Comparison of the transport kinetics of fexofenadine and its pH dependency among OATP1A2 genetic variants

Little is known about the influence of non-synonymous genetic variations in the organic anion-transporting polypeptide (OATP) 1A2 on the transport kinetics of its substrate fexofenadine. Moreover, the pH-dependency of fexofenadine uptake also remains unclear. This study aimed to evaluate the effects of genetic variants (Ile13Thr, Asn128Tyr, Glu172Asp, Ala187Thr, and Thr668Ser) on the OATP1A2-mediated uptake of fexofenadine at pH 6.3 and 7.4 and compare the pH dependency of OATP1A2-mediated uptake of fexofenadine and estrone 3-sulfate. The uptake clearances of 0.3 μM and 300 μM fexofenadine were compared with those of 0.3 μM and 300 μM estrone 3-sulfate at pH 6.3 and 7.4. Among the six variants examined, the Thr668Ser variant showed the highest fexofenadine uptake clearance (V_max/K_m); i.e., 4.53- and 6.28-fold higher uptake clearance than the wild type at pH 6.3 and 7.4, respectively. All variants exhibited significantly higher fexofenadine uptake at pH 6.3 than at pH 7.4. Compared with estrone 3-sulfate uptake, the uptake of 0.3 μM fexofenadine was less sensitive to pH. Our findings suggest that genetic variations in OATP1A2 may lead to altered intestinal absorption of fexofenadine, such as increased absorption in subjects bearing the Thr668Ser variant, which showed higher uptake activity.     

6-Hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor elevated in renal failure patients

The hepatic uptake transporter organic anion transporting polypeptide (OATP) 1B1 is inhibited by some uremic toxins; however, direct inhibition can only partially explain the delayed systemic elimination of substrate drugs in renal failure patients. This study aimed to examine the long-lasting inhibition of OATP1B1 by uremic toxins and their metabolites. Preincubation of HEK293/OATP1B1 cells with 21 uremic toxins resulted in almost no change in the uptake of a typical substrate [³H]estrone-3-sulfate (E₁S), although some directly inhibited [³H]E₁S uptake. In contrast, preincubation with an indole metabolite, 6-hydroxyindole, reduced [³H]E₁S uptake, even after the inhibitor was washed out before [³H]E₁S incubation. Such long-lasting inhibition by 6-hydroxyindole was time-dependent and recovered after a 3-h incubation without 6-hydroxyindole. Preincubation with 6-hydroxyindole increased the Km for [³H]E₁S uptake with minimal change in Vmax. This was compatible with no change in the cell-surface expression of OATP1B1, as assessed by a biotinylation assay. Preincubation with 6-hydroxyindole reduced [³H]E₁S uptake in human hepatocytes without changes in OATP1B1 mRNA. Plasma concentration of 6-hydroxyindole in renal failure patients increased as renal function decreased, but might be insufficient to exhibit potent OATP1B1 inhibition. In conclusion, 6-hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor with elevated plasma concentrations in renal failure patients.     

Phenolsulfonphthalein as a surrogate substrate to assess altered function of the prostaglandin transporter SLCO2A1

The prostaglandin (PG) transporter SLCO2A1 regulates PGE₂ signaling and interacts with many drugs, and SLCO2A1 defects is associated with PG metabolic disorders. This study aimed to characterize a non-metabolic phenolsulfonphthalein (PSP) transport mediated by SLCO2A1. PSP uptake by HEK293 cells expressing human SLCO2A1 (HEK/2A1 cells) was pH-independent and saturable with a K_m value of 54.5 ± 9.5 μM PGE₂ competitively inhibited PSP uptake with a K_i of 257.3 ± 22.8 nM. When PSP was intravenously (i.v.) injected, concentration-time curve showed a biphasic response. In Slco2a1-deficient (−/−) mice, AUC_inf tented to decrease and the central distribution volume (V₁) significantly increased, compared to wild-type (wt) counterparts. Intriguingly, Slco2a1-deficiency significantly reduced a ratio of tissue-to-plasma concentration in the lungs at 15 min after i.v. injection, suggesting that SLCO2A1 limits tissue distribution of PSP. In conclusion, these results prove that PSP is a potential surrogate for monitoring SLCO2A1 function, providing a new concept for diagnostics for the genetic diseases caused by defects in SLCO2A1 gene.     

