Eremantholide C from aerial parts of Lychnophora trichocarpha, as drug candidate: fraction absorbed prediction in humans and BCS permeability class determination

Abstract

Background

Lychnophora trichocarpha (Spreng.) Spreng. ex Sch.Bip has been used in folk medicine to treat pain, inflammation, rheumatism and bruises. Eremantholide C, a sesquiterpene lactone, is one of the substances responsible for the anti-inflammatory and anti-hyperuricemic effects of L. trichocarpha.

Objectives

Considering the potential to become a drug for the treatment of inflammation and gouty arthritis, this study evaluated the permeability of eremantholide C using in situ intestinal perfusion in rats. From the permeability data, it was possible to predict the fraction absorbed of eremantholide C in humans and elucidate its oral absorption process.

Methods

In situ intestinal perfusion studies were performed in the complete small intestine of rats using different concentrations of eremantholide C: 960 μg/ml, 96 μg/ml and 9.6 μg/ml (with and without sodium azide), in order to verify the lack of dependence on the measured permeability as a function of the substance concentration in the perfusion solutions.

Results

Eremantholide C showed P_eff values, in rats, greater than 5 × 10⁻⁵ cm/s and fraction absorbed predicted for humans greater than 85%. These results indicated the high permeability for eremantholide C. Moreover, its permeation process occurs only by passive route, because there were no statistically significant differences between the P_eff values for eremantholide C.

Conclusion

The high permeability, in addition to the low solubility, indicated that eremantholide C is a biologically active substance BCS class II. The pharmacological activities, low toxicity and biopharmaceutics parameters demonstrate that eremantholide C has the necessary requirements for the development of a drug product, to be administered orally, with action on inflammation, hyperuricemia and gout.

Graphical abstract

Introduction

In general, the treatment of gouty arthritis requires anti-inflammatory agents, such as non-steroidal anti-inflammatory drugs (NSAIDs), colchicine and corticosteroids. Among them, indomethacin, a NSAID, is commonly prescribed. However, due to renal and cardiovascular toxicity and gastrointestinal effects, its use must be carefully analyzed and individualized. Colchicine and corticosteroids are alternative therapies to NSAIDs. However, they have serious side effects and the continued use is potentially harmful and requires monitoring [1, 2].

Besides that, for the management of hyperuricemia, xanthine oxidase inhibitors (allopurinol and febuxostat) and/or uricosuric agents (probenecid, benzbromarone and lesinurad) are used. However, the use of these agents is also associated with several side effects, such as gastrointestinal intolerance, nausea, skin rashes, undesirable cardiac effects and renal problems [1, 2].

Lychnophora trichocarpha (Spreng.) Spreng. ex Sch.Bip (Asteraceae), popularly known as “Brazilian arnica”, is a native plant of the Brazilian “cerrado”. Aerial parts of L. trichocarpha macerated in alcoholic or hydroalcoholic preparations are used in folk medicine to treat pain, inflammation, rheumatism and bruises [3–5].

Previous studies showed that the ethanolic extract of L. trichocarpha was able to reduce the inflammation and serum urate levels in hyperuricemic mice. These activities must be related to eremantholide C (Fig. 1), one of the active chemical constituents of L. trichocarpha’s ethanolic extract [6, 7].

Fig. 1.

Chemical structure of eremantholide C

Eremantholide C showed anti-inflammatory effects in models of carrageenan-induced and monosodium urate crystals-induced paw edema in Swiss mice. In addition, eremantholide C inhibited, in vitro, the production of TNF-cytokines and stimulated the production of IL-10 in macrophages J774A.1 [6, 7].

Regarding the anti-hyperuricemic activity, eremantholide C, at doses of 5 and 10 mg/kg, reduced serum uric acid to normal levels in Wistar rats. The hypouricemic effect produced by this sesquiterpene lactone was due to the inhibition of hepatic xanthine oxidase and increased excretion of uric acid in urine [8].

The pharmacological potential of eremantholide C and the side effects caused by drugs used in the therapy of patients with arthritis justify the biopharmaceutics studies of eremantholide C, in order to analyze its viability to become a drug to treat inflammation, hyperuricemia and gout.

