A mechanistic investigation of the bioavailability enhancing potential of lysergol, a novel bioenhancer, using curcumin

Abstract

Lysergol (LYZ), a novel bioenhancer, has shown potential to enhance the bioavailability of some antibiotics. In the present investigation, the bioavailability enhancing potential of LYZ on curcumin (CUR) has been explored. We initially carried out in vivo pharmacokinetic and in situ permeation studies of CUR with and without LYZ coadministration. In presence of LYZ, the C_max, AUC and elimination half-life of CUR were significantly increased. A noteworthy decrease in the clearance of CUR was also observed. An enhancement (3.3-fold) in the effective permeability of CUR was observed. To delve into the mechanistic insights, the probable role of LYZ in inhibiting the metabolism of CUR was investigated. In vitro phase I and II metabolic stability studies of CUR following pre-incubation with LYZ using rat liver microsomes were performed. Also, its effect on major efflux transporters using human P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) membrane preparations was examined. The corroboration of results was provided by in situ permeation and in vivo pharmacokinetic study of digoxin (probe P-gp substrate) and sulfasalazine (probe BCRP substrate) in the presence and absence of LYZ. The results were compared with the inhibitory potential of verapamil (for P-gp) and pantoprazole (for BCRP). Furthermore, the studies ruled out the probability of P-gp inhibition but strongly evidenced the involvement of BCRP inhibition. A remarkable increase in in vitro half-life of CUR and 1.4-fold enhancement in intestinal permeation of sulfasalazine in presence of LYZ clearly revealed that bioavailability enhancing potential is attributed to the inhibition of metabolic enzymes and BCRP efflux transporters.

---

1. Introduction

Oral administration is the most common and convenient route for drug therapy. Bioavailability (BA) is defined as the extent and rate at which unchanged drug proceeds from the site of administration to systemic circulation.¹ BA is governed by the kinetic processes including passage of the drug from the gastrointestinal tract (through enterocytes) to hepatic portal vein followed by hepatic clearance and reaching the systemic circulation. Many high therapeutically efficacious drugs suffer from the disadvantage of low oral BA. Low BA takes the shine away from drug and makes it less favourable for use. Bioenhancers are a hot topic nowadays and gained significant importance in the scientific community in current scenario owing to their use in improving the pharmacokinetic (PK) parameters and enhancing BA.² A bioenhancer is said to be a substance which lacks its own inherent pharmacological activity but enhances the BA and efficacy of the co-administered drug at the specific dose. Some potent herbal bioenhancers have revealed promising ability to enhance the bioefficacy of different classes of drugs (antibiotics, antitubercular, antiviral and anticancer) at low doses.³ Lysergol {LYZ; (7-methyl-4,6,6a,7,8,9-hexahydro-indolo[4,3-fg]quinolin-9-yl)-methanol; Fig. 1} has shown potential to enhance BA of berberine (poorly water soluble herbal anticancer agent).⁴ It is obtained from the seeds of Ipomoea muricata, I. turbinate and Calonyction muricata belonging to the family Convolvulaceae. Seeds are commonly known as ‘Kaladana’ in trade and are being used as a purgative in India and Pakistan.⁵ LYZ is an indole alkaloid present in the microfungi of Claviceps purpurea as well.⁶ It is one of the minor constituents of the ancient Mexican hallucinogenic drug Ololiuqui, which is obtained from Rivea corymbosa seeds.⁷ LYZ has been used by the 17^th century midwives to induce labour and stop postpartum bleeding because of its ability to induce uterine contractions. It may also cause ergot poisoning, diarrhea, hallucinations, delirium, seizures, burning sensations, and gangrene in the limbs.^8,9 However, it is not being used presently in the clinic. Nevertheless, it has only a weak central stimulating activity at 20 mg kg⁻¹ dose in mice.¹⁰ The in vitro studies have investigated the bioenhancing potential of LYZ and reported that it facilitated the transport of the antibiotics across the membrane for better efficacy on the target site.⁴ However, the mechanistic investigation is not yet reported.

Fig. 1 Chemical structure of lysergol.

Curcumin (CUR), the principal curcuminoid from miracle Indian spice turmeric, has a plethora of medicinal capabilities. The bottlenecks in the use of CUR are its low aqueous stability, poor absorption from the gut, rapid elimination and poor oral BA. Many attempts have been made to enhance the BA of CUR so far. Several formulations of CUR (nanoparticles, liposomes, micelles and phospholipid complexes) have been prepared leading to improved BA, better permeability and resistance to metabolic processes. Still a dependable and easily applicable approach is up in the air. Apart from the modified formulation approaches, natural compounds have also been used to increase the BA of low bioavailable drugs and CUR.¹¹ In humans, BA of CUR was increased by 2000% on coadministration with piperine; whereas in rats, it has been found that concomitant administration of piperine (20 mg kg⁻¹) with CUR (2 g kg⁻¹) increased the serum concentration of CUR by 154% for a short period of 1–2 h post dose.¹² Piperine is the world's first herbal bioavailability enhancer and generally regarded as safe for the intended use as per US Food and Drug Administration.¹³ LYZ has also shown a similar effect when used with antibiotics. However, its bioenhancing ability for drugs other than antibiotics has not been explored. The revival of the interest in phytopharmaceuticals prompted us to explore the bioenhancing potential of LYZ. Also, its potential to inhibit phase I and II metabolic enzymes and major efflux transporters is scrutinized.