In vitro inhibitory effects of components from Salvia miltiorrhiza on catalytic activity of three human Arachidonic acid ω-hydroxylases

CYP4 enzymes are involved in the metabolism of xenobiotics and endogenous molecules. 20-Hydroxyeicosatetraenoic acid (20-HETE), the arachidonic acid (AA) ω-hydroxylation metabolite catalyzed by CYP4A/4F enzymes, is implicated in various biological functions. The goal of this investigation is to examine the inhibitory effects of components from Salvia miltiorrhiza(Danshen) on AA ω-hydroxylation using recombinant CYP4A11, CYP4F2, CYP4F3B, and microsomal systems. Tanshinone IIA had noncompetitive inhibition on CYP4F3B (K_i = 4.98 μM). Cryptotanshinone (K_i = 6.87 μM) and tanshinone I (K_i = 0.42 μM) had mixed-type inhibition on CYP4A11. Dihydrotanshinone I had mixed-type inhibition on CYP4A11 (K_i = 0.09 μM), and noncompetitive inhibition on CYP4F2 (K_i = 4.25 μM) and CYP4F3B (K_i = 3.08 μM). Salvianolic acid A had competitive inhibition on CYP4A11 (K_i = 19.37 μM), and noncompetitive inhibition on CYP4F2 (K_i = 15.28 μM) and CYP4F3B (K_i = 6.45 μM). Salvianolic acid C had noncompetitive inhibition on CYP4F2 (K_i = 5.70 μM) and CYP4F3B (K_i = 18.64 μM). In human kidney, human liver or rat heart microsomes, 20-HETE formation was significantly inhibited (P < 0.05) by dihydrotanshinone I (5 and 20 μM) and salvianolic acid A (20 and 50 μM). Given that low plasma concentrations of Danshen components after oral administration, Danshen preparations may not play pharmacological roles by inhibiting AA ω-hydroxylases; however, as Danshen components may reach high concentration in human intestine, drugs that have an important pre-systemic metabolism by these CYP4A/4F enzymes should avoid being co-administered with Danshen preparations.     

Discovery of [1,2,3]triazolo[4,5-d]pyrimidine derivatives as highly potent,selective, and cellularly active USP28 inhibitors

Ubiquitin specific peptidase 28 (USP28) is closely associated to the occurrence and development of various malignancies, and thus has been validated as a promising therapeutic target for cancer therapy. To date, only few USP28 inhibitors with moderate inhibitory activity have been reported, highly potent and selective USP28 inhibitors with new chemotypes remain to be discovered for pathologically investigating the roles of deubiquitinase. In this current study, we reported the synthesis and biological evaluation of new [1,2,3]triazolo[4,5-d]pyrimidine derivatives as potent USP28 inhibitors. Especially, compound 19 potently inhibited USP28 (IC₅₀ = 1.10 ± 0.02 μmol/L, K_d = 40 nmol/L), showing selectivity over USP7 and LSD1 (IC₅₀ > 100 μmol/L). Compound 19 was cellularly engaged to USP28 in gastric cancer cells. Compound 19 reversibly bound to USP28 and directly affected its protein levels, thus inhibiting the proliferation, cell cycle at S phase, and epithelial-mesenchymal transition (EMT) progression in gastric cancer cell lines. Docking studies were performed to rationalize the potency of compound 19. Collectively, compound 19 could serve as a new tool compound for the development of new USP28 inhibitors for exploring the roles of deubiquitinase in cancers.     

