journal of exploratory

JOURNAL OF EXPLORATORY RESEARCH IN PHARMACOLOGY

CONTENTS 2017 2(3):67–104

Original Articles

Chemical Analyses, Antimicrobial and Antioxidant Activities of Extracts from Cola nitidaJulius K. Adesanwo, Seun B. Ogundele, David A. Akinpelu, Armando G. McDonald. . . . . . . . . . . . . . 67

Bioequivalence Study of Generic Metformin Hydrochloride in Healthy Nigerian VolunteersAdebanjo Jonathan Adegbola, Olugbenga James Awobusuyi, Babatunde Ayodeji Adeagbo, Bolanle Stephen Oladokun, Adegbenga Rotimi Owolabi, Julius Olugbenga Soyinka . . . . . . . . . . . . . . . . . . . . . . . . . 78

Review Articles

Investigating the Role of the Endocannabinoid System in Early PsychosisAisling O’Neill, Sagnik Bhattacharyya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Cannabis: A Prehistoric Remedy for the Deficits of Existing and Emerging Anticancer TherapiesBakht Nasir, Humaira Fatima, Madiha Ahmed, Abdul-Rehman Phull, Ihsan-ul-Haq . . . . . . . . . . . . . 93

Journal of Exploratory Research in Pharmacology 2017 vol. 2 | 67–77

Copyright: © 2017 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Original Article

Chemical Analyses, Antimicrobial and Antioxidant Activities of Extracts from Cola nitida Seed

Julius K. Adesanwo1*, Seun B. Ogundele1, David A. Akinpelu2 and Armando G. McDonald3

1Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria; 2Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria; 3Department of Forestry, Rangeland and Fire Sciences, University of Idaho, Moscow, ID 83843, USA

Abstract

Background and Objectives: Medicinal plants are the richest, cheapest and most readily available source of drugs, nutraceuticals and food supplements. Pharmaceutical industries still rely largely on medicinal plants for interme-diates due to their chemical diversities. This study, therefore, investigated the chemical constituents, thermal decomposition products and biological activities of extract from seeds of Cola nitida (the ‘kola nut’).

Methods: The pulverized seed was sequentially extracted with dichloromethane and methanol CH3OH. The ex-tracts were analysed directly by Fourier Transform Infra-Red, electrospray ionization mass spectrometer and as fatty acid methyl ester and trimethylsilyl derivatives by gas chromatography-mass spectrometry (GC-MS). The CH3OH extract was analysed by high-performance liquid chromatography for sugars. The intact and extracted seed biomasses were analysed directly by pyrolysis GC-MS. For isolation of chemicals and assessment of biologi-cal activity, a large scale CH3OH extraction was performed and the extract partitioned with n-hexane, ethyl ac-etate (EtOAc) and butanol. Fractionation was done using various chromatographic techniques. Antimicrobial and antioxidant activities of the extract, fractions and isolated caffeine were respectively determined by the methods of agar-well diffusion and 2,2-diphenyl-1-picrylhydrazyl radical scavenging.

Results: Caffeine and hexadecanoic acid were isolated from the EtOAc fraction while theobromine, caffeine, cat-echins, procyanidins, proanthocyanidins, sugars, fatty acids, alcohols and sterols were identified in the extracts. Multitude (62) biomass degradation products were identified in pyrolysed seed samples. The extract and frac-tions showed varying activities against most of the tested microbes, except against Shigella species, for which neither the extract nor fractions elicited any response. The butanol fraction exhibited the highest antioxidant activity.

Conclusions: This report gives insight into the chemi-cal constituents in Cola nitida seed, details the ther-mal decomposition constituents and establishes the antimicrobial and antioxidant activities of the seed extract and fractions, thereby contributing to the knowledge on the chemistry and pharmacology of the genus.

Keywords: Cola nitida; Caffeine; n-Hexadecanoic acid; Catechin; Antimicrobial; Antioxidant; GC-MS; ESI-MS.Abbreviations: AGC, Acelerated Gradient Chromatography; 13CNMR, Carbon 13 Nuclear magnetic resonance spectroscopy; CND, Cola nitida dichloromethane extract; CNM, Cola nitida methanol extract; IC50 value, concentration of samples leading to 50% reduction of initial DPPH radical concentration; CDCl3, deuterated chloroform; DCM or CH2Cl2, dichloromethane; DPPH, 2,2-diphenyl-1-picrylhydra-zyl; ESI-MS, electrospray ionization mass spectrometer; EFAs, essential fatty acids; EtOAc, ethyl acetate; FAME, fatty acid methyl ester; FAs, fatty acids; FTIR, fourier transform infrared; GC-MS, gas chromatography-mass spectrometry; GRAM +ve, Gram positive; GRAM −ve, Gram negative; HPLC, high-performance liquid chroma-tography; IS, internal standard; CH3OH, methanol; 1HNMR, proton nuclear magnetic resonance spectroscopy; Pyro GC-MS or Py GC-MS, pyrolysis gas chromatography-mass spectrometry; Ag, silver; TLC, thin-layer chromatography; TMS, trimethylsilyl; UV, Ultra- violet; ZnSe, Zinc Selenium.Received: April 28, 2017; Revised: June 23, 2017; Accepted: June 28, 2017*Correspondence to: Julius K. Adesanwo, Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria. Tel: +234-8030821561, E-mail: [email protected] or [email protected] to cite this article: Adesanwo JK, Ogundele SB, Akinpelu DA, McDon-ald AG. Chemical Analyses, Antimicrobial and Antioxidant Activities of Extracts from Cola nitida Seed. J Explor Res Pharmacol 2017;2(3):67–77. doi: 10.14218/JERP.2017.00015.