2. Materials and methods

2.1 Materials

LYZ (purity ≥98.0%) was obtained as a gift sample from Chemical Resources (Haryana, India). CUR (purity ≥98.0%), phenacetin, verapamil, digoxin, propranolol, sulfasalazine, pantoprazole, phenol red, glucose, nicotinamide adenine dinucleotide phosphate reduced tetra sodium salt (NADPH), uridine 5′-diphosphoglucuronic acid trisodium salt (UDPGA), alamethicin, saccharolactone, ammonium acetate, tert-butyl methyl ether, sodium bicarbonate (NaHCO₃), sodium phosphate dibasic (NaH₂PO₄), sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl₂), calcium chloride (CaCl₂), acetonitrile (ACN) and methanol (MeOH) were purchased from Sigma Aldrich (St. Louis, USA). HPLC grade n-hexane and ethyl acetate were procured from Spectrochem (Mumbai, India). The Gentest™ ATPase assay kit was purchased from Corning (Woburn, USA). Rat liver microsomes were prepared by the method of Nelson et al.¹⁴ with slight modifications. Ultrapure water (18.2 Ωm) was from a Milli Q PLUS PF (Billerica, USA) water purification system. Krebs–Ringer buffer containing 20 μg mL⁻¹ of phenol red was used as perfusion buffer. The perfusion buffer contained 133.3 mmol L⁻¹ NaCl, 4.7 mmol L⁻¹ KCl, 0.2 mmol L⁻¹ MgCl₂, 3.3 mmol L⁻¹ CaCl₂, 2.7 mmol L⁻¹ NaH₂PO₄, 7.8 mmol L⁻¹ glucose, 16.3 mmol L⁻¹ NaHCO₃ and 56.4 μmol L⁻¹ phenol red in 1000 mL Milli Q water and was adjusted to pH 7.4 using concentrated phosphoric acid.

Drug-free blood was collected from adult and healthy male Sprague Dawley rats provided by the Laboratory Animal Services Division of the institute and serum was separated after centrifuging the collected blood at 3000g for 10 min. Following selectivity screening, the collected serum was pooled and stored at −80 °C till use. All experiments, euthanasia and disposal of carcasses were carried out as per the guidelines of local ethics committee for animal experimentation.

2.2 Instrumentation

2.2.1 Liquid chromatography equipment and conditions. An UFLC system (Shimadzu, Kyoto, Japan) consisting of LC-20AD UFLC pumps and DGU-20A3 online degasser was used to deliver the mobile phase [85% ACN in aqueous ammonium acetate buffer (AAB; 0.01 M, pH 4.5)] for CUR and sulfasalazine at a flow rate of 0.6 and 0.7 mL min⁻¹, respectively. The AAB was filtered through Ultipor 0.45 μm membrane filter (Pall, Michigan, USA) and degassed for 15 min in an ultrasonic bath (Bransonic, Danbury, USA) before use. For digoxin, MeOH : Milli Q water (80 : 20, %v/v) with 0.1% formic acid was used as mobile phase and delivered at a flow rate of 0.6 mL min⁻¹. Aliquots (20 μL) of the clear solution of the dry extractives reconstituted in mobile phase were injected through a temperature-controlled (10 °C) peltier-tray equipped with autosampler (SIL-HTc) onto the mass spectrometer. Chromatographic separation of CUR was achieved on a Zorbax SB-CN column (100 × 4.6 mm, 3.5 μm; Agilent, Santa Clara, USA) preceded by a guard column (20 × 4.0 mm, 3.5 μm; Agilent, Santa Clara, USA) packed with the same material using isocratic elution. The chromatographic separation of digoxin and sulfasalazine were achieved on a Discovery HS C-18 column (50 × 4.6 mm, 5 μm; Supelco Analytical, Bellefonte, USA) preceded by a guard column (20 × 4.6 mm, 5 μm; Supelguard, Discovery HS C-18, Bellefonte, USA) packed with the same material using isocratic elution. Column oven (CTO-10AS) temperature was set to 40 °C during analysis. Before and after injection of the sample, a rinsing solution [ACN : Milli Q water; 1 : 1] was injected to minimize the carryover, if any.

2.2.2 Mass spectrometry. A hybrid triple quadrupole/LIT (linear ion trap) mass spectrometer (API 4000 QTrap, Applied Biosystems, Toronto, Canada) equipped with Turbo V ion source using standard electrospray ionization (ESI) was used for quantification of the analytes and phenacetin (IS). The quantification was performed using an ESI in positive ion mode (ion spray voltage, 5500 V) coupled with the UFLC system. Optimized multiple reaction monitoring (MRM) parameters for analytes and phenacetin (IS) are summarized in Table 1. Zero air was used as source gas, while nitrogen was employed as both collision and curtain gas. Control of the equipment, data acquisition and analysis of data were controlled by Analyst™ (version 1.4.2; Applied Biosystems, Toronto, Canada).

Table 1 Optimized multiple reaction monitoring parameters for analytes and internal standard^a

	Analytes			Phenacetin (IS)
	Curcumin	Digoxin	Sulfasalazine
a Abbreviations: CE, collision energy; CXP, collision cell exit potential; DP, declustering potential; EP, entrance potential; ISV, ion spray voltage; GS1, nebulizer gas; GS2, heater gas; Q1, parent ion; Q3, product ion; T, temperature.
Compound parameters
Q1	369.3	798.7	399.1	180.1
Q3	177.1	651.4	381.1	138.2
DP (V)	75	60	135	60
EP (V)	10	11	10	10
CE (V)	30	18	30	25
CXP (V)	10	10	8	10
CG (psi)	10	10	10	10
Source parameters
ISV (V)	5500	5500	5500	5500
T (°C)	500	500	550	500
GS1 (psi)	45	45	50	45
GS2 (psi)	55	55	40	55

---

2.2.3 Sample cleanup. A simple two-step liquid–liquid extraction (LLE) was applied for extraction of the analytes and IS as well as the removal of endogenous components. Digoxin, sulfasalazine and CUR were extracted using ethyl acetate, tert-butyl methyl ether and n-hexane : ethyl acetate (60 : 40, %v/v), respectively. To the blank, spiked or test serum/intestinal perfusate samples (50 μL) in 1.5 mL microcentrifuge tubes, methanolic solution (10 μL) of IS (500 ng mL⁻¹) was added and vortexed for 15 s. Following the addition of the extraction solvent (1 mL) through Dispensette Organic (Brand, Wertheim, Germany) and vortexing for 5 min, it was centrifuged at 3000g at 4 °C for 10 min. The organic layer (0.9 mL) was separated and evaporated to dryness at 40 °C in Turbo vap LX (Caliper, Hopkinton, USA). The extraction procedure was repeated with another 1 mL of the extraction solvent for maximum recovery. The residue was reconstituted in 100 μL of the mobile phase, vortex-mixed for 5 min and centrifuged at 3000g at 4 °C for 10 min. Clear supernatant (70 μL) was transferred into HPLC vial and 20 μL was injected onto the analytical column and assayed using LC-MS/MS system.