Effect of OATP1B1 genotypes on plasma concentrations of endogenous OATP1B1 substrates and drugs,and their association in healthy volunteers

This study aimed to elucidate the impact of OATP1B1 genotype (*1b/*1b, *1b/*15, and *15/*15) on plasma concentrations of endogenous OATP1B1 substrates. Healthy volunteers with OATP1B1 *1b/*1b (n = 10), *1b/*15 (n = 7), or *15/*15 (n = 2) received oral administration of a cocktail of statins (atorvastatin, pitavastatin, rosuvastatin, and fluvastatin). Mean area under the plasma concentration of atorvastatin, pitavastatin, and rosuvastatin in OATP1B1 *15/*15 were 2.2, 1.7 and 1.58-times greater than the corresponding values in OATP1B1 *1b/*1b, respectively, whereas that of fluvastatin was identical to those in other OATP1B1 genotypes. OATP1B1 *15/*15 also showed higher mean plasma concentrations of OATP1B1 endogenous substrates compared with the other OATP1B1 genotypes, such as coproporphyrin I, glycochenodeoxycholate sulfate (GCDCA-S), lithocholate sulfate (LCA-S), glycolithocholate sulfate (GLCA-S) and taurolithocholate sulfate (TLCA-S), but not total or direct bilirubin, chenodeoxycholate-24-glucuronide, or ω-dicarboxylic long-chain fatty acids. Area under the plasma concentration-time curves of plasma coproporphyrin I and GLCA-S discriminated OATP1B1 genotype *15/*15 from the other genotypes. In combination with previously published clinical studies, these results support the notion that coproporphyrin I, and GLCA-S and GCDCA-S could be a surrogate probe for assessing human in vivo OATP1B1 activities.     

Evodiamine-inspired dual inhibitors of histone deacetylase 1 (HDAC1) and topoisomerase 2 (TOP2) with potent antitumor activity

A great challenge in multi-targeting drug discovery is to identify drug-like lead compounds with therapeutic advantages over single target inhibitors and drug combinations. Inspired by our previous efforts in designing antitumor evodiamine derivatives, herein selective histone deacetylase 1 (HDAC1) and topoisomerase 2 (TOP2) dual inhibitors were successfully identified, which showed potent in vitro and in vivo antitumor potency. Particularly, compound 30a was orally active and possessed excellent in vivo antitumor activity in the HCT116 xenograft model (TGI = 75.2%, 150 mg/kg, p.o.) without significant toxicity, which was more potent than HDAC inhibitor vorinostat, TOP inhibitor evodiamine and their combination. Taken together, this study highlights the therapeutic advantages of evodiamine-based HDAC1/TOP2 dual inhibitors and provides valuable leads for the development of novel multi-targeting antitumor agents.     

Inhibitory effects of sesamin on CYP2C9-dependent 7-hydroxylation of S-warfarin

A recent report demonstrated that sesamin strongly and non-competitively inhibits S-warfarin 7-hydroxylation activity in human liver microsomes with a K_i value of 0.2 μM. This finding suggests that sesamin predominantly binds to CYP2C9 at another site for which it has a higher affinity than its affinity for the active site, thereby inhibiting the activity of CYP2C9 non-competitively. In this study, we found that sesamin competitively inhibited the 7-hydroxylation activity of S-warfarin in human liver microsomes with a K_i value of 15.7 μM. In addition, the recombinant CYP2C9-dependent 7-hydroxylation activity of S-warfarin was competitively inhibited by sesamin with a Ki value of 13.1 μM. These results are consistent with the fact that sesamin is a good substrate of CYP2C9, and its activity follows Michaelis-Menten kinetics. As the plasma concentration of sesamin after its administration is usually lower than 0.01 μM, the inhibition of S-warfarin metabolism by sesamin does not appear to be severe.     

