ABSTRACT
Objectives:
To study the inhibitory effect of novel indole derivative (NID) from Indian toad skin (Bufo melanostictus) on permeability glycoprotein (P-gp).
Materials and Methods:
Dried Indian toad skin was used to isolate NID with column chromatography, and its structure was elucidated by infrared spectra, 13C nuclear magnetic resonance (NMR), 1H NMR spectra, and liquid chromatography-mass spectrometry. Female Wistar rats were used to determine LD50, in vitro permeability studies were done with the intestinal sac method, and in vivo pharmacokinetic studies were carried out to prove the P-gp inhibition using the rat model.
Results:
The NID has shown increased clear permeability Papp (x10-6 cm/sec) significantly (p<0.001) from 1.04±0.11 to 2.90±0.08 in ileum 1.44±0.14 to 3.92±0.13 in jejunum this in vitro results confirmed that P-gp inhibited, this was further confirmed by in vivo studies in in vivo studies observed increased oral bioavailability of digoxin (DIG) significantly in NID treated groups from 3.26±0.25 to 7.47±0.18 ng/mL, the volume of distribution decreased from 232.56±64.59 to 86.57±7.04 L/kg. Area under the curve increased from 37.89±1.13 to 64.62±0.70 ng/mL/hr. This demonstrates NID increased the oral bioavailability of DIG significantly.
Conclusion:
Many compounds were isolated from the Indian toad skin. This NID was not reported earlier. Results demonstrate NID increased the oral bioavailability of DIG significantly. The isolated NID from Indian toad skin proved as a potent P-gp inhibitor in both in vitro and in vivo studies, and further studies are needed to develop as a possible new drug candidate.
INTRODUCTION
Toxic animals are widely distributed throughout the globe.1,2 Venomous animals are recognized as a new emerging source of new drug discovery and therapeutics.3 Recently many new bioactive compounds from different toads were reported.4 Toads belong to amphibians and Anura family. The toad skin and parotid glands play an essential role in the survival of amphibians from diverse conditions and predators.5,6 Toads possess two types of glands beneath their skin, mucous glands and granular glands. Mucous glands secrete thick mucus secretions, which are important to keep toad skin moist.7 Granular glands secrete acrid, toxic substances, which provides protection from predators.8 This acrid, toxic substance, when comes intact, induces inflammation, irritation, and vomiting sensations in toad predators.9 This glandular secretion chemically belongs to potent substances like steroids, alkaloids, peptides, proteins, and biogenic amines.10 New drug discovery is a challenge many active compounds extracted from plants, animals, fungi, other sources. There is still to discover new compounds from the above sources.11 Toad skin extracts have been widely used for treating many types of ailments in China and other countries as traditional alternative medicine. The chemical composition and pharmacological activities of toad skin remain unclear.12 Permeability glycoprotein (P-gp) is an important transporting protein present on the cell membrane that effluxes many xenobiotic substances like drug molecules out of cells.13 P-gp has a significant impact on drug absorption, distribution, metabolism, excretion and is associated with drug-drug interactions.14,15,16 P-gp is over-expressed on the surface of cancer cells and prevents drug entry into the tumor due to rapid and prolonged efflux mechanism.17,18 P-gp induces resistance to anticancer drugs, which leads to therapeutic failure. There are many phytochemicals and drugs reported as P-gp inhibitors but associated with severe side effects.19,20 An alternate approach is needed to overcome this issue by exploring new compounds from new sources,21,22 in this study toad skin extract studies for inhibitory action on P-gp. In this study, digoxin (DIG) was used as probe substrate,23 and verapamil (VER)24 was taken as standard inhibitor. The isolated novel indole derivative (NID) inhibited P-gp and enhanced the oral bioavailability of DIG in vivo studies.
MATERIALS AND METHODS
RESULTS
DISCUSSION
In this study, we isolated a new compound from toad skin and spectral data accessed the structure. Some other studies reported different compounds from toad skin,34 but this compound was not reported earlier. The MTD was determined according to OECD guidelines and found 5 mg/kg; others reported the LD50 of TSE as 400 mg/kg.35 In vitro studies proved that NID inhibited P-gp and enhanced the clear permeability of DIG. Some plant extracts have shown P-gp inhibition in in vitro studies, and several studies are reported as P-gp inhibitors.36 Few more studies reported that VER increases the oral bioavailability of DIG up to 60% but reported side effects;37 in our study, NID has shown better oral bioavailability of DIG. In a similar study, the compound from the Asiatic toad (Bufo gargarizans) inhibited P-gp and decreased the expression.38 Some studies explained that the herbal compounds inhibited P-gp and enhanced the oral bioavailability of its substrate drugs.39 A similar study reported that toad parasitoid gland secretion inhibited P-gp and increased the bioavailability of substrate drug.40 Our study also achieved P-gp inhibition and improved the bioavailability of DIG with NID.