2.3 In situ single pass intestinal perfusion (SPIP) study of curcumin with and without lysergol

Preliminary experiments were carried out to check any adsorption of the drugs (CUR and LYZ) with the tubing and syringe employed in the study. Drugs solutions were perfused through the tubing for the intended time of the experiment and the concentrations in the perfusate were determined and compared to the inlet concentration of the drug. The study revealed that no considerable adsorption of the compounds on the tubing and syringe took place. The stability of the compounds was tested in the perfusion solution and the blank perfusate obtained from rat intestine by incubation at 37 ± 0.5 °C for 2 h. The blank perfusate was obtained by passing the blank perfusion buffer (Krebs–Ringer buffer, pH 7.4) through jejunum in situ at a flow rate of 0.2 mL min⁻¹. The samples were analyzed by LC-MS/MS method. There was no sign of degradation of the compounds during this period of time. LYZ is reported to be labile to degradation when exposed to light for longer durations and, therefore, the samples were protected from light. However, no stability issues were observed during the intended duration of the experiment.

The study was conducted in two groups of rats (each n = 3), namely group I (CUR only) and group II (CUR with LYZ). Briefly, young and healthy male Sprague Dawley rats were kept for overnight fasting with free access to water prior to the commencement of the study. Urethane (1 g kg⁻¹) was used as an anesthetic agent and given intra-peritoneally. A heating pad was used to maintain the body temperature of rat. A midline longitudinal abdominal incision was made using a surgical blade and the jejunum part of the intestine was held out and a 10 cm length was marked. The proximal end of the lumen was catheterized with an inlet polypropylene tube, which was connected to a peristaltic perfusion pump (Gilson, Minipuls 3, France). The distal end of the jejunum was also catheterized with an outlet polypropylene tube to collect intestinal effluent. The part of jejunum was flushed with a pre-warmed (at 37 °C) perfusion buffer until a clear buffer passes out from the distal end to make sure the part of intestine intended to be used is clean. The whole procedure was carried out in a neat environment and extra care was taken to minimize the surgery and to avoid any damage to the mesenteric blood circulation. The intestinal parts other than the 10 cm under study were put back into the abdominal cavity cautiously and the excised part was covered with an absorbable cotton soaked with perfusion buffer pre-warmed at 37 °C to avoid any dehydration. The cotton pad was replaced with a fresh one after certain time interval. The perfusion buffer at a flow rate 0.2 mL min⁻¹ was allowed to run for 30 min to attain an equilibrium state. To keep a check on intra- and inter-subject variability and steady-state, phenol red was used as non-absorbable marker. After attaining steady-state outlet concentrations of phenol red, the outlet perfusate were collected after every 15 min upto 2 h. Finally, the animals were euthanatized with a cardiac injection of saturated solution of urethane. Rats of the group I were perfused with CUR (2 μg mL⁻¹) dissolved in perfusion buffer. The solution was kept on a magnetic stirrer with a temperature regulator (at 37 °C) to maintain homogeneity throughout the experiment. Group II rats were perfused with perfusion buffer containing both CUR (2 μg mL⁻¹) and LYZ (10 μg mL⁻¹). The collected samples were stored at −80 °C until analysis.

The net water flux (NWF) per cm of jejunum and corrected outlet concentration of CUR (C_out(corr)) were calculated using the following equations:¹⁵

where, C_out = outlet concentration of CUR, CPR_in = concentration of phenol red entering the intestinal segment and CPR_out = concentration of phenol red exiting the intestinal segment. NWF value will be negative, if intestinal lumen absorbs water, while NWF is positive if intestinal lumen secretes water. The steady-state effective permeability was calculated using the following equation:
where, P_eff = effective permeability coefficient, Q = perfusion flow rate, C_in = inlet CUR concentration, r = radius of intestinal segment and l = length of perfused intestinal segment.

2.4 Pharmacokinetic study of curcumin on coadministration with lysergol in rats

The effect of pre-treatment with LYZ at a single 20 mg kg⁻¹ dose on the oral PK of CUR (100 mg kg⁻¹) in Sprague Dawley rats was examined. Young (10–12 weeks) and healthy male rats (body weight, 225 ± 25 g) were procured from the National Laboratory Animal Centre, CDRI (Lucknow, India). All experiments, euthanasia and disposal of carcasses were carried out as per the guidelines and the study protocols submitted to the local ethics committee at CSIR-Central Drug Research Institute for animal experimentation (IAEC approval no.: IAEC/2015/14 dated 15 April 2015).

To investigate the PK profile, overnight-fasted (14–16 h; free access to water) rats were divided into two groups (each n = 4) namely, CUR only (group I) and CUR with LYZ (group II). Formulations for oral dosing of CUR and LYZ were prepared as suspensions in Milli Q water using gum acacia (1% w/v). The group I rats were administered CUR suspension (100 mg kg⁻¹) per oral and the group II rats were given LYZ suspension (20 mg kg⁻¹) 15 min prior to the CUR dose. Blood (∼100 μL) was collected in microtubes (Axygen, California, USA) from the caudal vein by excising the tail. Blood samples were withdrawn at 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12 and 24 h post dose. The blood was allowed to clot, centrifuged at 3000g for 10 min at 4 °C and the serum was separated and stored at −80 °C until analysis. Test samples (10 μL) were assayed along with the calibration standards and QC samples prepared in rat serum using the developed and validated LC-MS/MS method and the levels of CUR were calculated using Analyst™ (version 1.4.2; Applied Biosystems, Toronto, Canada).