Organic anion-transporting polypeptide (OATP) 2B1 contributes to the cellular uptake of theaflavin

Organic anion-transporting polypeptide (OATP) 2B1 has been reported in the apical membranes of the human small intestinal epithelium, where it contributes to the intestinal absorption of pharmacologically active drugs. To investigate the potential for OATP2B1-mediated drug–food interactions, the effects of several polyphenolic compounds on OATP2B1-mediated estrone-3-sulfate (E3S) transport were studied by using OATP2B1-expressing HEK293 cells. Our results showed that some compounds, especially theaflavin, were strong inhibitors of OATP2B1-mediated E3S uptake. Theaflavin showed a significantly higher uptake into the OATP2B1-expressing HEK293 cells than the control cells. The concentration dependence of the uptake of theaflavin was determined over a range of concentrations (0.5–100 μM) and the kinetic parameters (K_m and V_max) of theaflavin uptake were found to be 5.12 ± 0.67 μM and 41.6 ± 1.3 pmol/mg protein/min, respectively. The OATP2B1-mediated theaflavin uptake was inhibited by known OATP2B1 substrates such as E3S, bromsulphthalein (BSP), dehydroepiandrosterone-3-sulfate (DHEAS), and fluvastatin. Our results indicate that theaflavin is a novel substrate of OATP2B1. The results of this study might be helpful to predict the potential OATP2B1-mediated drug–theaflavin interactions and to avoid undesirable clinical consequences.     

A Novel Fluorescence-Based Method to Evaluate Ileal Apical Sodium-Dependent Bile Acid Transporter ASBT

This study aimed to demonstrate usefulness of the fluorophore-labeled bile acid derivative, N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2′-aminoethane sulfonate (tauro-nor-THCA-24-DBD) as a substrate of apical sodium-dependent bile acid transporter (ASBT, SLC10A2), which is expressed at distal ileum for reabsorption of bile acids and to find a novel fluorescence-based method to evaluate ASBT activity. In HPLC analysis, chromatogram of tauro-nor-THCA-24-DBD showed double peaks: R- and S-isomers of the compound. When ASBT was expressed in Xenopus laevis oocytes, their uptakes were higher than those by control oocytes, demonstrating both are transported by ASBT. Therefore, results were analyzed separately as peak 1, peak 2 and sum of them. Concentration dependent uptake of tauro-nor-THCA-24-DBD in ASBT-expressing oocytes was saturable with Km 122 μM and Vmax 1.49 pmol/oocyte/30 min for peak 1, 30.7 μM and 1.34 pmol/oocyte/30 min for peak 2, and 40.6 μM and 2.36 pmol/oocyte/30 min for sum, respectively. These uptakes were decreased in the presence of taurocholic acid and in the Na⁺ free condition. Furthermore, in Caco-2 cells, tauro-nor-THCA-24-DBD uptake was also Na⁺-dependent and saturable. Additionally, these uptakes were decreased by elobixibat, a selective ASBT inhibitor. Accordingly, it was concluded that tauro-nor-THCA-24-DBD is a substrate of ASBT and useful to evaluate the intestinal ASBT transport activity.     

Functional role of serine 318 of the proton-coupled folate transporter in methotrexate transport

This study revealed the importance of serine 318 (S318) residue for proton-coupled folate transporter (PCFT, SLC46A1) functioning. Substitution of S318 with arginine or lysine impaired transport of methotrexate (MTX), but substitution with alanine (has a simple side chain structure), or cysteine (structurally similar to serine), had no significant effect on MTX transport. The initial uptake rate of MTX by S318A and S318C mutant at pH 5.0, followed by Michaelis–Menten kinetics with a K_m value of approximately 2.3 μM (for S318A) and 2.9 μM (for S318C), was similar to that of the wild-type. The normalized V_max value of the S318A mutant, calculated by dividing the V_max value by the Western blot protein band's relative intensity, was approximately 2-fold greater than that of the wild-type. The normalized V_max value of the S318C mutant was approximately 0.8-fold smaller than the wild-type. Results obtained showed that the substitution of S318 with basic amino acid residues results in the loss of transport activity, even though PCFT mutants are expressed at the cell membrane. Alternatively, the substitution of S318 with neutral amino acids did not significantly affect the transport function of PCFT.     