The evaluation of absorption mechanisms of potential drug candidates can be a predictor of the in vivo behavior. Then, to elucidate the absorption process, helping to reach better oral bioavailability and reducing time and financial investment, several alternatives have been developed. Among them, stands out the Biopharmaceutics Classification System (BCS), which reports that solubility and permeability parameters can be used to determine whether a drug is a good candidate for oral administration [9].

According to the BCS, drugs are classified into four classes considering low/high solubility and low/high permeability [10]. Regarding the permeability, a drug is highly permeable when systemic bioavailability or extent of absorption in humans is equal to or greater than 85% and is not associated with any instability in the gastrointestinal tract [11–13].

Alternatively, methods, which do not involve humans, are able to predict the oral drug absorption. Among them, in situ intestinal perfusion in rats [12, 13].

In situ intestinal perfusion in rats shows good relationship with human intestinal absorption [14–16]. It has been increasingly used and improved in order to predict the oral absorption of compounds derived from plants, in their purified forms or in the context of herbal mixtures [17]. This type of study can be used to obtain critical biopharmaceutics knowledge about chemical markers [18] and it has been a tool in the decision-making process, supporting the selection of drug candidates in the early stages of discovery and development studies [19, 20].

Previous studies have indicated the oral absorption potential of eremantholide C with respect to solubility [21, 22]. However, regarding the permeability, only transcellular passive permeation data, obtained from Parallel Artificial Membrane Permeability Assay (PAMPA), are available [21].

Therefore, the purpose of this work was to evaluate the permeability of eremantholide C using in situ intestinal perfusion in rats. This study allowed to provide information about the absorption process of eremantholide C and if this substance would be a substrate for both influx and efflux transporters, which are directly related to the oral bioavailability. In addition, from permeability data, it was possible to predict the fraction absorbed of eremantholide C in humans and classify the substance according to the BCS.

Materials and methods

Materials

Acyclovir and propranolol hydrochloride were provided from the Brazilian Pharmacopeia/Fiocruz/INCQS (Rio de Janeiro, Brazil).

Sodium azide from Química Moderna (Barueri, SP, Brazil) was kindly donated by Drª Daniela Caldeira Costa, from Programa de Pós-Graduação em Ciências Biológicas (CBIOL)/NUPEB/UFOP.

Chloroform, hexane, ethyl acetate and dichloromethane were purchased from Qhemis (Indaiatuba, SP, Brazil). Monobasic potassium phosphate and sodium hydroxide from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide was obtained from Proquimios (Rio de Janeiro, Brazil). Acetonitrile HPLC grade was purchased from JTBaker (Xalostoc, Edo. de Mex., Mexico). Water was obtained from a Millipore purification system (Darmstadt, Germany). All other chemicals were of analytical grade or higher.

Eremantholide C

In July 2015, aerial parts of Lychnophora trichocarpha (Spreng.) Spreng. ex Sch.Bip. were collected in Ouro Preto, Minas Gerais, Brazil (20°23,053″S; 43°30,042″W) with the permission of Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA – licence number 49183–1).

Voucher specimens were deposited at Instituto de Ciências Exatas e Biológicas of Universidade Federal de Ouro Preto, reference number 20635.

The scientific research is registered in the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SISGEN) under number AF50E17.

Extraction and isolation of eremantholide C were performed according to described at Caldeira et al. [22], which allowed to obtain 10.2 g of eremantholide C (colourless crystalline powder, melting point: 234.7–241.6 °C; purity: 89.9% (HPLC analysis) and optical rotation: [α]²⁵_D = −10.0° (methanol)).

The chemical structure of eremantholide C was identified by analyses of melting point, optical rotation and NMR spectra. The data obtained are in agreement with those described by Le Quesne et al. [23], Saúde et al. [4] and Saúde-Guimarães et al. [24].

Perfusion solutions

Perfusion solutions were prepared using simulated intestinal fluid without enzymes (pH 6.8) as solvent. This buffer solution was prepared according to the United States Pharmacopeia 37th edition [25]: 250 ml of monobasic potassium phosphate 0.2 mol/l, 112 ml of sodium hydroxide 0.2 mol/l and enough water to complete 1000 ml, adjusted to pH 6.8.

