European Journal of Pharmaceutical Sciences 148 (2020) 105313

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

The effect of on oral absorption of following acute vs. chronic administration T ⁎ Dvora Izgelov, Abraham J. Domb, Amnon Hoffman

Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120, Israel

ARTICLE INFO ABSTRACT

Keywords: Piperine is an alkaloid naturally found in with a myriad of pharmacological attributes. Piperine's Piperine most far reaching indication is drug absorption enhancment, with supportive data regarding its ability to inhibit Cannabidiol first pass effect mechanisms. However, alongside these findings, the role of piperine as an absorption enhancer is Oral absorption undermined with publications stating an apparent effect of a metabolic inducer. The aim of this work is to First pass effect investigate the effect of repeated administration of piperine in a lipid-based formulation ,on oral absorption of SNEDDS cannabidiol (CBD), compared to acute piperine dosing. The effect of piperine on CBD absorption was determined pre-clinically in the freely moving rat model. Results of this work demonstrated that there was no significant difference in piperine's effect, when given chronically or in a single dose regimen. Both groups resulted in approximate 2.5-fold increase in oral bioavailability of CBD compared to control group without piperine.

1. Introduction Cherniakov et al., 2017). The model molecule in the current investigation was CBD. Although Piperine is the major bioactive constituent found in black pepper CBD is not reported as a substrate of P-gp (Alhamoruni et al., 2010), it is (Piper nigrum L., family Piperaceae) (Piper longum, family known for its extensive phase I and phase II Pre-systemic metabolism Piperaceae) and other Piper spices. This compound has a plethora of (Huestis, 2007; Ujváry and Hanuš, 2016). pharmacological properties such as anti-oxidative, anti-carcinogenic, In light of conflicting data that piperine causes increased first pass anti-inflammatory effects and more (Lee et al., 2018; Srinivasan, 2017). effect following repeated administration, the question of piperine's Nonetheless, piperine is mostly known for its effects as an absorption chronic effect was raised. Thus, the purpose of this paper is to examine enhancer of other compounds. It may enhance oral drug absorption via the effect of piperine on oral absorption of CBD following acute and inhibition of first pass effect mechanisms (FPE) of phase I such as cy- repeated administration to verify if the suggested induction is plausible. tochrome P450 (CYP 450) enzymes, phase II metabolism of Uridine 5′- While the rational for piperine as an absorption modulator has been diphospho-glucuronosyltransferase (UGT) enzymes and the efflux established, there are challenges in its oral administration, due to poor transporter P-glycoprotein (P-gp)(Athukuri and Neerati, 2017; solubility in water (McNamara et al., 2005). Therefore, researchers Bedada et al., 2017; Vijay Singh et al., n.d.). utilized the concept of a lipid-based drug delivery system for oral ad- On the other hand, there are also contradictory findings regarding ministration of piperine in a solubilized form (I. Cherniakov et al., piperine's role as an absorption enhancer, indicating that it has in- 2017). The developed formulation is a self-emulsifying drug delivery ductive properties of these mechanisms, resulting in up-regulation of system (SNEDDS), termed Pro-nano lipospheres (PNL). PNL serves as a enzymatic activity (Wang et al., 2013) and P-gp expression (Han et al., delivery platform for both the lipophilic drug and piperine. It is a 2008; Qiang et al., 2012). It ought to be noted, that the induction of all mixture of a lipid, surfactants and co-solvent that dissolve the lipophilic these FPE mechanisms may be coordinated and linked to one another molecules. Upon contact with an aqueous environment, the formulation when exposed to different xenobiotics(Manikandan and Nagini, 2017; spontaneously forms a thermodynamically stable o/w nano dispersion Schuetz et al., 1996; Xu et al., 2005). with nano particles of 50 nm or less. Particles entrap lipophilic mole- Previous investigation of our group, proved piperine's effect as an cules such as CBD and piperine in their lipid core and remaine stabi- absorption enhancer of major Δ9- lized in the GI with the aid of surfactants. As a result, solubility of the (THC) and cannabidiol (CBD) based on single dose administration(I. compounds is increased and they are delivered in a solubilized state