Development and evaluation of febuxostat solid dispersion through screening method

Febuxostat (Febux) is a BCS II drug and has a very low solubility. In order to overcome this shortcoming, the purpose of study is to increase the in vitro dissolution (%) and drug release (%) of Febux by using a screening method. The Febux-SD formulation was prepared by screening solubilizers, pH agents, and carriers using with a solvent evaporation method.The novel Febux SD formulation was successfully developed. The dissolution (%) of Febux of optimal formulation (SD3) was higher than that of Feburic® tab in pH 1.2, distilled water (DW), and pH 6.8 buffer by 6.3-, 2.6-, and 1.1-fold, respectively, at 60 min. The in vitro drug release (%) and permeability (μg/cm²) of SD3 formulation were improved compared to those of Feburic® tab in the pH shifting method and PBS (7.4), respectively. The SD3 formulation was well maintained the stability for 6 months, and that of physicochemical properties were altered. In conclusion, the Febux solubilization study with meglumine was first attempted and successfully performed. Through the improved dissolution (%) of Febux, high bioavailability of SD3 formulation is expected in animal and human studies.     

Characterization of organic anion transporting polypeptide 1b2 knockout rats generated by CRISPR/Cas9: a novel model for drug transport and hyperbilirubinemia disease

Organic anion transporting polypeptide 1B1 and 1B3 (OATP1B1/3) as important uptake transporters play a fundamental role in the transportation of exogenous drugs and endogenous substances into cells. Rat OATP1B2, encoded by the Slco1b2 gene, is homologous to human OATP1B1/3. Although OATP1B1/3 is very important, few animal models can be used to study its properties. In this report, we successfully constructed the Slco1b2 knockout (KO) rat model via using the CRISPR/Cas9 technology for the first time. The novel rat model showed the absence of OATP1B2 protein expression, with no off-target effects as well as compensatory regulation of other transporters. Further pharmacokinetic study of pitavastatin, a typical substrate of OATP1B2, confirmed the OATP1B2 function was absent. Since bilirubin and bile acids are the substrates of OATP1B2, the contents of total bilirubin, direct bilirubin, indirect bilirubin, and total bile acids in serum are significantly higher in Slco1b2 KO rats than the data of wild-type rats. These results are consistent with the symptoms caused by the absence of OATP1B1/3 in Rotor syndrome. Therefore, this rat model is not only a powerful tool for the study of OATP1B2-mediated drug transportation, but also a good disease model to study hyperbilirubinemia-related diseases.     

Development and evaluation of niacinamide transdermal formulation by artificial membrane permeability

Despite many efforts to improve the transdermal permeability of drugs, most of them are blocked by the skin barrier. Niacinamide (NAC) is a Biopharmaceutics Classification System class I drug with high aqueous solubility and intestinal permeability. Due to the high solubility and intestinal permeability of NAC, the development of new formulations is insufficient as transdermal, injection etc. Thus, this study aimed to develop the novel NAC formulation with improved skin permeability and secured stability. The NAC formulation approach is to first select a solvent that improves skin permeability, and then select a second penetration enhancer to determine the final formulation. All formulations were evaluated for skin permeability using an artificial membrane (Strat-M®). The optimal formulation (non-ionic formulations (NF1) consisted of NAC/Tween®80 = 1:1 wt ratio in dipropylene glycol [DPG]) showed the highest permeability in all formulations in PBS buffer (pH 7.4). The thermal properties of NF1 were altered. Moreover, NF1 maintained a stable drug content, appearance, and pH value for 12 months. In conclusion, DPG had an excellent effect in increasing the NAC permeation, and Tween®80 played a boosting role. Through this study, an innovative NAC formulation was developed, and good results are expected for human transdermal research.     