CONCLUSION
The isolated compound from Indian toad skin is confined as a NID and unreported earlier; the compound significantly inhibited P-gp mediated transportation. In vivo studies revealed that NID increased the oral bioavailability of DIG. Co-administration of a drug with potent molecules like NID can alter transporter function to improve drug bioavailability.
Experimental
Sample collection and preparation
Adult live toads (45 to 50 g) were collected from the near places in Warangal and University surroundings. After collecting the toads, the skins were isolated carefully and shade dried at room temperature (27°C); after complete dryness soaked in methanol for 30 days in an amber-colored bottle, the supernatant was collected, evaporated to dryness using rotary evaporator, at the end, dark brown solid mass methanolic extract (44 g) was obtained. The methanol extract was extracted further with ethyl acetate; this ethyl acetate fraction (EAF) was collected. EAF was subjected to column chromatography on silica gel (100-200 mesh-Merck), eluted slowly in increasing polarity mixture of solvents like n-hexane, chloroform, ethyl acetate, ethanol, methanol, and water to obtain different fractions. five fractions were collected; fraction-2 was obtained as a pale gray colored compound, which on TLC produced a single spot. Further purification was done with acetone and methanol.25,26 The final isolated compound yield was found to be 800 mg.
Animals
Female and male Wistar rats were procured from Vyas Enterprises, Hyderabad, acclimatized for 10 days, then used housed in standard laboratory conditions.27 All experimental animal protocols were approved by the Ethics Committee of IAEC (approval number: IAEC/02/UCPSc/KU/2016).
Chemicals and other requirements
Acetonitrile (Merck-Mumbai), methanol (Merck-Mumbai), EA (Merck-Mumbai), DIG (Sigma Aldrich-Bangalore), VER (Lupin Pvt Labs-Pune-India) Equipment used are, n-hexane (Merck-Mumbai), chloroform (Merck- Mumbai), ethanol (Merck- Mumbai), high-performance liquid chromatography (HPLC) (Schimadzu, with phenominex C-18 column), biofuge-centrifuge (Heraeus instrument- Germany), chromatography column-borosilicate made, TLC aluminum Plates-Sigma Aldrich, ultra sonicator (Ramsit scientific equipement-Hyd), rotavapor-R-300 (Mumbai-India), oral feeding needle, syringe filters-minisart (sartorius stedim Biotech-Germany).
Toxicity studies
According to the OECD-423 guidelines maximum, tolerated dose (MTD) was determined using 15 female Wistar rats. Rats were divided into 5 groups (n=3), control group treated with normal saline, the second group given NID (5 mg/kg, p.o.), third group NID (50 mg/kg, p.o.), the fourth group NID (300 mg/kg, p.o.), fifth group NID (2000 mg/kg, p.o.). Toxic effects were recorded for 14 days during the period observed for mortality, physiological parameters like body weight changes, food intake, water intake, and behavioral changes in each animal noted.28
Characterization of NID using spectral data
Spectral analysis was done using liquid chromatography-mass spectrometry (LC-MS) analysis 2.6.1. The components were identified using mass spectral libraries The 1H spectra were recorded at 300 K on a spectrometer operating at 600.13 MHz (14.1 T) using a 5 mm inverse probe equipped with a z-shielded gradient. Nuclear magnetic resonance (NMR) samples were prepared by dissolving extract in 500 µL dimethyl sulfoxide and 1 µL dimethyl formamide as an internal standard. 13C NMR spectral reports were made by comparison of the observed chemical shift values with the reported values. An infrared (IR) spectrophotometer is used, and spectral data is used to find functional groups. ChemDraw pro 8.0 (Perkin Elmer) was used for structure assessment.