2.5 Pharmacokinetic analysis

The serum concentration–time data were subjected to one-, two- and three-compartmental models using Phoenix WinNonlin (version 6.3; Certara Inc., St. Louis, USA). The maximum serum concentration (C_max) and its time of occurrence (T_max) were observed directly from concentration–time profile. The PK models were compared according to the maximal correlation between observed and predicted concentration, minimal sum of squared residuals, Akaike's Information Criterion (AIC) and Schwarz Bayesian Criterion (SBC).^16,17 The PK parameters (AUC = area under the serum concentration–time curve, T_1/2 = elimination half-life, Cl/F = clearance, V_d/F = volume of distribution) were subjected to statistical analysis using GraphPad Prism (version 5.01; GraphPad Software Inc., La Jolla, CA, USA). The relative BA (%) of CUR suspension on coadministration with LYZ to the CUR suspension (reference formulation) was calculated with the following equation:

2.6 In vitro metabolic stability studies

In vitro phase I metabolic stability study of CUR (1 μM) was conducted in a bench-top Lab-Line shaker (Julabo SW23, Germany) for 1 h at 37 ± 0.5 °C following preincubation with and without LYZ for 30 min. An aliquot (1.84 μL) of stock solution of CUR (100 μg mL⁻¹) was spiked in pre-incubated metabolic reaction mixture [50 mM Tris buffer (pH 7.4), 0.5 mg mL⁻¹ protein of rat liver microsomes, 20 mM MgCl₂ and 2 mM NADPH] resulting in a final volume of 500 μL. Testosterone was used as positive control and the metabolic reaction mixture without NADPH was used as negative control under similar experimental conditions. Aliquots (50 μL) were withdrawn at 0, 2, 5, 10, 15, 30, 45 and 60 min and quenched with 150 μL of ice cold ACN.¹⁸ Samples were vortex-mixed (Maxi-mix, Thermolyne, USA) for 1 min followed by centrifugation (Sigma, 3-16K, Germany) at 4 °C for 10 min at 3000g. The clear supernatant (100 μL) was transferred into HPLC vials and 20 μL was injected onto the analytical column and assayed using LC-MS/MS system. The study was carried out at different LYZ concentrations ranging from 1 to 80 μM.

Glucuronidation has already been established as the major phase II metabolic pathway of CUR. Therefore, in vitro phase II metabolic stability study of CUR (1 μM) was carried out for 1 h at 37 ± 0.5 °C following pre-incubation with and without LYZ for 30 min. The rat liver microsomes (0.5 mg mL⁻¹), 50 mM Tris buffer (pH 7.4) and 25 mg of alamethicin were mixed and placed on ice for 15 min. To this, MgCl₂ (20 mM), saccharolactone (5 mM), UDPGA (5 mM) were added and the mixture was preincubated at 37 °C for 3 min. To initiate the reaction, 1.84 μL of stock solution of CUR (100 mg mL⁻¹) was added to give a 500 μL final volume. Blank incubations were performed without UDPGA. Aliquots (50 μL) were withdrawn at 0, 2, 5, 10, 15, 30, 45 and 60 min and the reaction was stopped with addition of 150 μL of ice-cold ACN.¹⁹ The clear supernatant (100 μL) obtained after vortex mixing and centrifugation was injected onto the analytical column and assayed using LC-MS/MS system. The study was carried out at different LYZ concentrations ranging from 20 to 80 μM. The rate constant of metabolism in liver microsomes (k) was calculated directly from the slope of log of % drug remaining versus time profile. The in vitro half-life (t_1/2) was calculated from t_1/2 = 0.693/k.

2.7 ATPase assay for P-gp and BCRP

As per the manufacturer's protocol, drug stimulated P-gp/BCRP ATPase activity was estimated by measuring the inorganic phosphate released from ATP. Briefly, 20 μg of human P-gp/BCRP membrane (20 μL of 1 mg mL⁻¹) was preincubated at 37 °C for 5 min in a 40 μL reaction mixture with each test compound in the absence or presence of 300 μM sodium orthovanadate in 96-well plates. The reaction was initiated by the addition of 20 μL of 12 mM Mg ATP solution and was terminated by the addition of 30 μL of stop solution (10% sodium dodecyl sulfate solution). The incubation time for P-gp and BCRP membrane were 20 and 10 min, respectively. Two hundred μL of detection reagent (8% ascorbic acid, 0.8% ammonium molybdate, 3 mM zinc acetate) was added and the mixture was incubated at 37 ± 0.5 °C for 20 min to allow color development. The inorganic phosphate complex was detected by its absorbance at 800 nm and was quantified by comparing the absorbance with that of a phosphate standard. The validity of the P-gp assay was assessed by using verapamil and propranolol as positive and negative controls, respectively. The validity of the BCRP assay was assessed by using sulfasalazine and propranolol as positive and negative controls, respectively. The vanadate-sensitive ATP hydrolysis was determined by subtracting the vanadate-free membrane fraction value from the vanadate-coincubated membrane fraction values.

2.8 In situ single pass intestinal perfusion and pharmacokinetic studies of digoxin (P-gp substrate)

To assess the effect of LYZ on digoxin, in situ SPIP study of digoxin was carried out in presence and absence of verapamil (proven P-gp inhibitor). The similar study was conducted with LYZ to explore its potential as P-gp inhibitor. The study was conducted in three groups of rats [group I, digoxin only (20 μM); group II, digoxin (20 μM) with LYZ (200 μM) and group III, digoxin (20 μM) with verapamil (200 μM); each n = 3]. The protocol for the study and calculations were same as that for the in situ SPIP study of CUR. Propranolol (100 μM) and phenol red (50 μM) were perfused along with the digoxin. Propranolol was used as a high permeation marker and phenol red was used as non-absorbable marker.

To confirm the results obtained from SPIP study, in vivo PK study of digoxin on coadministration with verapamil or LYZ in rats was carried out. The effect of pre-treatment with LYZ (20 mg kg⁻¹) or verapamil (20 mg kg⁻¹) on the oral PK of digoxin (0.4 mg kg⁻¹) in Sprague Dawley rats was examined. Suspension formulations of digoxin, verapamil and LYZ were prepared separately using 1% w/v gum acacia and Milli Q water. The rats were divided into three groups (each n = 4), namely group IV (digoxin only), group V (digoxin with LYZ) and group VI (digoxin with verapamil). Group IV rats were administered digoxin suspension per oral and group V rats were given LYZ suspension 15 min prior to the digoxin dose. Group VI rats were administered verapamil suspension 15 min prior to the digoxin dose. Blood samples were withdrawn at 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12 and 24 h post dose. Serum was harvested and stored at −80 °C until analysis. Test samples (10 μL) were assayed along with calibration standards and QC samples prepared in rat serum using validated LC-MS/MS method.