Inhibitory kinetics of fruit components on CYP2C19 activity

It has been suggested that the fruit components resveratrol (RSV), 6′, 7′-dihydroxybergamottin (DHB), and bergamottin (BG) might inhibit cytochrome P450 2C19 (CYP2C19) activity, but the mode and potency of such inhibition are yet to be investigated. This study aimed to investigate the mode and kinetics of the inhibition of CYP2C19-based omeprazole metabolism by RSV or grapefruit juice components (DHB or BG). RSV and DHB reduced CYP2C19 activity in a preincubation time-dependent manner, suggesting that they inactivated CYP2C19 via mechanism-based inhibition (MBI). Although BG inactivated CYP2C19 in a preincubation time- and concentration-dependent manner, suggesting that both MBI and reversible inhibition contributed to these effects, the concentration required to achieve 50% inhibition was 26-fold higher for reversible inhibition than for MBI (0.859 and 0.0331 μM, respectively), indicating that the inhibition of CYP2C19 by BG is primarily attributable to MBI. Based on the estimated intestinal concentrations of these components, it is considered that >90% of CYP2C19 would be inactivated after the consumption of normal amounts of grapefruit juice or RSV-containing substances. In conclusion, these findings suggest that food containing these components has the potential to evoke drug-food interactions caused by the MBI of intestinal CYP2C19 activity in the clinical setting.     

Dual kinetics of OATP2B1: Inhibitory potency and pH-dependence of OATP2B1 inhibitors

Organic anion transporting polypeptide (OATP) 2B1 is expressed in the intestine and liver, and OATP2B1-mediated transport of estrone 3-sulfate is pH-dependent and consists of: the high-affinity component (Hc) and low-affinity component (Lc). This study aimed to evaluate the influence of pH on the transport kinetics of each component, along with the inhibitory nature of ten OATP2B1 inhibitors. The Michaelis constants (K_m) were 4-fold and 1.5-fold lower at pH 6.3 than at pH 7.4, for Hc and Lc respectively. The inhibitory potencies of diclofenac, indomethacin, and ibuprofen towards Hc were 1.5–4.3 fold lower at pH 6.3 than at pH 7.4. Contrastingly, inhibitory potencies towards Lc were 9.0–52 fold lower at pH 7.4. Similarly, the inhibitory effect of naproxen was stronger towards Hc at pH 6.3 and towards Lc at pH 7.4. On the other hand, celecoxib selectively inhibited Lc transport at pH 7.4. Rifampicin inhibited both components at pH 6.3 and 7.4 to a similar extent, while bromosulphophthalein, naringin, and gefitinib selectively inhibited Hc irrespective of pH. Fexofenadine inhibited neither component. In conclusion, the transport affinities of both Hc and Lc were enhanced under acidic conditions. The influence of pH on the inhibitory potency towards each component varied among the inhibitors.     

Bioactivity and molecular docking of lactones isolated from Centaurea pseudosinaica Czerep

Two cytotoxic sesquiterpene lactones, 17-epichlorohyssopifolin A (1) and chlorjanerin (2), and a monoterpene lactone, loliolide (3) were isolated from Centaurea pseudosinaica. The cytotoxicity of the total extract and terpenoids 1–3 were evaluated against three human cancer cells (HepG2, PC-3, and HT-29), along with the human normal primary epidermal keratinocytes (HEKa) cells. With IC₅₀ values ranging between 0.6 ± 0.04 and 5.0 ± 0.61 μg/mL against HepG2; 0.2 ± 0.01 and 11.9 ± 1.31 μg/mL against PC-3, and 0.04 ± 0.013 and 8.9 ± 0.97 μg/mL against HT-29, the total extract, and lactones 1–3 demonstrated cytotoxic effects. Compound 1 displayed the strongest impact on all cancer cells and a slightly safe effect on the normal cells HEKa. Compound 1 caused accumulation of HepG2 and HT-29 cells in G1 phase as displayed cell cycle analysis. On the other hand, the cell distributions were increased in the S phase in PC-3 cells. Furthermore, 1 caused apoptosis in PC-3 and HePG2 cells with 91.50%, and 79.72 %, respectively. A higher fraction of necrotic cells was observed in HT-29 cells amounting to 23.60%. These results suggested that the promising cytotoxicity exhibited by 1 is brought by the apoptosis induction in the cancer cells, which were evaluated. As the compounds showed antiproliferative effect against the HT-29 cells, the docking simulation was performed aiming at determining how they would interact with the EGFR enzyme, whose PDB: 4I23 is considered one of the two distinct wild types of EGFR enzymes. The antibacterial activity results revealed that 3 showed the most remarkable antibacterial effects, especially against the examined Gram-positive bacteria. The total extract exhibited potent activity against all examined bacteria. The total extract showed a potent antifungal effect against two Candida and two Aspergillus pathogens. The antioxidant activity revealed the potency of the total extract and 3 as antioxidant candidates. The obtained results refer to the importance of Centaurea pseudosinaica as a source of potent antiproliferative agents and the whole plant as an antipathogenic and antioxidant agent.     