Introduction

Cola nitida (Vent) Schott et Endl, family Malyaceae, is a tree na-tive to the rainforest of tropical West Africa. The seed or nut com-monly called ‘kola nut’ is a popular stimulant in West Africa. It is called “Obi gbanja” by Yorubas in western Nigeria, “Goro” by Hausas in the north, and “Oji” by Ibos in the east.1 It is mainly used for nutritional purpose, eaten across cultures in West Africa, and

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has great social, religious and medicinal importance as well.2 Kola nut extracts are used in the manufacture of various non-alcoholic beverages, soft drinks, wines, chocolate and sweets.3–5

Previous researchers have reported a number of secondary me-tabolites from the kola nut, including caffeine, theobromin, cate-chin, epicatechin, procyanidins and proanthocyanidins.6–8 Aliphat-ic and heterocyclic amines in different species of cola have been reported as well,9 and quinic, tannic and chlorogenic acids have been found as present in the kola nut.10 Caffeine was the major alkaloid identified in Cola seeds and was considered as one of the signature compounds due to its concentration range.6

Knebel and Higler showed that fresh kola nut contained a glu-coside named “kolanin”, reporting that it is readily hydrolysed or split into glucose and caffeine in ripe or dried fruit.7 Kola nuts are rich in xanthine alkaloids, such as theobromine, caffeine, kolatin and kolanin. While caffeine stimulates the body, kolatin stimu-lates the heart.11,12 These constituents (caffeine, theobromine, and theophylline) in kola nut extract have been shown to contribute to the anti-photodamage effect, including wrinkles, and to elicit antioxidant and anti-aging activities, including suppression of UV-induced erythema, and to decrease skin roughness and scaling by topical or oral application.13

Nowadays, pathogenic microorganisms are developing resist-ance to existing antibiotics at an alarming rate. Extract of the leaves, root and stem bark of Cola nitida are extensively used in folk medicine.6 Different parts of Cola nitida have been exploit-ed for different biotechnological applications. For instance, cola pods have been used for production of fructosyltransferase, an important enzyme for the synthesis of fructooligosaccharides,14 and most recently for the biosynthesis of silver (Ag) nanoparticles and silver-gold alloy nanoparticles for diverse biomedical appli-cations,15–19 such as antimicrobial, antioxidant, anticoagulant and thrombolytic activities.

Similarly, the seed and seed shell extracts of Cola nitida have been used for green synthesis of Ag nanoparticles, showing pro-found antibacterial activities.20 In Uganda, extracts of the seed and leaves are used in treatment of sexual and erectile dysfunctions.21 In Nigeria, methanol extract of the seed is used against emesis and migraine.22 The aqueous extract of the seed has also been used as flavouring in carbonated drinks.23 Kola nut extract also demon-strates good protective property against red cell degradation.9

The aims of this study were to: (i) determine the composition of kola nut dichloromethane (CH2Cl2) and CH3OH extracts by a com-bination of electrospray ionization-mass spectrometry (ESI-MS) and gas chromatography-mass spectrometry (GC-MS) analyses; (ii) determine the antimicrobial and antioxidant activity of the ex-tracts; and (iii) isolate the active principle component(s).

Experimental

General

All thin-layer chromatography (TLC) analyses were performed at room temperature using pre-coated plates (silica gel 60 F254 0.2 mm; Merck). Detection of spots was achieved by staining with iodine crystals and exposure to ultraviolet light (254 and 366 nm). Melting point determination was carried out using a Gallen-kamp apparatus. Accelerated gradient chromatography (Baeck-strom Separo AB) was carried out using silica gel (Kieselgel 60G 0.040–0.063 mm) and column chromatography using silica gel, with a 60–200 mesh for fractionation. Proton Nuclear magnetic resonance (1HNMR) spectroscopic data were recorded on a NMR

machine (Agilent Technologies) at 400 MHz and at 100 MHz for 13CNMR. Chemical shifts of signals were reported in parts per mil-lion (ppm).

Collection of plant material

The plant material (kola nut) was collected at Alaro Farm Settle-ment, Ile-Ife, identified and authenticated at the Department of Pharmacognosy, Obafemi Awolowo University (Voucher Number FPI 2052).