From the list of drugs provided in US FDA BCS Guidance [13], acyclovir (1600 μg/ml) and propranolol hydrochloride (320 μg/ml) were selected as control drugs of low (fraction absorbed less than 50%) and high permeability (fraction absorbed equal or higher than 85%), respectively.

Eremantholide C was evaluated at three concentration levels, in geometric progression: 960 μg/ml, 96 μg/ml and 9.6 μg/ml, in order to verify the lack of dependence on the measured in situ permeability due to the drug concentration in the perfusion solutions [13].

Concentrations of control drugs and eremantholide C were obtained by dividing the highest single dose administered: 400 mg for acyclovir; 80 mg for propranolol hydrochloride and 240 mg for eremantholide C, by 250 ml (volume of water that drug product are administrated in bioequivalence studies). For being a drug candidate without a defined dose for humans, the dose of 240 mg for eremantholide C was obtained by allometric extrapolation reported by Caldeira et al. [21].

For acyclovir and eremantholide C (at three concentration levels), 1% of dimethyl sulfoxide (DMSO) was used to achieve complete dissolution of these substances in simulated intestinal fluid without enzymes (pH 6.8). This concentration of DMSO does not affect the intestinal membrane integrity of the rat [26, 27].

Perfusion solutions were maintained at 37 °C until use.

Animals

Male Wistar rats (250–300 g) were supplied by the Centro de Ciência Animal (CCA) of Universidade Federal de Ouro Preto. The animals were used in the in situ intestinal perfusion studies according to all the guidelines established by the Conselho Nacional de Controle de Experimentação Animal (CONCEA). The experimental protocol was approved by ethics committee (Comitê de Ética no Uso de Animais - CEUA) of Universidade Federal de Ouro Preto under protocol number 2018/07.

Intestinal perfusion

Effective permeability of eremantholide C, acyclovir and propranolol hydrochloride was determined in the complete small intestine of rats using in situ closed-loop perfusion based on Doluisio method [28], according to described in Caldeira et al. [29].

Male Wistar rats (n = 4–6) were anesthetized by intraperitoneal injection of ketamine and xylazine (90 mg/kg and 10 mg/kg, respectively). After complete sedation, the animals were placed on surgical plaques in dorsal decubitus position.

Complete small intestine (about 100 cm) was identified from an abdominal incision. After this, small incisions with 45° angle were performed at the extremities of the intestinal segment. Glass cannulas coupled to syringes with three-way stopcock were introduced at the extremities and fixed using nylon threads. The bile duct was blocked to prevent the release of bile salts into the intestinal segment.

Cannulated intestinal segment was washed, carefully, with perfusion solution free of drugs. After complete removal of the contents present in the intestinal lumen, 10 ml of the perfusion solution containing the control drugs or eremantholide C was introduced directly into the intestinal segment. Every 5 min until 30 min, aliquots of 200 μl were collected without replacement and alternating the extremities of the intestinal segment.

After collecting the last sample, the animals were euthanized by intraperitoneal administration of an overdose of ketamine and xylazine (270 mg/kg and 30 mg/kg, respectively).

The samples were filtered through of filtering units of PVDF 0.45 μm and immediately analyzed by chromatographic method [21, 30]. Besides that, the residual perfusion solution presents in the intestinal segment was collected and measured.

Analysis of results

In order to calculate the effective permeability and to predict the fraction absorbed, it was necessary to correct the experimental concentration from the water reabsorption presented by the animal [31, 32].

After this, from the experimental concentration corrected at each time (C_t), it was possible to determine the absorption rate coefficient (k_a) and, consequently, the effective permeability in rats (P_{eff rats}), using Eq. 1 [16, 31] and Eq. 2 [16, 32], respectively.

$$C_t=C_0.e^{-k_a.t}$$ 1

$${\mathrm{P}}_{\mathrm{eff}\text{rats}}=\frac{k_a.r}{2}$$ 2

where r = 0.18 cm (radius value estimated considering the intestinal segment as a cylinder – volume = πr²L).