2.9 In situ single pass intestinal perfusion and pharmacokinetic studies of sulfasalazine (BCRP substrate)

For assessment of the effect of LYZ on sulfasalazine, in situ SPIP study was carried out in presence and absence of pantoprazole (known BCRP inhibitor). The similar study was conducted with LYZ to explore its potential as a BCRP inhibitor. The study was conducted in three groups of rats [group I, sulfasalazine only (100 μM); group II, sulfasalazine (100 μM) with LYZ (40 μM) and group III, sulfasalazine (100 μM) with pantoprazole (40 μM); each n = 3]. The protocol for the study was same as that for the in situ SPIP study of digoxin.

The effect of pre-treatment with LYZ (20 mg kg⁻¹) or pantoprazole (20 mg kg⁻¹) on the oral PK of sulfasalazine (60 mg kg⁻¹) in Sprague Dawley rats was also examined. Suspension formulations of sulfasalazine, pantoprazole and LYZ were prepared separately using gum acacia (1%, w/v) and Milli Q water. The rats were divided into three groups [group IV, sulfasalazine only (60 mg kg⁻¹); group V, sulfasalazine (60 mg kg⁻¹) with LYZ (20 mg kg⁻¹) and group VI, sulfasalazine (60 mg kg⁻¹) with pantoprazole (20 mg kg⁻¹); each n = 4]. Group IV rats were administered sulfasalazine suspension per oral and group V rats were given LYZ suspension orally 15 min prior to the sulfasalazine dose. Group VI rats were administered pantoprazole suspension 15 min prior to the sulfasalazine dose. Blood samples were withdrawn at 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12 and 24 h post dose. Serum was harvested and stored at −80 °C until analysis. Test samples were analyzed using the developed and validated LC-MS/MS method.

3. Results and discussion

3.1 Effect of lysergol on intestinal permeation of curcumin

Intestinal permeability of CUR and permeability markers was determined in jejunum segment of rat intestine using the in situ SPIP technique. CUR and phenol red levels were analyzed using the developed LC-MS/MS method and calorimetrically, respectively. Effective permeability (P_eff) values were calculated by measuring the concentrations of compounds in the perfusate collected from the intestine outlet.

The net water flux (NWF) and P_eff values of CUR and permeability markers were determined as the average of three sampling points from the three rats after the steady-state had been achieved (30 min). The NWF was found to be consistent and near zero signifying the viability, intactness of the intestinal membrane and transport system. The NWF of the phenol red was found to be 0.21 ± 0.02 μL h⁻¹ cm⁻¹ and 0.27 ± 0.06 μL h⁻¹ cm⁻¹ in groups I and II, respectively. The P_eff 10⁻⁴ (cm s⁻¹) of group I (CUR only) and group II (CUR with LYZ) rats were found to be 0.68 ± 0.21 cm s⁻¹ and 2.41 ± 0.18 cm s⁻¹ which was in agreement with the available values in literature.²⁰ A significant difference in the P_eff values between the two groups was observed, which infers that LYZ can enhance the BA of CUR by improving its permeability and absorption from the gut lumen (jejunum). The relative increase in the P_eff of group II rats was found to be 3.3-fold than that in group I rats.

The increase in the P_eff of CUR in presence of LYZ infers that LYZ facilitates the transport of the CUR across the gut membrane, thus increasing its absorption. Alike other bioenhancers, increased blood flow to the gastrointestinal tract and modifications in the permeability of the epithelial cells lining the intestinal membrane can be the possible mechanism of permeation enhancement by LYZ. Also, the probability of inhibition of intestinal efflux transporters like P-gp and BCRP by LYZ cannot be ruled out. To statistically analyze the effect of LYZ on the effective permeability of CUR, a comparison between the P_eff of groups I and II rats was made by employing two tailed unpaired t-test using GraphPad Prism. The statistical variations were tested at a confidence level of 95% and the results were found to be highly significant. The representative bar plot showing the difference between the P_eff values of the CUR in presence and absence of LYZ group is shown in Fig. 2. A significant difference was observed between the two group of rats, which infers that the LYZ has a significant effect on the P_eff of CUR.

Fig. 2 Effect of lysergol on the jejunal permeation of curcumin in rats. Data are shown as the mean ± SEM (each n = 3).

3.2 Pharmacokinetics of curcumin coadministered with lysergol

The PK study of CUR in male Sprague Dawley rats at 100 mg kg⁻¹ was performed with and without coadministration of LYZ (20 mg kg⁻¹) and the results revealed that the animals tolerated the treatment as no peculiarities in their behavior were observed. The CUR was monitored upto 24 and 48 h in both the group of rats. The serum concentration–time profile of CUR in group I and II rats is illustrated in Fig. 3 and the calculated PK parameters are listed in Table 2. For statistical analysis, an unpaired t-test was applied and the results revealed highly significant (AUC, Cl and T_1/2) and significant (C_max and V_d) differences between the PK parameters of the two group of rats. It is obvious from the Table 2 that the LYZ has a considerable effect on the PK profile of CUR, which otherwise poses encumbrance in its PK. A substantial increase in C_max and the AUC of CUR in presence of LYZ implies the BA enhancing potential of LYZ. This can be attributed to its potential of possessing the inhibitory action on intestinal efflux transporters and increasing blood flow to the GIT tract. The highly significant (p < 0.001) decrease in the Cl and increase in the T_1/2 of the CUR clearly indicate the alleviation in the metabolism of the otherwise rapidly metabolised CUR. By virtue of their metabolism inhibition properties, bioenhancers increase the sojourn of the drug and its metabolites in the body. Thus, the increased T_1/2 of CUR in presence of LYZ is explanatory and can be attributed to the intestinal and hepatic metabolic inhibitory action of LYZ. The decrease in V_d might be a consequence of drug interactions at the hepatic transporters level.²¹ The percent relative bioavailability of CUR in presence of the bioenhancer (LYZ) was found to be 1607.3 ± 419.8% affirming the bioenhancing potential of LYZ for drugs other than antibiotics confirming our hypothesis.

Fig. 3 Serum concentration–time plot of curcumin with and without coadministration of lysergol in male Sprague Dawley rats (n = 4 per group). Error bars represent ± SEM.