Sample preparation and extraction

The seeds were dried at 60 °C for 48 h, and milled to particle size of 1 mm. Moisture content was determined in duplicate before extraction. The plant material was Soxhlet extracted (40 g, in du-plicate) first with dichloromethane (DCM) (150 mL) for 24 h and then with CH3OH (150 mL) for 48 h. The extracts were concen-trated in vacuo at 40 °C to give yield of 0.77% and 17.62% for the DCM and CH3OH extracts, respectively.

Fourier transform infrared (FTIR) spectroscopic analysis

The functional groups in the milled samples and extracts, and the extracted biomasses were determined by FTIR spectroscopy using a Nicolet iS5 spectrometer (ThermoScientific) using a ZnSe at-tenuated total reflection probe. Spectra were collected in duplicate. The absorbance spectra were baseline-corrected and averaged us-ing Omnic v9.0 software (ThermoScientific).24,25

ESI-MS experiment

The samples (about 1.0 mg each of DCM and CH3OH extracts, in duplicate) were added to CH3OH (1 m:) and acetic acid (10 µL). The mixture was subjected to sonication to ensure total dissolu-tion. Electrospray mass spectrometric analyses were performed on a 5989A device (Hewlett-Packard) equipped with an electrospray interface 59987A. Nitrogen was used as nebulizing gas, at a pres-sure of 50 psi and a temperature of 300 °C. Sample analysis was performed by direct infusion in ESI-MS using a syringe pump (Harvard Apparatus) at a flow-rate of 10 mL/min. Mass spectra were acquired in scan mode detection, and ESI-MS conditions were optimized using available standards.

GC-MS of fatty acid methyl ester (FAME) derivatives

Extracts (about 2.0 mg, in duplicate) were prepared by adding a solution (2 mL) of CH3OH/H2SO4/CHCl3 (1.7:0.3:2.0 v/v) in which the CHCl3 contained 1-naphthaleneacetic acid (100 µg/mL) as an internal standard. The mixture was heated for 90 min at 90 °C in a sealed vial. Water was added to the mixture and the CHCl3 layer was removed, dried and transferred to a GC vial. The prepared FAME derivatives were analysed by electron impact ionization GC-MS on a Focus ISQ (ThermoScientific) with a ZB5 column (30 m × 0.25 mm; Phenomenex) and a temperature pro-file of 40 °C (1 min) to 320 °C (10 min) at 5 °C/min. The eluted compounds were identified with authentic standards (C12 to C20 fatty acids) and by spectral matching with the NIST 2008 spectral library.

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GC-MS of extracts for trimethylsilyl (TMS) derivatives

Extracts (about 1.0 mg, in duplicate) were weighed in GC vials, to which CH2Cl2 (1 mL) containing anthracene as an internal stand-ard (IS; 50 µg/mL) was added. The samples were silylated with addition of N,O-bis(trimethylsilyl)-trifluoro-acetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS; 50 µL) and pyridine (50 µL) and heated for 30 min at 70 °C or longer until the solu-tion became clear. The prepared TMS derivatives were analysed by GC-MS (as described above).

Analytical pyro (Py)-GC-MS of samples

Analytical Py-GC-MS was carried out on the milled sample before and after extraction using a Pyrojector II (SGE Analytical Science) at 500 °C in He coupled to a GC-MS (FOCUS-ISQ; ThermoScien-tific) instrument operating in the electron impact ionization mode. The compounds were separated on the ZB5-MS capillary column (30 mm × 0.25 mm; Phenomenex) with temperature programmed to be 50 °C to 3000 °C, at 5 °C min−1. The eluted compounds were identified by their mass spectra, authentic standards, and with NIST 2008 library matching.

High-performance liquid chromatography (HPLC) analysis

Sugars were quantified by HPLC using a Rezex ROA column (7.8 mm × 30 cm; Phenomenex) and a Waters HPLC (Waters Corp.) equipped with differential refractive index detector (Shodex SE61; Showa Denko America, Inc.), on elution with 0.01 M H2SO4 (0.5 mL/min) at 65 °C. The CH3OH extract (10 mg) was dissolved in 0.01 M H2SO4 (5 mL), centrifuged and the supernatant filtered (0.45 µm). Data was acquired and analysed using the N2000 chro-matography software (Science Technology Inc.). The sugar con-tents were determined from peak area using the external standard method with standard sugars (glucose, fructose, xylose, myo-ino-sitol, sucrose, maltose and turanose).

Isolation of chemical compounds

The seeds (2 kg) were extracted with CH3OH and concentrated in vacuo to obtain CH3OH extract (80 g, 4% yield). The crude extract was suspended in water and partitioned with n-hexane, ethyl acetate (EtOAc) and n-butanol to give respective fractions. The EtOAc fraction was subjected to Acelerated Gradient Chro-matography (AGC) fractionation. Fractions 24 through 76 were bulked together and separated by column chromatography (silica gel 60–200 mesh) and elution was monitored by TLC. Fractions 36 through 67 were evaporated to yield a white crystalline solid (caf-feine) with melting point of 230–233 °C. Pooled AGC fractions 8 through 23 were concentrated and a yellow viscous solid was obtained, which was further purified by column chromatography, yielding a colourless solid (hexadecanoic acid) with minor impuri-ties as accessed by TLC. Both compounds were characterized by NMR spectroscopy (400 MHz; Agilent).