⁎ Corresponding author. E-mail address: [email protected] (A. Hoffman). https://doi.org/10.1016/j.ejps.2020.105313 Received 26 November 2019; Received in revised form 13 February 2020; Accepted 17 March 2020 Available online 19 March 2020 0928-0987/ © 2020 Elsevier B.V. All rights reserved. D. Izgelov, et al. European Journal of Pharmaceutical Sciences 148 (2020) 105313 through the unstirred water layer (UWL) attached to the enterocyte quantification(Hoffman and Levy, 1989). During the recovery period, (Elgart et al., 2013, 2012). The effect of piperine-SNEEDS on drug ab- food, but not water, was deprived. Throughout the experiment, free sorption was investigated in the case of THC and CBD. These com- access to food was available 4 h post-oral administration. pounds are extremely lipophilic, thus they have low solubility and (5 mg/kg, 1 mg/ml) was given subcutaneously to rats before surgery undergo extensive first pass metabolism, rendering molecules with low and on the day of the experiment as part of analgesia protocol. oral bioavailability (estimated 6%)(Deutman, 2018; WHO, 2018). In- corporation of piperine into SNEDDS increased oral bioavailability of 2.3.2. Experimental protocol THC and CBD compared to administration in SNEDDS The small volume of gastric fluids existing in rats is insufficient for alone in pre-clinical and clinical studies (I. Cherniakov et al., 2017; proper self-emulsification required for the lipid based PNL. Thus, to Cherniakov et al., 2017). The added effect of piperine in the SNEDDS enable proper formation of a nano particles, SNEDDS formulations were constellation demonstrates that it affects the first pass metabolism dispersed in-vitro in pre-heated water (1:9 v/v) and vortex mixed for barriers to which cannabinoids are susceptible. The effect of piperine in 30 seconds. This technique results in an o/w nano dispersion that we SNEDDS formulation on the Phase II metabolism process of glucur- term “dispersed SNEDDS.” Dispersed P-PNL was administered to ani- onidation, was further validated with its effect on the oral bioavail- mals (piperine10 mg/kg , 2 mg\ml) on days 1 through 9 via a feeding ability of raloxifene, a model molecule that undergoes extensive in- tube. On the 9th day, rats underwent jugular vein cannulation. On the testinal glucuronidation(Izgelov et al., 2018). Here, Investigation was 10th day, dispersed CBD-P-PNL (CBD 15 mg/kg, piperine 10 mg/kg) elbaorated beyoned single dose, with CBD absorption examined in the was administered to animals. freely moving rat model, administered in PNL and piperine-PNL fol- All preparations were freshly prepared on the day of the trial and on lowing acute and repeated administration. each day of administration in the experimental group (n = 4). The same composition of CBD-piperine-SNEDDS dispersion (n = 6) or CBD- 2. Materials and methods SNEDDS dispersion (n = 4) were administered to control groups. Systemic blood samples (0.30 ml) were obtained periodically via the 2.1. Chemicals intravenous cannula and replaced with equal volumes of saline solu- tion. Blood samples were collected into heparin-containing test tubes. Ethyl lactate, Polysorbate 20 (Tween® 20), Sorbitane monooleate 80 Plasma was separated by centrifugation (3220 g, 10 min, 4°C) and (Span® 80), food grade piperine, (CBG) and ammonium stored at -20°C pending analysis. acetate were purchased from Sigma Aldrich, Israel. Pharmaceutical grade Lecithin was purchased from Cargill, USA. Pharmaceutical grade 2.4. Assay and analysis of CBD concentration Polyoxyl 40-hydroxy castor oil (Kolliphor® RH40) was purchased from BASF The Chemical Company, Germany. Pharmaceutical grade The quantitative measurements of the CBD plasma concentrations Tricaprin (CremerCOOR®; MCT C10-95) was purchased from CREMER were determined as previously described via a plasma extraction Oleo Division, France. HPLC grade Acetonitrile (ACN), ethanol, me- method.9 thanol were purchased from J.T. Backer, Phillipsburg, NJ, USA. CBD was purchased from Ai Fame GmbH, Switzerland. CBD was plant ex- 2.5. PK analysis tracted pharmaceutical grade compound of 94% purity, related sub- stance THC was less than 0.05% The concentration vs. time data and pharmacokinetic parameters of C T ,T , V/F, CL/F and area uber the curve (AUC) were calcu- 2.2. SNEDDS and piperine-SNEDDS preparation max, max 1/2 lated using non-compartmental analysis with WinNonlin® (version 5.2, Pharsight, Mountain View, CA). SNEDDS was prepared by a method previously detailed(I. Cherniakov et al., 2017). Briefly, lecithin and ethyl lactate were weighed in a glass vial and heated to 37°C until completely dissolved. 2.6. Statistical analysis Tricaprin, Tween® 20, Span® 80 and Kolliphor® RH40 were added se- quentially. The mixture was heated to 40°C until a homogenous, clear All values are expressed as mean ± standard error of the mean solution was formed. This SNEDDS formulation is termed PNL (SEM) if not stated otherwise. To determine statistically significant To obtain piperine-SNEDDS , piperine (2% w/w) was added to the differences between the experimental groups, one-way ANOVA, fol- SNEDDS formulation, stirred, and heated to 37°C until completely lowed by Tukey`s test, was used. P value less than 0.05 was termed dissolved, creating piperine-PNL (P-PNL). For CBD-piperine-SNEDDS significant. formulation, CBD (3% w/w) and piperine (2% w/w) were added to the SNEDDS formulation, stirred, and heated to 37°C until completely 3. Results dissolved. Single oral administration of CBD in the SNEDDS formulation PNL 2.3. In-vivo studies with piperine resulted in a statistically significant 2-fold increase in