Table 2 Pharmacokinetic parameters of curcumin with and without coadministration of lysergol in male Sprague Dawley rats^ab

Parameters	Curcumin only	Curcumin with lysergol
a Values of pharmacokinetic parameters are mean ± SEM (n = 4).b Abbreviations: AUC, area under the serum concentration–time curve; Tmax, time to Cmax; Cl/F, clearance; Vd/F, volume of distribution; T1/2, elimination half-life, *p < 0.05, **p < 0.01 and ***p < 0.001.
Cmax (ng mL^−1)	21.6 ± 3.6	53.2 ± 2.1**
Tmax (h)	1.0 ± 0.0	1.9 ± 0.1
AUC (h ng mL^−1)	41.8 ± 3.1	1073.0 ± 86.0***
Cl/F (L h^−1 kg^−1)	2421.7 ± 188.6	102.9 ± 9.8***
Vd/F (L kg^−1)	5607.3 ± 190.2	2359.0 ± 711.0*
T1/2 (h)	4.1 ± 0.3	18.9 ± 0.8***
Relative bioavailability (%)	—	1607.3 ± 419.8

---

3.3 Effect of lysergol on phase I and II metabolism of curcumin

The in vivo PK parameters have proved to be an explanation in the role of LYZ in inhibiting the phase I and II metabolism of CUR. Further, in vitro phase I metabolic stability study of CUR (1 μM) using pooled rat liver microsomes revealed that preincubation with LYZ decreased the metabolism rate of CUR. The plot of the logarithm of % drug remaining versus preincubation concentration of LYZ is shown in Fig. 4. On increasing the preincubation concentration of LYZ from 1 to 80 μM, the in vitro t_1/2 of CUR increased from 12.1 to 21.1 min. However, preincubation with 20 to 80 μM concentrations of LYZ, the t_1/2 of CUR was almost similar (18.6–21.1 min) indicating the saturation of the inhibitory potential of LYZ above 20 μM concentration for phase I metabolic enzymes.

Fig. 4 Phase I (A) and phase II (B) metabolic depletion of curcumin in rat liver microsomes following preincubation with different concentrations of lysergol.

The in vitro phase II metabolic stability study of CUR (1 μM) also revealed the remarkable decrease in its metabolism rate on preincubation with LYZ. The in vitro t_1/2 of CUR increased from 6 to greater than 60 min on increasing the preincubation concentration of LYZ from 20 to 80 μM. Moreover, preincubation with 40 to 80 μM concentration of LYZ, the t_1/2 of CUR was almost similar (57.2–66.0 min) indicating the saturation of inhibitory potential of LYZ for phase II metabolic enzymes above 40 μM concentration. This immense potential of LYZ for inhibiting glucuronidation might be the major reason behind increased BA of CUR on coadministration with LYZ.

3.4 Human P-gp and BCRP ATPase assay

The enhanced effective permeability values of CUR obtained during SPIP studies guided our study to explore the potential of LYZ in inhibiting the activity of efflux transporters. P-gp and BCRP are the two major transporters involved in the intestinal efflux of xenobiotics. The affinity of LYZ, verapamil (positive control) and propranolol (negative control) for human P-gp and LYZ, sulfasalazine (positive control) and propranolol (negative control) for human BCRP were assessed by using ATPase activity assay. Monolayer based efflux assays were not used here because they are labour-intensive (due to cell culture) with sophisticated analytical requirements (limiting assay throughput). On the contrary, ATPase assays have higher throughput, a generic readout (release of inorganic phosphate) and are readily automated, but these assays are not capable of distinguishing between substrates and inhibitors.²²

The vanadate-sensitive ATPase activity of the tested compounds is shown in Fig. 5. On incubation with P-gp membrane, verapamil (20 μM) showed 3.27-fold increase in basal activity than that of the drug-free membrane, while the change in basal fold activity in the presence of negative control (propranolol, 20 μM) and (LYZ, 20 μM) was 0.78- and 0.83-fold, respectively, than that of the reference (drug-free membrane). The highly significant (p < 0.001) increase in ATPase activity in the presence of verapamil indicates efficient working of the system. Almost similar increase in basal activity in presence of LYZ as compared to negative control and an insignificant change than that of the drug-free membrane indicating no or very less affinity of LYZ for P-gp. However, it is a well-known fact that several compounds reported to be transported by P-gp do not stimulate the vanadate-sensitive ATPase activity of membrane preparations containing P-gp. It is hypothesized that these compounds are transported by P-gp with a low turnover rate that does not yield detectable amount of inorganic phosphate in the ATPase assay.²³ Therefore, further studies are warranted using LYZ with specific P-gp substrate and inhibitor before deriving any conclusion.

Fig. 5 Human P-gp ATPase activities of verapamil, propranolol and lysergol (A) and human BCRP ATPase activities of sulfasalazine, propranolol and lysergol (B). Data are shown as the mean ± SEM (each n = 3), *p < 0.05, **p < 0.01 and ***p < 0.001.

On incubation with human BCRP membrane, sulfasalazine (20 μM) and LYZ (20 μM) showed 9.9- and 7.9-fold increase respectively, in basal activity, than that observed for the drug-free membrane, while the increase in basal activity was only 2.9-fold in the presence of the negative control (propranolol, 20 μM). In the presence of sulfasalazine (proven BCRP substrate), highly significant (p < 0.001) increase in the ATPase activity indicates efficient working of the BCRP membrane system. The significant increase in basal activity in presence of LYZ indicates its affinity for BCRP. Moreover, further confirmatory studies are required to corroborate the role of LYZ as a BCRP inhibitor.

3.5 Effect of lysergol on intestinal permeation and pharmacokinetics of digoxin

Digoxin is a well-known P-gp substrate and verapamil is a proven P-gp inhibitor. Therefore, in situ SPIP study of digoxin in presence and absence of verapamil was carried out and the results were compared with the permeation study of digoxin in presence of LYZ. Table 3 summarizes the P_eff of digoxin in the presence and absence of verapamil or LYZ. The coperfusion of verapamil with digoxin resulted in significant (p < 0.05) increase (3.5-fold enhancement) in jejunal permeation of digoxin (Fig. 6). The increase in the P_eff of digoxin in the presence of LYZ was found to be statistically insignificant (p > 0.05). These results are in strong agreement with the results obtained from ATPase assay indicating no role of inhibition of P-gp mediated efflux in intestinal permeation enhancing effect of LYZ.