Antimicrobial assay

The antimicrobial activity of CH3OH extract, solvent fractions and isolated caffeine were determined using the agar-well diffusion method.26,27 The bacteria were grown in a nutrient broth before

use, while the fungi were grown on potato dextrose agar medi-um until they sporulated, at which time they were harvested. The standardized bacteria suspension was spread on Muller-Hinton agar and allowed to set. Wells were then bored with a 6-mm borer and filled with the respective sample solutions at 10 mg/mL, with ampicillin and nystatin added at 1 mg/mL, which was followed by incubation at 37 °C for 24 hrs. The fungal plates were incubated at 25 °C for 96 hrs.

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging an-tioxidant assay

Quantitative antioxidant activity was determined spectropho-tometrically as described.28 Reactions were carried out in test tubes, and each of the solvent fractions and CH3OH extracts were tested at varying concentrations (µg/mL). Initial stock solutions of 1 mg/mL were prepared for the various plant extracts. The following final concentrations were prepared based on the pre-liminary qualitative TLC antioxidant screening: 60, 30, 15, 7.5, 3.75 and 1.875 µg/mL for the crude extract; 50, 25, 12.5, 6.25 and 3.13 µg/mL for n-butanol fraction; 30, 15, 7.5, 3.75 and 1.88 µg/mL for the EtOAc fraction; and 2000, 1000, 500, 250, 125 and 62.5 µg/mL for the n-hexane extract and caffeine from the stock solution. A 0.1 mM DPPH radical solution in CH3OH (1 mL) was added to 1 mL of the concentration series for each sample tested, in triplicate, and allowed to react at room temperature in the dark for 30 min. The negative control was prepared by adding 0.1 mM DPPH radical solution (1 mL) to 1 mL of methanol, in triplicate, and absorbance was measured at 517 nm. The percentage anti-oxidant activity (%AA) values of test samples were calculated from the absorbance using the formula:% AA={Xcontrol−XtestXcontrol}×100where Xcontrol is the absorbance of the negative control, Xtest is the absorbance of test samples concentrations. Ascorbic acid (vitamin C) was used as the standard antioxidant

Fig. 1. Fourier transform infrared spectra of Cola nitida seed. (A) Pow-dered unextracted material, (B) Cola nitida dichloromethane (CND) ex-tract, (C) Cola nitida methanol (CNM) extract, (D) Extracted biomass.



agent.The IC50 value (i.e. the concentration of the test samples leading

to 50% reduction of the initial DPPH radical concentration) was calculated from the separate linear regression of plots of the mean percentage of antioxidant activity against the concentration of the test samples in µg/mL.

Results and discussion

Extraction techniques

Soxhlet extraction of the kola nut with CH2Cl2 and CH3OH af-forded yields of 0.77% and 17.6%, respectively. In comparison, large-scale cold extraction of the kola nut afforded a low yield of 4%. Therefore, only the Soxhlet extracts were analysed in detail by a combination of GC-MS and ESI-MS. However, the batch CH3OH extract was used for antioxidant and antimicrobial assess-ment.

FTIR spectroscopic analysis

FTIR spectroscopy was used to investigate the functional groups

in the sample and extracts. The spectra (Fig. 1) showed the pres-ence of strong O–H stretching vibration at 3340 cm−1 correspond-ing to hydroxyl groups in all, but quite pronounced in Figure 1A, C, and D. This band is less pronounced in Figure 1B, because the extract contained a majority of low polar compounds, whereas Figure 1C and D contained high polar phenolic compounds and lignocellulose respectively and containing multiple H-bonding. The absorption at just above 3000 cm−1 in Figure 1a is due to an aromatic C–H stretch of polyphenols and aromatics; it is virtu-ally absent in Figure 1b but noticeable in Figure 1C and D. Simi-larly, the substituted aromatic C–H bend band at 600–900 cm−1 is almost absent in Figure 1b but very conspicuous in Figure 1A, C and D, indicating a low proportion of aromatics in the CND extract.

On the other hand, the C–H sp3 stretching vibration, just below 3000 cm−1 and characteristic of long chain fatty acids [–(CH2)n–CH3], is more conspicuously prominent in Figure 1B than in Figure 1C and D. Carbonyl stretching absorption of carboxylic acid aldehydes and ketones is very low in Figure 1D, as most car-bonyl compounds had already been removed through the process of extraction. The absorption band at 1000–1200 cm−1 that was due to C–O stretch of esters (glycosides and cellulose) was most intense in Figure 1C and D but low in Figure 1B. These data show that FTIR can be used to monitor and assess the effectiveness of the extraction process, apart from facilitating quantitative evalu-

Fig. 2. Electrospray ionization mass spectra of Cola nitida extracts positive mode. (A) Cola nitida methanol extract, (B) Cola nitida dichloromethane extract.



ations.