AUC and a 1.4 in Cmax compared to CBD-PNL. Administration of P-PNL 2.3.1. Animals and surgery for 10 consecutive days resembling chronic administration did not alter All surgical and experimental procedures were approved by the the enhancing effect of the piperine component in PNL on the absorp- Animal Experimental Ethics Committee of the Hebrew University, tion of CBD. Chronic administration of P-PNL did not diminish the pi- Hadassah Medical School, Jerusalem. perine effect and resulted in an approximately 2.8 increase in AUC and

Rats in the group of chronic P-PNL administration (male Wistar, a 1.3 increase in Cmax compared to CBD-PNL. There was no statistically Harlan, Israel) weighed 240-260 g on the first day of dosing and 270- significant difference between oral bioavailability of CBD administered

300 g on the day of the trial (CBD-P-PNL adminstartion). In the control in P-PNL given once and P-PNL given for 10 days. Tmax was similar for group of acute P-PNL or PNL administration, male Wistar rats weighed all three groups, the group of P-PNL given for 10 days had a wider range

275-300 g. The rats were kept under a 12 h light/dark cycle with free of time points. T1/2 is very similar for all groups, indicating that the access to food (standard rat chow) and water prior to trial. An in- elimination phase was not affected. V/F was not statistically different dwelling cannula was implanted in the right jugular vein as previously between groups, however, CL/F was higher for the group in which CBD described, enabling blood sampling for drug concentration was administered with blank PNL (Fig. 1, Table 1).

2 D. Izgelov, et al. European Journal of Pharmaceutical Sciences 148 (2020) 105313

Fig. 1. Plasma CBD concentration vs. time plot following PO administration of acute CBD-PNL, acute CBD-piperine-PNL and CBD-piperine-PNL after chronic pi- perine-PNL adminstration, CBD 15 mg/kg, piperine 10 mg/kg (n = 4-6).