The effective permeability for humans (P_{eff humans}) was calculated, from P_{eff rats} values, using Eq. 3 [33, 34].

$${\mathrm{P}}_{\mathrm{eff}\text{humans}}=3.6{\mathrm{P}}_{\mathrm{eff}\text{rats}}+0.03x{10}^{-4}$$ 3

Besides that, the P_{eff rats} values allowed to predict the fraction absorbed (F_abs) in rats using the Eq. 4 [16, 35] and in humans using the Eq. 5 [34, 36].

$${\mathrm{F}}_{\mathrm{abs}\text{rats}}=1-e^{-{\mathrm{P}}_{\mathrm{eff}\text{rats}}.\frac{2}{\mathrm{r}}.\mathrm{t}}$$ 4

$${\mathrm{F}}_{\mathrm{abs}\text{humans}}=1-e^{-38450\mathrm{x}{\mathrm{P}}_{\mathrm{eff}\text{rats}}}$$ 5

where P_{eff rats} is the effective permeability value obtained in rats (in cm/s); r is 0.18 cm and t is the intestinal transit time on the rat, previously estimated to be 3600 s [35].

Chromatographic conditions

HPLC system (Waters Alliance® e2695, Milford, MA, USA) coupled to UV (Waters 2489), PDA (Waters 2996) and FLU (Waters 2475) detectors, was used to analyze eremantholide C [21], acyclovir and propranolol hydrochloride [30], respectively.

Chromatographic conditions to quantify eremantholide C and propranolol hydrochloride were described, previously [21, 30]. Eremantholide C was quantified at 267 nm using column C18 (150 × 4.6 mm; 3 μm), acetonitrile and water (50:50) under 1.0 ml/min and 30 °C. The injection volume was 25 μl [21]. Propranolol hydrochloride was analyzed using 290 nm and 358 nm as excitation and emission wavelength, respectively, column C18 (50 × 4.6 mm; 5 μm), acetonitrile and phosphate buffer 40 mmol/l (28:72) pH 3.5 under 1.0 ml/min and 25 °C. The injection volume was 8 μl [30].

Lastly, acyclovir was quantified at 250 nm using column C18 (150 × 4.6 mm; 5 μm), acetonitrile and acidified water 0.1% (for this, acetic acid was used) (3:97) under 1.0 ml/min and 30 °C. The injection volume was 25 μl (unpublished data).

All analytical methods were validated, showing adequate selectivity, linearity (R > 0.99), precision and accuracy (coefficient of variation <5%). In addition, LLOQ of 0.35 μg/ml for eremantholide C and LLOQ of 14.98 μg/ml and 3.99 μg/ml for acyclovir and propranolol hydrochloride, respectively.

Statistical analysis

Absorption rate coefficient and effective permeability values were expressed as mean ± standard deviation (SD). Statistical analysis, using analysis of variance (ANOVA) and Tukey test, were performed by GraphPad Prism version 5.01 (GraphPad Software, INC., La Jolla, CA, USA). A p value <0.05 was considered statistically significant.

Results and discussion

Eremantholide C is a sesquiterpene lactone highly lipophilic, showing cLogP equal to 2.00 and effective permeability in PAMPA of 30.40 × 10⁻⁶ cm/s, value about 3 times higher than the permeability of propranolol hydrochloride (9.23 × 10⁻⁶ cm/s) [21].

PAMPA refers to the passive permeability presented by the substance, because only the transcellular passive route is evaluated in this experiment [37].

Therefore, it was necessary to evaluate the permeability of eremantholide C using a method that are as close as possible to what occurs in the organism in vivo.

A standardized protocol for in situ intestinal perfusion, besides to evaluate the permeability of drugs to allowing its biopharmaceutics classification, can elucidate the absorption processes that occur in the organism, in order to characterize, effectively, the permeation mechanism of the substance [19, 20]. This includes to evaluate the molecule affinity for transporters, verifying if it would be a substrate for uptake and/or efflux carriers, which have a direct impact on the fraction absorbed of the substance and, consequently, in its oral bioavailability [14, 38].