Electrospray mass spectrometric analysis of extracts

ESI-MS is a powerful analytical tool to rapidly analyse extracts and isolated fractions without chromatographic separation.29–31 Moreover, ESI-MS makes it possible to discriminate between var-ious flavonoid classes, provide information on the glycosylation position and characterize saponins.32–34 Therefore, ESI-MS was employed to directly analyse the CH2Cl2 and CH3OH extracts in positive (Fig. 2) and negative (Fig. 3) ion modes.

The positive ion ESI-MS for both extracts showed the larg-est peak at m/z 195 (100% intensity), which was due to caffeine ([M+H]+). In the MeOH extract (Fig. 2A), the tentative peak assign-ments are as follows: 181 ([M+H]+, theobromine); 295 (unknown); and 391 (unknown). For the CH2Cl2 extract (Fig. 2B), the tentative peak assignments are: 193 ([M+H]+, quinic acid); 313 ([M+Na]+, catechin), 355 ([M+H]+, chlorogenic acid); and m/z 281, 294, 377 and 426 (unknowns). The negative ion ESI-MS for both extracts

(Fig. 3) showed a multitude of peaks. For the CH3OH extract (Fig. 3A), the tentative assignments are: m/z 289([M-H]−, catechin); m/z 577 and 578 (procyanidins B1, B2 [oligomeric catechins] and naringin flavonoids); and m/z 1153 and 1154 (proanthocyanidins). There were many unidentified peaks. Niemenak et al.6 also reported that HPLC of Cola nitida extract had 11 unidentified compounds.

GC-MS of extracts of FAME derivatives

It is well known that fatty acids (FAs), especially the essential fat-ty acids (EFAs), are important for optimal functioning of the body. They regulate such bodily functions as heart rate, blood pressure, blood clotting and fertility. They also participate in immune sys-tem inflammation against harmful waste products. Balance of EFAs is important for good health and normal development of humans.35,36 FAs occur widely in natural fats and dietary oils and they play important roles as nutritious substances and metabolites in living organisms.37 With these important biological activities of FAs, those present in the extracts as free or glycerides were con-

Fig. 3. Electrospray ionization mass spectra of Cola nitida extracts negative mode. (a) Cola nitida methanol extract, (b) Cola nitida dichloromethane extract.



verted to FAME derivatives for identification and quantification.The CH2Cl2 and CH3OH extracts were derivatised into FAME

and analysed by GC-MS. The CH2Cl2 extract contained FAs rang-ing from C16 to C19. Palmitic acid (C16: 0.447 mg/g), linoleic acid (C18: 0.198 mg/g), oleic acid (C18: 0.553 mg/g), stearic acid (C18: 0.034mg/g), 8,9-methylene-heptadec-8-oic acid (C18: 0.112 mg/g), 10,12-octadecadienoic acid (C18: 0.040 mg/g), 8-oxohexa-decanoic acid (C16: 0.275 mg/g) and 9,10-methylene-octadec-9-oic acid (C19: 0.032 mg/g) were the major FAs detected. The FAs constitute about 73% of the extract. Furthermore, caffeine, fatty alcohols and sterols were also detected (Table 1). The CH3OH extract did not contain any FAs, only caffeine.

GC-MS of extracts of TMS derivatives

Trimethylsilylating reagent is routinely used to derivatise rather

non-volatile compounds, such as certain alcohols, phenols, or car-boxylic acids, by substituting a TMS group for a hydrogen in the hydroxyl groups on the compounds. Both extracts were deriva-tised to TMS ethers/esters to conduct the analyses for non-polar and polar components using GC-MS. The results for the CH2Cl2 and CH3OH extracts as their TMS derivatives are given in Tables 2 and 3.

Caffeine (17.9% and 3.85% in CH3OH and CH2Cl2 extracts, re-spectively) was predominant, as observed by Niemenak et al.6 Cat-echin was the dominant flavonoid in the kola seed. Other identified constituents of CH3OH extract included: sugars (45%); catechin (13%); malic acid and glycerol (Table 2). The presence of glucose, fructose and sucrose in the CH3OH extract was detected by both GC-MS and HPLC analyses. The CH2Cl2 extract consist mainly of FFAs (Table 3), and this observation is consistent with the results of FAME analysis. Other compounds found in this extract were alkaloids and sterols.