Table 1 The enzymes involved in the oxidation of THC are CYP450 2C9, Pharmacokinetic parameters derived from CBD oral administration in PNL, 2C19, 3A4. Metabolism of CBD is similar to that of THC, with CYP2C19 piperine-PNL acute and chronic dose regimen. and CYP3A4 being the main enzymes responsible for its first pass effect. CBD-piperine-PNL CBD-piperine-PNL CBD-PNL acute There also are reports for CBD being prone to direct phase II metabo- acute chronic lism, specifically , glucuronidation via UGT 1A9 (Jiang et al., 2011). Single oral administration of THC and CBD in SNEDDS in male AUC0-∞ (h*ng/ 593 ± 17(*) 809 ± 87(*) 286 ± 87 Wistar rats resulted in a 6-fold and 4-fold increase in AUC, respectively. ml/kg) Incorporation of piperine led to an additional increase in AUC of 1.5- Cmax (ng/ml) 178 ± 11 168 ± 27 130 ± 50 ¥ Tmax(h) 2, [0.67-2] 1.8, [1-3] 1.2, [0.67-2] fold for THC and 2-fold for CBD (I. Cherniakov et al., 2017). The pi- V/F (L/kg) 65 ± 16 55 ± 5 71 ± 18 perine compound itself has relatively poor water solubility, the SNEDDS CL/F (L/h/kg) 24 ± 0.6(*)19±2(*) 66.5 ± 16 formulation dissolves the piperine and increases solubility in an aqu- T (h) 1.8 ± 0.5 2 ± 0.2 2.2 ± 1.3 1/2 eous environment via thermodynamically stable nano particles. Thus, ⁎ ff A statistical difference was not found between acute and chronic admin- enabling the piperine to a ect the metabolic process cannabinoids are istration of piperine with a statistical difference compared to acute CBD-PNL of prone to at the intestinal level. p < 0.01. In light of findings suggesting that repeated doses of piperine may ¥ Results are presented as median, range. have an inductive effect on enzymatic and efflux activity, our aim was to research the effect of chronic piperine administration on cannabinoid 4. Discussion absorption compared to the previously reported enhanced absorption, following a single dose. Piperine has a multitude of medicinal properties, however, it has Oral administration of CBD in the experiment group of rats pre- been investigated predominantly for its effect as an absorption en- treated with piperine-PNL resulted in increased absorption of CBD hancer. S. K. Bedada et al (2017) researched the effect of piperine on compared to administration of CBD-PNL without piperine. Upon com- pharmacokinetics and bioavailability of the drug carbamazepine in paring oral bioavailability of CBD following single or repeated admin- healthy volunteers receiving piperine once daily for 10 days. Treatment istration of piperine, a statistical difference was not found. with piperine significantly enhanced AUC and Cmax of the drug, which Thus, pre-treatment with piperine did not diminish absorption of is subjected to CYP3A4 metabolism (Bedada et al., 2017). However, CBD, as hypothesized basing on various findings regarding piperine's alongside these findings and many others in favor of piperine as an inductive rather than inhibitory effect on pre-systemic metabolism absorption enhancer, there are publications indicating the opposite constituents. Following a 10 day treatment with piperine-PNL, the ad- effect. Wang Y. et al. (2013), revealed the dichotomous effect of pi- ditive effect of piperine on CBD absorption remained compared to PNL perine and reported that piperine activates human pregnane X receptor alone with no trend indicating an opposite effect. Overall exposure of (PXR), causing induction of the of both CYP3A4 and P-gp (Wang et al., CBD increased following the addition of piperine. This may be result of 2013). the effect piperine has on the phase I and phase II metabolism These findings were in correlation with other reports, such as by (Izgelov et al., 2018; Lee et al., 2018). As shown by the CL/F parameter Qiang et al. (2012), which established that repeated dosing of piperine which is significantly higher for the CBD-PNL group, compared to CBD- induced gene expression of P-gp through PXR activity thus decreasing P-PNL groups (acute and chronic). Assuming that piperine has a local oral bioavailability of diltiazem in rats (Qiang et al., 2012). Relaying on effect on intestinal metabolism, the CL of the drug ought to remain the the correlation between P-gp expression and CYP450 via the PXR, the same. Especially, considering that elimination half-life and the derived effect of piperine as an inducer for P-gp levels strengthens the potential liner terminal slope are not changed. However, the CL/F parameter effect on CYP3A4 and other enzymes of this family (Schuetz et al., differs because of the difference in the bioavailability of CBD in each 1996; Sinz, 2013). vehicle. Administration of CBD without piperine, results in lower When administered orally, THC and CBD readily penetrate the en- bioavailability and as a result an increased value of CL/F. terocyte layer due to their lipophilic nature. Their absorption, however, The time length of the experiment was set on 10 days, since the time is slow and more erratic with reported bioavailability of approximately needed for an inductive effect is variable among researchers, ranging 6%%(Deutman, 2018; Zhornitsky and Potvin, 2012). The low and in- from 3 to 14 days and beyond (Chen et al., 2018; Xu et al., 2018). consistent extent of absorption derives from low solubility and sig- Results of repeated dosing of piperine do not demonstrate a different PK nificant first pass metabolism (Huestis, 2007). behavior or overall absorption compared to single dose, without an

3 D. Izgelov, et al. European Journal of Pharmaceutical Sciences 148 (2020) 105313 inclination towards decreased absorption of CBD, rendering the limit of pharmacokinetic evaluation in healthy volunteers in comparison to buccal spray time less relevant. administration. J. Control. Release 266, 1–7. https://doi.org/10.1016/j.jconrel.2017. 09.011. Although CBD is not a classical substrate of P-gp, which is the focus Cherniakov, I., Izgelov, D., Domb, A.J., Hoffman, A., 2017. The effect of pro nano lipo- of many reports regarding piperine's inductive effect, CBD is prone to spheres formulation containing natural absorption enhancers on the oral bioavail- CYP3A4 metabolism, which is closely linked to P-gp activity and ex- ability of delta-9-tetrahydrocannabinol and cannabidiol in a rat model. Eur. J. Pharm. ff Sci. 109. https://doi.org/10.1016/j.ejps.2017.07.003. pression. Piperine's controversial e ect on CYP3A4 expression raised Deutman, R., 2018. 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