Intestinal permeability of a substance has a direct impact on the drug and formulation development process, and, consequently, in the success of the drug product. Therefore, permeability data are requested during the drug development process and it is a critical factor for the pharmacotechnical development and to ensure safety and efficacy of the potential new drug [38, 39].

The permeability of eremantholide C was evaluated throughout the complete small intestine in rats using the in situ intestinal perfusion technique. Absorption rate coefficient (k_a) and effective permeability in rats (P_{eff rats}) obtained for eremantholide C as well as control drugs of low and high permeability, acyclovir and propranolol hydrochloride, respectively, are shown in Table 1.

Table 1.

Absorption rate coefficient (k_a) and effective permeability in rats (P_{eff rats}) obtained for eremantholide C, acyclovir and propranolol hydrochloride, from in situ intestinal perfusion studies (mean ± SD)

Substance/Concentration	ka (min−1)	Peff rats (cm/s)
Acyclovir 1600 μg/ml	0.007 ± 0.002	1.09 × 10−5 ± 0.38 × 10−5
Propranolol hydrochloride 320 μg/ml	0.073 ± 0.011	10.94 × 10−5 ± 1.67 × 10−5
Eremantholide C 9.6 μg/ml	0.052 ± 0.021	7.81 × 10−5 ± 3.16 × 10−5
Eremantholide C 96.0 μg/ml	0.051 ± 0.010	7.71 × 10−5 ± 1.48 × 10−5
Eremantholide C 960.0 μg/ml	0.049 ± 0.003	7.28 × 10−5 ± 0.52 × 10−5
Eremantholide C + Sodium azide 9.6 μg/ml + 65.0 μg/ml	0.049 ± 0.009	7.31 × 10−5 ± 1.29 × 10−5

The results showed that the experimental protocol used for in situ intestinal perfusion technique was suitable to determine the effective permeability of drugs and/or biologically active substances, since high and low permeability were confirmed for propranolol hydrochloride and acyclovir, respectively. Propranolol hydrochloride presented the highest k_a value and consequently the highest effective permeability. Acyclovir, on the other hand, exhibited the lowest values of k_a and P_{eff rats}.

Absorption rate coefficient (k_a) is the parameter that regulates the rate at which a compound is absorbed under standard and reproducible conditions. This process is conditioned to factors characteristic of the technique, such as membrane surface area, pH of the perfusion solution, temperature and agitation, as well as parameters inherent to the drug, such as lipophilic character and molar mass [31]. In this way, the higher the k_a, the greater the amount of substance that crosses the intestinal membrane.

In order to determine the BCS permeability class and to evaluate if the permeation process occurs only by the passive transport mechanism, the intestinal perfusion of eremantholide C was evaluated using three different concentrations: 9.6 μg/ml, 96.0 μg/ml and 960.0 μg/ml.

For the three concentrations, effective permeability values were very close, with no statistically significant differences (p > 0.05) between them (Fig. 2).

Fig. 2.

P_{eff rats} values obtained for control drugs and eremantholide C from in situ intestinal perfusion studies (mean ± SD). *** p < 0.0001 compared to acyclovir

During BCS-based permeability determination, according to the FDA, an apparent passive transport mechanism can be admitted when in situ permeability measured, in an appropriate animal model, is independent of the administered dose, i.e., when lack of dependence is detected using 0.01, 0.1 and 1 times the highest dose dissolved in 250 ml in the perfusion solution [13].

In addition, when the absorption process occurs by passive diffusion, the first-order absorption kinetics is observed at any dose of the drug and the limiting factor is the solubility [40].

Besides the P_{eff rats}, no significant differences between the k_a values for eremantholide C concentrations were found. Therefore, it can be assumed that absorption of eremantholide C is a linear process over the range of concentrations studied. Consequently, the absorption mechanism of eremantholide C is a passive process and the absorption rate coefficient is not modified by a high or low substance concentration in the perfusion solution.