Table 2. GC-MS of Cola nitida CH3OH extracts of TMS derivatives

Compound Molecular formula Class Molecular weight Retention time (min) % Extract

Glycerol TMS C12H32O3Si3 Alcohol 308 18.11 0.2

Malic acid TMS C13H30O5Si3 Acid 350 23.65 1.5

Anthracene (IS) C14H10 178 30.14

Fructose TMS5 C19H46O6Si4 Sugar 540 30.70 2.2

Caffeine C8H10N4O2 Alkaloid 194 31.23 17.9

Glucose-TMS-5 C21H52O6Si5 Sugar 540 32.77 0.7

D-Turanose-TMS-7 C33H78O11Si7 Sugar 846 45.65 2.9

Sucrose-TMS8 C36H86O11Si8 Sugar 918 45.75 39.5

Catechin-TMS-5 C30H54O6Si5 Flavonoid 650 48.74 12.9

Table 1. GC–MS FAME derivatives of CND extract

S/No Compound Molecular formula Class Molecular weight

Retention time (min) % Extract

1 Naphthalene acetic acid (IS) C13H12O2 200 27.342 Caffeine C8H10N4O2 Alkaloid 194 30.43 1.73 Palmitic acid C17H34O2 FA 270 31.83 19.44 Linoleic acid C19H34O2 FA 294 35 8.65 Oleic acid C19H36O2 FA 296 35.11 24.06 Stearic acid C19H38O2 FA 298 35.6 1.57 8,9-Methylene-8-heptadecenoic acid C19H34O2 FA 294 36.24 4.98 10,12-Octadecadienoic acid C19H34O2 FA 294 36.39 1.79 7-(Tetrahydro-2H-pyran-2-yloxy)-2-octyn-1-ol C13H22O3 Alcohol 226 36.63 1.310 2-Octylcyclopropene-1-heptanol C18H34O Alcohol 266 36.79 1.711 8-Oxohexadecanoic acid C17H32O3 FA 284 37.25 12.012 Tetrahydropyran-2-yl ether of 7-dodecynol C17H30O2 Alcohol 266 37.46 1.213 9,10-Methylene-9-octadecenoic acid C20H36O2 FA 308 37.99 1.414 1-Ethyl-(1,1-dimethylethyl)-methoxycyclohexan-1-ol C13H26O2 Alcohol 214 38.97 3.215 Stigmastan-3,5-diene C29H48 Sterol 396 50.12 0.216 Sitosterol acetate C31H52O2 Sterol 456 52.16 0.717 Sitosterol C29H50O Sterol 414 52.86 0.6



Analytical Py-GC-MS of samples

Direct analysis of the kola nut and extracted nut were performed by analytical Py-GC-MS. The identities of the products are given in Table 4. The main compounds in the kola nut were caffeine (22.5%), CO2 (12.8%), methyl acetate (6.3%), acetic formic anhy-dride (5.9%), levoglucosan (5.4%), N-methyl ethylamine (4.9%), 1,2-cyclopentanedione (3.4%) and pyrocatechol (3.0%). Aliphatic and heterocyclic (pyrolidine, alstonine and xanthenes) amines were identified. This finding is in accordance with the report by Atawodi et al.9 Py-GC-MS of the extracted kola nut showed the presence of 22 compounds (Table 4). The main compounds identified were: CO2 (17%), acetol (11%), 1,2-benzenediol (11%), 1,2-cyclopentan-edione (10%), acetic acid (7%), butylamine (5%) and caffeine (5%).

It is noteworthy that alkanes, alkenes ethers and steroids/sterols were not identified by the Py-GC-MS of the extracted biomass; this may be explained as due to exhaustive removal of the relative-ly low polar compounds during the process of extraction. Analyti-cal pyrolysis experiment showed caffeine as the major constituent of Cola nitida. Maltol, a naturally occurring organic compound, is primarily used as a flavour enhancer.10 Its presence in the Cola nitida seed may be contributory to the application of the seed in the manufacture of soft drinks.3–5

Structure elucidation

Compound 1 (caffeine) was obtained as a white crystalline solid, with a melting point of 230–233 °C. 1HNMR (400 MHz, CDCl3, δ ppm): 7.46 (1Hs, 8H), 3.95, 3.54, 3.36 (s, 3H at 1,3,7). 13CNMR (100 MHz, CDCl3, δ ppm): 155.3 and 151.6 (C6 and C2 respec-tively), 148.6, 141.5, 107.5 (olefinic C8, C4 and C5), 33.5, 29.7 and 27.9 (methyl groups). These correlate with the literature data for caffeine.

Compound 2 (n-Hexadecanoic acid): 1HNMR (400 MHz, CDCl3, δ ppm): 2.24–1.21 (CH2 protons), 0.78 (CH3 protons). 13CNMR (100 MHz, CDCl3, δ ppm): 179 (C=O); 34.15–22.64 (CH2 carbon atoms); 15.02 CH3 group.

Antimicrobial activity

The kola nut CH3OH extract and fractions, and isolated caffeine showed varying degrees of inhibitory activities against the tested

bacterial and fungal strains (Fig. 4 and Supplementary Table S1). Their activity was more pronounced against Gram-positive bacte-ria than Gram-negative bacteria. This could be a result of the mor-phological differences between these microorganisms. The Gram-positive bacteria have an outer peptidoglycan layer, which is not an effective permeability barrier, making these microorganisms more susceptible to the compounds under investigation; meanwhile, the Gram-negative bacteria have an outer phospholipidic membrane that contains LPSs, making the cell wall of these microorganisms impermeable.38–40.