Table 3 Permeation of digoxin and sulfasalazine in presence and absence of verapamil or lysergol through isolated perfused rat jejunum

Drugs	Peff 10^−5 (cm s^−1)
Digoxin (20 μM)	0.43 ± 0.12
Digoxin (20 μM) + verapamil (200 μM)	1.49 ± 0.24
Digoxin (20 μM) + lysergol (200 μM)	0.63 ± 0.15
Sulfasalazine (100 μM)	0.64 ± 0.03
Sulfasalazine (100 μM) + pantoprazole (40 μM)	0.72 ± 0.02
Sulfasalazine (100 μM) + lysergol (40 μM)	0.89 ± 0.03

---

Fig. 6 Effect of verapamil or lysergol on the jejunal permeation of digoxin in rats. Data are shown as the mean ± SEM (each n = 3), *p < 0.05, **p < 0.01 and ***p < 0.001.

For authentication of the above findings, the in vivo PK studies of digoxin were carried out with and without LYZ coadministration in male Sprague Dawley rats. The serum concentration–time plot of digoxin with and without coadministration of verapamil or LYZ are shown in Fig. 7 and the PK parameters are summarized in Table 4. The verapamil treated rats (group VI) showed the highest concentration of digoxin in serum among the three groups followed by the LYZ treated rats (group V). It was notable that the C_max of digoxin from the LYZ co-treated group of rats (35.9 ± 2.0 ng mL⁻¹) was although lower than the verapamil co-treated group of rats, but it was significantly higher (p < 0.01) than the values (14.6 ± 0.8 ng mL⁻¹) obtained from the digoxin treated group of rats. The Cl/F of digoxin (27.7 ± 1.3 L h⁻¹ kg⁻¹) also got significantly decreased (p < 0.001) on co-treatment with verapamil as well as LYZ. The significant decrease in V_d/F might be a consequence of drug interactions at the hepatic transporter level.²¹ Thus, on comparison of PK parameters of digoxin obtained from verapamil or LYZ co-treated group of rats, it can be concluded that LYZ enhances the BA of digoxin (most evident from increased AUC and decreased Cl/F of digoxin) but the increased BA may not be fully attributed to the inhibition of P-gp. There are some other key factors like metabolism playing a crucial role that further need to be explored.

Fig. 7 Serum concentration–time plot of digoxin with and without coadministration of verapamil or lysergol in male Sprague Dawley rats (n = 4 per group). Error bars represent ± SEM.

Table 4 Pharmacokinetic parameters of digoxin with and without coadministration of verapamil or lysergol in male Sprague Dawley rats^ab

Parameters	Digoxin only	Digoxin with lysergol	Digoxin with verapamil
a Values of pharmacokinetic parameters are mean ± SEM (n = 4).b Abbreviations: AUC, area under the serum concentration–time curve; Tmax, time to Cmax; Cl/F, clearance; Vd/F, volume of distribution; MRT, mean residence time, *p < 0.05, **p < 0.01 and ***p < 0.001.
Cmax (ng mL^−1)	14.6 ± 0.8	35.9 ± 2.0*	71.0 ± 8.2***
tmax (h)	1.5	1.0	1.0
AUC (ng h mL^−1)	32.2 ± 3.3	62.5 ± 2.1*	120.4 ± 10.8***
Vd/F (L kg^−1)	16.0 ± 0.4	4.5 ± 0.2***	2.5 ± 0.2***
MRT (h)	2.0 ± 0.1	1.8 ± 0.1	2.2 ± 0.1
Cl/F (L h^−1 kg^−1)	12.7 ± 1.3	6.4 ± 0.2***	3.4 ± 0.4***

---

3.6 Effect of lysergol on intestinal permeation and pharmacokinetics of sulfasalazine

To confirm the potential of LYZ as a BCRP inhibitor as indicated by ATPase assay, in situ SPIP study of sulfasalazine was carried out with and without LYZ. A similar study was done on coperfusion of sulfasalazine with pantoprazole (a proven BCRP inhibitor²⁴) and the results were compared with LYZ coperfusion (Table 3). The coperfusion of LYZ with sulfasalazine resulted in highly significant increase (p < 0.001; Fig. 8) in jejunal permeation (1.4-fold enhancement) of sulfasalazine. However, the increase in the P_eff of sulfasalazine in the presence of pantoprazole was found to be just statistically significant (p < 0.05). These results are in agreement with the results obtained from ATPase assay and provided strong evidence regarding the role of LYZ as a BCRP inhibitor.

Fig. 8 Effect of pantoprazole or lysergol on the jejunal permeation of sulfasalazine in rats, *p < 0.05, **p < 0.01 and ***p < 0.001.

For the final testimony of the results obtained from ATPase and SPIP studies, in vivo PK studies of sulfasalazine with and without LYZ/pantoprazole were carried out. The serum concentration–time profile of sulfasalazine alone and on coadministration with pantoprazole or LYZ in male Sprague Dawley are shown in Fig. 9. The LYZ treated rats showed the highest concentration of sulfasalazine in serum among the three groups followed by pantoprazole treated rats.

Fig. 9 Serum concentration–time profiles of sulfasalazine with and without coadministration of verapamil or lysergol in male Sprague Dawley rats (n = 4 per group). Error bars represent ± SEM.

The PK parameters are summarized in Table 5. It was notable that the C_max of sulfasalazine from the LYZ co-treated (1910.0 ± 77.8 ng mL⁻¹) group of rats was significantly higher (p < 0.001) than the values (656.0 ± 19.9 ng mL⁻¹) obtained from the sulfasalazine treated group of rats. The Cl/F of sulfasalazine (3.8 ± 0.2 L h⁻¹ kg⁻¹) also got decreased on co-treatment with LYZ as well as pantoprazole. A highly significant increase (p < 0.001) in AUC and mean residence time of sulfasalazine was observed on coadministration with LYZ. The results obtained are rather more prominent than that obtained with a well-established BCRP inhibitor (pantoprazole) co-treatment. Therefore, PK studies are convincing the activity of LYZ as a BCRP inhibitor.