However, in order to confirm the lack of dose-dependent permeability, and to verify the possible influence of intestinal transporters, the in situ intestinal perfusion of eremantholide C at the concentration of 9.6 μg/ml in the presence of sodium azide 65.0 μg/ml was performed. The results obtained for the effective permeability of eremantholide C in the presence of sodium azide, as well as the comparison with the other P_{eff rats} values acquired for the three different concentrations of eremantholide C, are described in Table 1 and in Fig. 3.

Fig. 3.

P_{eff rats} values obtained for eremantholide C 9.6 μg/ml, 96.0 μg/ml, 960.0 μg/ml and 9.6 μg/ml with sodium azide 65.0 μg/ml (mean ± SD) from in situ intestinal perfusion studies (mean ± SD). No statistically significant differences were observed (p > 0.05)

Active transport system of substances in rats is ATP-dependent. Sodium azide, as a metabolic inhibitor, interferes the electron transport chain in the mitochondrial matrix, decreasing ATP levels. Therefore, the use of sodium azide in intestinal perfusion studies, in the rat model, promotes the inhibition of substance transport by both types of carriers (uptake and efflux) and, in this case, the P_eff value obtained is only a result of the passive permeability of the compound [41, 42].

As observed in Fig. 3, there were no statistically significant differences (p > 0.05) between the effective permeability values obtained for the three concentrations of eremantholide C and for eremantholide C in the presence of sodium azide. This fact confirms that the absorption process of eremantholide C occurs only through the passive route, and no affinity was observed for uptake and efflux transporters.

In the literature, there have been reported several variations in the experimental protocol of the in situ intestinal perfusion technique. Among them, it should be highlighted the single-pass intestinal perfusion (SPIP) [43] and closed-loop perfusion based on Doluisio method [28].

Both variations of the technique are widely used and a high correlation is obtained for the P_eff values using SPIP and closed-loop perfusion based on Doluisio method [16]. Regarding the fraction absorbed for humans, an excellent correlation can be observed comparing the P_{eff rats} values obtained using SPIP and closed-loop method with the humans data [16, 36]. Specifically, Zakeri-Milani et al. [36] showed coefficient of determination (R²) of 0.91 and 0.92, respectively, for the relationship between P_{eff rats} values and fraction absorbed for humans, and between observed and predicted fraction absorbed in humans. These results confirm a close relationship between rat and human permeability values, especially for passively absorbed substances. Therefore, consequently, fractions absorbed predicted from the permeability values obtained in rats, using both variations of the technique, shows a good correlation with intestinal permeation data of humans.

According to Zakeri-Milani et al. [44], drugs with P_eff values greater than 5.0 × 10⁻⁵ cm/s in rats or P_{eff humans} greater than 4.7 × 10⁻⁵ cm/s demonstrate F_abs greater than 85.0%. Thus, these values were defined as the cut-off points for the classification of highly permeable substances.

P_{eff humans} values predicted for eremantholide C, acyclovir and propranolol hydrochloride, from P_{eff rats} values obtained in the in situ intestinal perfusion studies are shown in Table 2.

Table 2.

P_{eff humans} and fraction absorbed (F_abs) in rats and humans predicted for eremantholide C, acyclovir and propranolol hydrochloride

Substance	Peff humans (cm/s)	Fabs in rats	Fabs in humans
Acyclovir Propranolol hydrochloride Eremantholide C	4.22 × 10−5 39.68 × 10−5 26.51 × 10−5	35.3% 98.7% 94.6%	34.2% 98.5% 93.9%

Both P_{eff rats} and P_{eff humans} values obtained for eremantholide C were higher than the established cut-off points [44], indicating that this biologically active substance has high permeability. In addition, no statistically significant differences were observed between the effective permeability obtained for propranolol hydrochloride and for eremantholide C, at the three concentrations evaluated (Fig. 2).

However, regulatory agencies recommend the classification of permeability according to the fraction absorbed of the drug in humans [11–13].

From the effective permeability results, it was possible to predict the fraction absorbed for eremantholide C and control drugs, in rats and humans. The results are also shown in Table 2.

As expected, acyclovir and propranolol hydrochloride showed F_abs in humans of 34.2% and 98.5%, respectively. Eremantholide C, on the other hand, presented fraction absorbed of 93.9%. This result is in agreement with the P_{eff rats} and P_{eff humans} values obtained for eremantholide C.