These microorganisms are implicated in the pathogenesis of hu-man infections. The result obtained showed the EtOAc fraction as having the highest antimicrobial activity against most of the organisms compared to other fractions. However, the EtOAc frac-tion was not active against Pseudomonas Spp., Clostridium sporo-genes, Corynebacterium pyogenes, Shigella Spp. and Candida albicans. The activity of the extract indicated CH3OH as a good solvent for preparation of extracts for antimicrobial assay.41,42 The antimicrobial activity of the CH3OH extract can be attributed to synergistic effect of compounds in the extract. Isolated caffeine (compound 1) demonstrated higher activity than n-butanol against Bacillus Spp., Escherichia coli and Aspergillus niger. Extract and fractions did not show activity against Shigella Spp. Caffeine dem-onstrated appreciable antimicrobial activity against Bacillus cere-us, Escherichia coli, Pseudomonas vulgaris and the fungi.

Antioxidant activity

The DPPH radical antioxidant activity showed the ability of the kola nut CH3OH extracts and fractions, and isolated caffeine to reduce DPPH radicals through the transfer of acidic labile protons by a free radical mechanism. None of the extracts compares sig-nificantly with that of ascorbic acid. However, the EtOAc and bu-tanol fractions exhibited good DPPH antioxidant activity. The IC50 values decreased in this order: ascorbic acid (3.2 ± 0.05 µg/mL), butanol extract (9.8 ± 0.5 µg/mL), EtOAc extract (15.1 ± 0.7 µg/mL), CH3OH extract (22.7 ± 1.7 µg/mL), hexane extract (321 ± 7 µg/mL) and caffeine (1370 ± 19 µg/mL).

Future research direction

A number of chemical compounds have been reported from the

Table 3. GC-MS of Cola nitida CH2Cl2 extracts of TMS derivatives

Compound Molecular formula Class Molecular weight Retention time (min) % ExtractNonanoic acid TMS C12H26O2Si FA 230 20.14 0.82Octanedioic acid TMS C14H30O4Si2 FA 318 28.27 0.26Anthracene C14H10 178 30.14Caffeine C8H10N4O2 Alkaloid 194 31.32 2.95Palmitic acid TMS C19H40O2Si FA 328 35.07 1.64Linoleic acid TMS C21H40O2Si FA 352 37.94 5.95Oleic acid TMS C21H42O2Si FA 354 38.11 1.89Stearic acid TMS C21H44O2Si FA 356 38.58 0.14Linolenic acid TMS C21H38O2Si FA 350 39.57 1.08Stigmasterol TMS C32H56OSi Sterol 484 53.46 0.20Sitosterol TMS C32H58OSi Sterol 486 54.13 2.05



Table 4. Py-GC-MS of unextracted and extracted powdered Cola nitida seed

Compound Molecular formula

Molecular weight

Retention time (min)

Kola nut, %

Extracted nut, %

CO2 CO2 44 1.12 12.8 17.4

N-Methyl ethylamine C3H9N 59 1.30 4.9

Butylamine C4H11N 72 1.38 4.9

Acetic acid C3H4O3 60 1.64 5.9 6.5

Pentanone C5H10O 86 1.65 4.6

Acetol C3H6O2 74 1.97 6.3 11.3

Unknown 92 3.17 1.5

Butanedial C4H6O2 86 3.36 1.7 3.4

Methyl pyruvate C4H6O3 102 3.49 1.7 1.1

Butanedial C4H6O2 86 3.63 2.7

Unknown C4H4O2 84 3.66 1.0 1.1

2-Oxo-3-cyclopentene-1-acetaldehyde? C7H8O2 124 4.37 1.3 3.2

2-Furfuryl alcohol C5H6O2 98 4.90 2.2 4.4

Unknown C5H8O3 116 5.16 1.1

2(5H)-Furanone C4H4O2 84 6.34 2.1 3.9

1,2-Cyclopentanedione C5H6O2 98 6.67 3.4 9.9

1-Methyl-1-cyclopenten-3-one C6H8O 96 7.85 1.3

4-Methyl-5H-furan-2-one + Unknown C5H6O2 98 + 110 8.09 0.6

Phenol C6H6O 94 8.28 XX

2 Hydroxy-3-methyl-2-cyclopenten-1-one C6H8O2 112 9.54 1.4 5.4

2,3 Dimethyl-2-cyclopenten-1-one C7H10O 110 9.85 0.3

Unknown 116 10.00 0.8

2-Methyl phenol C7H8O2 108 10.40 0.7

3-Methyl-phenol C7H8O2 108 10.99 0.7

Unknown 57? 11.51 1.9 0.9

3 Hydroxy-2-methyl-4H-pyran-4-one (Maltol) C6H6O3 126 12.03 0.5 0.7

3-Ethyl-2-hydroxy-2-cyclopenten-1-one C7H10O2 126 12.21 0.4 1.5

4-Hydroxy-3-methyl-(5H)-furanone or 3-methyl-2,4(3H,5H)-furandione C5H6O3 114 12.33 0.5

Dihydro-2H-pyran-3(4H)-one + unknown C5H8O2 100+128 13.16 0.8

Ethyl/dimethyl phenol + 3,5-dihydroxy-2-methyl-(4H)-pyran-4-one C11H18O7 122+142 13.66 0.7

Benzene diol + 3,5 dihydroxy-2 methyl-4-pyrone C6H6O2 + C6H6O4 110 + 142 14.13 0.7