In vitro studies
Intestinal sac study was conducted according to the previously described methods.29 Rats were grouped and sacrificed using anesthetic ether; the intestine was surgically removed, flushed with 50-mL saline (5%). The small intestine was cut into two segments jejunum and ileum of equal length (5 cm). The probe drug (DIG 500 µg/mL) was dissolved in pH 7.4 isotonic Dulbecco’s phosphate buffered saline (D-PBS) containing 25 mM glucose. Similarly, DIG + VER (100 µg/mL), DIG + NID 2 mg/mL and DIG + NID 4 mg/mL loaded. And both ends of the sac were ligated tightly with surgical sutures. The sacs were placed in a beaker containing 40 mL of D-PBS, containing 25 mM glucose. The medium was pre-warmed at 37°C and pre-oxygenated with 5% CO2/ 95% O2 under bubbling with mixture gas, the transport of the DIG from apical to basolateral and basolateral to apical samples was collected periodically for 120 min periodically, the collected samples were stored at -20°C until analysis. The samples were analyzed by HPLC.
Calculation of clear permeability coefficient
The clear permeability coefficient (Papp) of DIG was calculated from the following equation:
Where dQ/dt: Transport rate of the drug in the serosal medium, A: is the surface area of the intestinal sacs, and C°: Initial concentration inside the sacs.30
Sample preparation for intestinal sac sample analysis
Samples were extracted using a simple protein precipitation method by adding acetonitrile (200 µL) to samples (100 µL). Samples were vortexed for 10 min and centrifuged at 6,000 rpm for 15 min. The resultant clean supernatant (20 µL) was injected and analyzed using HPLC (Supplementary Figure 1). The mobile phase consists of acetonitrile: Water 65:35. Flow rate: 1 mL/min, pressure: 115 kg.f/cm2, ultraviolet-detection at the following: 220 nm.
In vivo studies
Male Wistar rats were used kept one week for acclimatization during the period supplied with normal ad libitum and free access for water. After one week they were divided into 4 groups (n=6) the first group treated with DIG (0.5 mg/kg, p.o.). Second group treated with DIG (0.5 mg/kg, p.o.) +VER (2 mg/kg, p.o.), third group treated with DIG (0.5 mg/kg, p.o.) + NID (2 mg/kg, p.o.) fourth group DIG (0.5 mg/kg, p.o.) + NID (4 mg/kg.p.o). Blood sample were collected by picturing lateral tail vein31 at 0, 0.5, 1, 2, 4, 6, 8, 12 and 24 h time points. Samples were centrifuged and supernatant extracted with acetonitrile precipitation methods Samples were stored at -4°C until used for analyzed by HPLC (Supplementary Figure 2).32
Statistical analysis
All the pharmacokinetic parameters were analyzed using Phoenix WinNonlin version 8.3 kinetic software. The statistical analysis was performed using One-Way ANOVA followed by Bonferroni post-test and Graph Pad Prism version (8.0.2).
Structure assessment using spectral analysis
Based on the spectral analysis using LC-MS (Supplementary Figure 3), 1H NMR (Supplementary Figure 4) (Table 1) 13C NMR (Supplementary Figure 5) IR spectra (Supplementary Figure 6) and values the structure of NID is elucidated (Figure 1).
Toxicity assessment and determination of MTD
The mortality was found in three animals at 50 mg/kg treated groups, according to OECD-423 guidelines, comes under category-2,33 (LD 50 cut-off dose 25 mg/kg). At 5 mg/kg, the animals remained alive after the administration of NID. Body weight slightly decreased in NID 5 mg/kg, compared to control. Water intakes decreased somewhat in NIA 5 mg/kg, compared to control, and locomotor activity was not changed significantly (Figure 2A-D) (Supplementary Table 1). The MTD found to be 25 mg/kg.
In vitro studies
The Papp (x10-6 cm/sec) significantly increased (p<0.001) in NID treated groups from 1.04±0.11 to 2.90±0.08 in ileum, and 1.44±0.14 to 3.92±0.13 in jejunum compare to control group (Table 2) (Supplementary Figure 1).
In vivo studies
The plasma drug concentration of DIG significantly increased in NID treated groups compared to control and positive control groups (Figure 3) (Supplementary Table 2). Cmax increased >from 3.26±0.254 to 7.47±0.186 ng/mL, Tmax decreased from 27.17±13.85 to 9.88±1.13 h, AUMC from 371.27±18.16 to 530.57±16.52 ng.h2/mL, area under the curve increased from 37.89±1.132 to 64.62±0.70 ng.h2/mL, CL from 6.09±0.24 to 7.87±0.22 L/h/kg, volume of distribution decreased from 232.56±64.59 to 86.57±7.049 L/kg, mean residual time from 9.79±0.27 to 8.20±0.19 h significantly (p<0.001) (Table 3).