Table 5 Pharmacokinetic parameters of sulfasalazine with and without coadministration of lysergol or pantoprazole in male Sprague Dawley rats^ab

Parameters	Sulfasalazine only	Sulfasalazine with lysergol	Sulfasalazine with pantoprazole
a Values of pharmacokinetic parameters are mean ± SEM (n = 4).b Abbreviations: AUC, area under the serum concentration–time curve; Tmax, time to Cmax; Cl/F, clearance; Vd/F, volume of distribution; MRT, mean residence time, *p < 0.05, **p < 0.01 and ***p < 0.001.
Cmax (ng mL^−1)	656.0 ± 19.9	1910.0 ± 77.8***	1107.5 ± 47.1***
tmax (h)	1.8 ± 0.1	3.0 ± 0.5	2.0 ± 0.0
AUC (μg h mL^−1)	3.5 ± 2.3	15.9 ± 1.0***	4.7 ± 0.1
Vd/F (L kg^−1)	39.3 ± 1.9	12.7 ± 0.2***	31.0 ± 0.8**
MRT (h)	4.0 ± 0.1	4.7 ± 0.1***	3.8 ± 0.1
Cl/F (L h^−1 kg^−1)	17.3 ± 1.2	3.8 ± 0.2***	12.6 ± 0.3**

---

4. Conclusion

Poor intestinal absorption and rapid metabolism are the major culprits behind low oral BA of drugs including CUR.²⁵ Our studies showed remarkable bioenhancing potential of LYZ for CUR, which has low BA due to high phase I and II metabolism in liver.²⁵ Thus, the clinically relevant dose of CUR could be significantly reduced on coadministration with LYZ. The present work hints at the use of LYZ as a bioenhancer once drug–drug interactions have been fully explored. This could open new avenues for delivery strategies to improve the therapeutic performance of CUR and other such drugs.

Declaration of interest

The authors declare no conflicts of interest in this work.

Acknowledgements

The authors MS and SJ acknowledge Council of Scientific and Industrial Research (CSIR) for providing research fellowship for this CDRI communication (9255).

References

1. United States Food and Drug Administration, Guidance for industry: bioavailability and bioequivalence studies for orally administered drug products-general considerations, 2003, Guidance Compliance Regulatory Information, Accessed on 10 Aug 2015, http://www.fda.gov/Drugs/.
2. G. B. Dudhatra, S. K. Mody, M. M. Awale, H. B. Patel, C. M. Modi, A. Kumar, D. R. Kamani and B. N. Chauhan, Sci. World J., 2012, 2012, 1–33.
3. Ajazuddin, A. Alexander, L. Qureshi, P. Kumari, M. Vaishnav, S. Sharma and S. Saraf, Fitoterapia, 2014, 97, 1–14.
4. S. Patil, R. P. Dash, S. Anandjiwala and M. Nivsarkar, Biomed. Chromatogr., 2012, 26(10), 1170–1175.
5. C. I. Abou-Chaar and G. A. Digenis, Nature, 1966, 212(5062), 618–619.
6. A. Stoll, A. Hofmann and F. Troxler, Helv. Chim. Acta, 1949, 32(2), 506–521.
7. A. Hofmann and H. Tscherter, Experientia, 1960, 16(9), 414.
8. A. Hofmann, Bull. Narc., 1971, 23(1), 3–14.
9. A. Hofmann, Pharmacol. Toxicol., 1978, 16(1), S1–S11.
10. T. Yui and Y. Takeo, Jpn. J. Pharmacol., 1958, 7, 157–161.
11. P. Anand, A. B. Kunnumakkara, R. A. Newman and B. B. Aggarwal, Mol. Pharm., 2007, 4(6), 807–818.
12. S. Prasad, A. K. Tyagi and B. B. Aggarwal, Cancer Res. Treat., 2014, 46(1), 2–18.
13. United States Food and Drug Administration, GRAS notices, GRN No. 474, 2013, http://www.accessdata.fda.gov/scripts/fdcc/?set=GRASNotices%26id=474, Accessed on 25 May 2016.
14. A. C. Nelson, W. Huang and D. E. Moody, Drug Metab. Dispos., 2001, 29(3), 319–325.
15. P. Neerati, Y. A. Sudhakar and J. R. Kanwar, J. Cancer Sci. Ther., 2013, 5, 313–319.
16. K. Yamaoka, T. Nakagawa and T. Uno, J. Pharmacokinet. Biopharm., 1978, 6(2), 165–175.
17. G. Schwarz, Ann. Stat., 1978, 6(2), 461–464.
18. N. J. Hewitt, K. U. Buhring, J. Dasenbrock, J. Haunschild, B. Ladstetter and D. Utesch, Drug Metab. Dispos., 2001, 29(7), 1042–1050.
19. M. B. Fisher, K. Campanale, B. L. Ackermann, M. VandenBranden and S. A. Wrighton, Drug Metab. Dispos., 2000, 28(5), 560–566.
20. H. Ji, J. Tang, M. Li, J. Ren, N. Zheng and L. Wu, Drug Delivery, 2016, 23(2), 459–470.
21. A. Grover and L. Z. Benet, AAPS J., 2009, 11(2), 250–261.
22. J. W. Polli, S. A. Wring, J. E. Humphreys, L. Huang, J. B. Morgan, L. O. Webster and C. S. Serabjit-Singh, J. Pharmacol. Exp. Ther., 2001, 299(2), 620–628.
23. H. Glavinas, E. Kis, A. Pal, R. Kovacs, M. Jani, E. Vagi, E. Molnar, S. Bansaghi, Z. Kele, T. Janaky, G. Bathori, O. von Richter, G. J. Koomen and P. Krajcsi, Drug Metab. Dispos., 2007, 35(9), 1533–1542.
24. S. Kunimatsu, T. Mizuno, M. Fukudo and T. Katsura, Drug Metab. Dispos., 2013, 41(8), 1592–1597.
25. M. Metzler, E. Pfeiffer, S. I. Schulz and J. S. Dempe, Biofactors, 2013, 39(1), 14–20.

---

Footnote

† Authors contributed equally.

---