential and the chemical gradient for Na+. The relationship between D-mannose transport and external D-mannose concentration was described by an equation that represented the superposition of a saturable component (Michaelis-Menten constant Km 12.5 microM) and another component unsaturatable up to 80 microM D-mannose. D-Mannose uptake was inhibited by various substances in the following order of potency: D-mannose>>D-glucose>phlorizin>phloretin>D-fructose. For the uptake of alpha-methyl-glucopyranoside the order was D-glucose=phlorizin>>phloretin=D-fructose=D-mannose. The initial rate of D-mannose uptake increased as the extravesicular [Na+] increased, with a Hill coefficient of 1, suggesting that the Na+:D-mannose cotransport stoichiometry is 1:1. It is concluded that the intestinal apical membrane has a saturable, electrogenic and concentration- and Na+-dependent mannose transport mechanism that differs from the sodium-dependent glucose transporter SGLT1.

Phenylglucosides and the Na+/glucose cotransporter (SGLT1): analysis of interactions.

Phenylglucosides are transported by the intestinal Na+/glucose cotransporter (SGLT1) and phlorizin, the classical competitive inhibitor of SGLT1, is also a phenylglucoside. To investigate the structural requirements for binding of substrates to SGLT1, we have studied the interactions between phenylglucosides and the cotransporter expressed in Xenopus oocytes using tracer uptake and electrophysiological methods. Some phenylglucosides inhibited the Na(+)-dependent uptake of 14C-alpha-methyl-D-glucopyranoside (alpha MDG) with apparent Kis in the range 0.1 to 20 mM, while others had no effect. Electrophysiological experiments indicated that phenylglucosides can act either as: (1) transported substrates, e.g., arbutin; (2) nontransported inhibitors, e.g., glucosylphenyl-isothiocyanate; or (3) noninteracting sugars, e.g., salicin. The transported substrates (glucose, arbutin, phenylglucoside and helicin) induced different maximal currents, and computer simulations showed that this may be explained by a difference in the translocation rates of the sugar and Na(+)-loaded transporter. Computational chemistry indicated that all these beta-phenylglucosides have similar 3-D structures. Analysis showed that among the side chains in the para position of the phenyl ring the -OH group (arbutin) facilitates transport, but the -NCS (glucosylphenyl-isothiocyanate) inhibits transport. In the ortho position, -CH2OH (salicin) prevents interaction, but the aldehyde (helicin) permits the molecule to be transported. Studies such as these may help to understand the geometry and nature of glucoside binding to SGLT1.

Antimalarial action of hydrophilic drugs: involvement of aqueous access routes to intracellular parasites.

The antimalarial action and intracellular distribution of the hydrophilic agents phloridzin (PHL) (a bioflavonoid glycoside) and desferrioxamine (DFO) (an iron chelator) were studied in cultures of Plasmodium falciparum-infected human erythrocytes. When added to cultures, these agents arrested parasite growth with IC50 values of 12 microM (PHL) and 22 microM (DFO). At 37 degrees, PHL (40 microM) was virtually impermeant to uninfected cells but permeated with a mean t1/2 of 1.5 hr in trophozoites (30% accessible cell volume) and 8 hr in rings (10% of accessible cell volume). PHL, in analogy with DFO, was demonstrably permeant to infected cells harboring mature forms of the parasites. Permeation was restricted to only a fraction of the infected cell volume. PHL elicited inhibition of nucleic acid synthesis within 1 hr of exposure of trophozoites to PHL (40 microM) and in > 8 hr of exposure of rings. Red cell containers into which millimolar concentrations of PHL or DFO were encapsulated demonstrably supported parasite invasion and subsequent parasite growth and maturation (48-hr incubation). Under culture conditions, uninfected or parasite-infected red cell containers that were loaded with either agent retained the drugs for at least 42 hr at hundred-micromolar concentrations. The agent present in the cells was fully active after release from cells and administration to test cultures of parasites. PHL added to parasite cultures was active at micromolar concentrations, but when present intracellularly it was virtually inactive even at millimolar concentrations. The data presented are consistent with direct access of hydrophilic agents from medium to parasite, a process referred to as fenestration. Permeation into parasites might constitute the rate-limiting step in drug uptake and drug-mediated arrest of parasite growth by PHL and DFO. The putative role of the parasitophorous duct in providing aqueous access routes from medium to parasites is discussed.