Evaluation of transporter-mediated hepatobiliary transport of newly developed 18F-labeled pitavastatin derivative,PTV-F1, in rats by PET imaging

Quantitative evaluations of the functions of uptake and efflux transporters directly in vivo is desired to understand an efficient hepatobiliary transport of substrate drugs. Pitavastatin is a substrate of organic anion transporting polypeptides (OATPs) and canalicular efflux transporters; thus, it can be a suitable probe for positron-emission tomography (PET) imaging of hepatic transporter functions. To characterize the performance of [¹⁸F]PTV-F1, an analogue of pitavastatin, we investigated the impact of rifampicin (a typical OATP inhibitor) coadministration or Bcrp (breast cancer resistance protein) knockout on [¹⁸F]PTV-F1 hepatic uptake and efflux in rats by PET imaging. After intravenous administration, [¹⁸F]PTV-F1 selectively accumulated in the liver, and the radioactivity detected in plasma, liver, and bile mainly derived from the parent PTV-F1 during the PET study (∼40 min). Coadministration of rifampicin largely decreased the hepatic uptake of [¹⁸F]PTV-F1 by 73%. Because of its lower clearance in rats, [¹⁸F]PTV-F1 is more sensitive for monitoring changes in hepatic OATP1B function that other previously reported OATP1B PET probes. Rifampicin coadministration also significantly decreased the biliary excretion of radioactivity by 65%. Bcrp knockout did not show a significant impact on its biliary excretion.[¹⁸F]PTV-F1 enables quantitative analysis of the hepatobiliary transport system for organic anions.     

Curative efficacy of purified serine protease inhibitor PTF3 from potato tuber in experimental visceral leishmaniasis

To overcome the drug toxicity and frequent resistance of parasites against the conventional drugs for the healing of human visceral leishmaniasis, innovative plant derived antileishmanial components are very imperative. Fuelled by the complications of clinically available antileishmanial drugs, a novel potato serine protease inhibitor was identified with its efficacy on experimental visceral leishmaniasis (VL). The serine protease inhibitors from potato tuber extract (PTEx) bearing molecular mass of 39 kDa (PTF1), 23 kDa (PTF2) and 17 kDa (PTF3) were purified and identified. Among them, PTF3 was selected as the most active inhibitor (IC₅₀ 143.5 ± 2.4 µg/ml) regarding its antileishmanial property. Again, intracellular amastigote load was reduced upto 83.1 ± 1.7% in pre-treated parasite and 88.5 ± 0.5% in in vivo model with effective dose of PTF3. Protective immune response by PTF3 was noted with increased production of antimicrobial substances and up-regulation of pro-inflammatory cytokines. Therapeutic potency of PTF3 is also followed by 80% survival in infected hamster. The peptide mass fingerprint (MALDI-TOF) results showed similarity of PTF3 with serine protease inhibitors database. Altogether, these results strongly propose the effectiveness of PTF3 as potent immunomodulatory therapeutics for controlling VL.