Therefore, using the cut-off point from Zakeri-Milani et al. [44] and the criteria described in the US FDA BCS Guidance for highly permeable drugs, in addition to the low solubility reported by Caldeira et al. [21], this work considers eremantholide C a biologically active substance BCS class II.

BCS class II drugs have high lipophilicity and, consequently, they are highly permeable through the intestinal membrane, being able to enter into the enterocytes by passive diffusion, without uptake transporters action. These drugs are characterized by absorption rate higher than the dissolution rate (controls the absorption rate in vivo), thus the dissolution process in vivo is the limiting step of absorption [10, 44].

Therefore, for an efficient drug and formulation development to be administered orally, it is essential to know about solubility, permeability and dissolution process. In view of the results, the proposed formulation to deliver, orally, eremantholide C, should contain excipients that assist the dissolution process and increase the degree of absorption throughout the intestine. Micronization, the use of surfactants and co-solvents, and complexation with cyclodextrins are resources that can be used during the formulation development to improve the solubility and the dissolution rate in the absorption process [45, 46].

In situ intestinal perfusion in rats also represents an efficient and reliable experimental tool in the verification of the presence of intestinal segment dependent absorption, mechanisms of permeability and drug interactions related to absorption [16].

Membrane transporters may be the major determinants of pharmacokinetics, safety, and drug efficacy profiles, because they control the uptake and efflux transport of important compounds, such as drugs, not only in the intestine but also throughout the organism [47].

BCS class II compounds, due to the high permeability, in general, not show variability in bioavailability when uptake transporters are inhibited. However, the low solubility presented by these substances will limit the concentrations that reach in the enterocytes. Thus, the saturation of efflux transporters is avoided and, consequently, drugs that are substrates for this type of transporter may have reduced oral bioavailability [47, 48].

Especially with respect to biologically active substances from plants, many of these, such as the sesquiterpenes isocalamenediol [49], isoalantolactona and alantolactona [50], are substrates of efflux transporters (e.g. P-glycoprotein or Multidrug Resistance-Associated Protein 2), and may have impaired oral bioavailability when administered alone, or induce toxicity, when administered in combination with substances that inhibit these transporters.

From the permeability results and due to moderate correlation in transporter expression levels between rat and human [14], it is possible to infer that eremantholide C exhibiting absorption only through the passive permeation route, probably, will not have impaired oral bioavailability due to the action of transporters. This fact, also, directly affects the proposed formulation to be used to deliver eremantholide C orally.

Conclusion

In situ intestinal perfusion in rats provided important information about the absorption process of eremantholide C. Besides confirming the high permeability of the substance, it showed that its permeation process occurs by passive route. Thus, the oral bioavailability of eremantholide C, probably, will not be impacted due to the action of efflux transporters, a fact commonly reported for BCS class II drugs, due to low solubility that limits the substance concentration in the intestine.

Therefore, the pharmacological activities presented for eremantholide C, in particular, anti-inflammatory and anti-hyperuricemic activities, low toxicity, and solubility and permeability characteristics comparable to several drugs, demonstrate that this substance has the necessary requirements for the development of a drug product, to be administered orally, with action on inflammation, hyperuricemia and gout.

Authors’ contributions

Tamires Guedes Caldeira performed the experiments and wrote the manuscript. Dênia Antunes Saúde-Guimarães supervised and guided some experimental activities and revised the manuscript. Isabel González-Álvarez and Marival Bermejo helped in the calculation and treatment of experimental data and gave suggestions on the revision of the manuscript. Jacqueline de Souza coordinated, supervised and guided the experimental activities and revised and gave suggestions on the revision of the manuscript. The manuscript has been read and approved by all authors.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethics approval

The experimental protocol was approved by ethics committee (Comitê de Ética no Uso de Animais - CEUA) of Universidade Federal de Ouro Preto under protocol number 2018/07. The animals were supplied by the Centro de Ciência Animal (CCA) of Universidade Federal de Ouro Preto and used, in studies, according to all the guidelines established by the Conselho Nacional de Controle de Experimentação Animal (CONCEA).