5-Hydroxymethyldihydrofuran-2-one C5H8O3 116 14.42 0.8

1,2-Benzenediol C6H6O2 110 14.67 3.1 11.3

1,4:3,6-Dianhydro-hexose C6H8O4 144 14.86 1.0 1.9

Coumaran C8H80 120 15.13 0.5

5 Hydroxymethyl-2-furaldehyde C6H6O3 126 15.47 0.6

4-Methyl catechol C7H8O2 124 17.21 1.5 2.0

Syringol C8H10O3 154 18.69 0.5

Levoglucosan C6H10O5 162 22.80 5.4

Caffeine C8H10N4O2 194 30.36 22.3 5.2

Theobromine C7H8N4O2 180 30.75 1.8 1.0



seed of Cola nitida; however, current findings revealed there are yet more compounds that remain to be identified. The present work has identified more chemical compounds from Cola nitida. The presence of a number of FAs revealed in this publication serves as evidence of the potential varied biological applications of the seed. Recent reports on the biotechnological/nanotechnological applica-tions of products of Cola nitida have opened the door to a new di-mension of research activities involving the genus, especially with respect to waste management. The kola nut pod, which hitherto was considered a waste product, is being converted to useful products – turning waste into wealth. With the aid of well-planned quantita-tive HPLC analysis of the methanol extract of Cola nitida, some of the unknown compounds can be isolated and structural elucidation carried out. The chemicals identified in the kola nut can be deriva-tised by functional group inter-conversion to more potent and less harmful compounds for both industrial and medicinal applications.

Conclusions

Previous research on Cola nitida have established the presence of

xanthine alkaloids, catechins, epicatechins, anthocyanidins and its oligomers (proanthocyanidins). However, from the ESI-MS experiments, we discovered there are more compounds yet to be identified and structurally elucidated. In this report, the FAs and sugars present in Cola nitida have been identified using the GC-MS (FAME and TMS derivatives of the extracts). With analytical Py-GC-MS experiment, the constituents of thermal decomposi-tion of Cola nitida were also identified and compared with those of extracted biomass. In the antimicrobial assay, CH3OH extract and solvent fractions showed no activity against Shigella Spp. but were effective against most of the tested microbes. The N-hexane fraction was ineffective in both antimicrobial and antioxidant as-says. Caffeine was ineffective in antioxidant assay, but exhibited appreciable activity against Bacillus cereus, Escherichia coli and Aspergillus niger. The butanol fraction displayed higher antioxi-dant activity than the CH3OH extract, which might be due to the higher concentration of phenolic compounds in the butanol frac-tion.

Acknowledgments

The corresponding author wishes to acknowledge the Tertiary Education Trust Fund (TETFund) Conference grant from Obafemi Awolowo University to present part of this report at the 4th Interna-tional Pharma & Clinical Pharmacy Congress, November 07–09, 2016, Las Vegas, Nevada, USA.

Compound Molecular formula

Molecular weight

Retention time (min)

Kola nut, %

Extracted nut, %

Palmitic acid C16H32O2 256 32.36 1.5 0.8

Linoleic acid C18H32O2 280 35.44 1.0

Oleic acid C18H34O2 282 35.62 0.9 0.5

Stearic acid C18H36O2 284 36.01 0.3

C18:2 C18H32O2 280 36.67 0.3

C19:2 C19H34O2 294 37.16 0.4

Methyl-2,3-dicyano-3- [4-dimethylamino)phenyl]-2-propenoate C14H13N3O2 255 43.57 0.6

Squalene C30H50 410 46.34 0.4

Cholestadiene C27H44 368 47.28 0.1

Unknown steroid 344 47.50 0.1

Stigmastan-3,5-diene C29H48 396 49.89 0.3

Nortrachelogenin C20H22O7 374 50.77 0.2

6,9,10-Trimethoxy-12H-[1]benzopino[2,3,4-ij]isoquinoline (oxocularine) C19H17NO4 323 51.17 0.4

6,16 Dimethylpregna-1,4,6-triene-3,20-dione C23H30O2 338 51.58 0.3

Stigmasterol C29H48O 412 51.94 0.2

3,4,5,6-Tetrahydro alstonine C21H24N2O3 352 52.26 0.4

Sitosterol C29H50O 414 52.67 0.5

Table 4. Py-GC-MS of unextracted and extracted powdered Cola nitida seed - (continued)

Compound 1. 1,3,7-trimethyl-1 H-purine-2,6(3H,7H)-dione (Caffeine). Compound 2. n-Hexadecanoic acid.



Conflict of interest

The authors have no conflict of interests related to this publication.

Author contributions

Originator of the research idea and supervising the work (JKA), involving in isolation and antimicrobial experiments (SBO), as-sisting in the antimicrobial study (DAA), involving in the chemi-cal/instrumental analyses (AGM).

Supporting Information

Supplementary material for this article is available at https://doi.org/10.14218/JERP.2017.00015.

Table S1. In vitro antimicrobial activity of the extracts and com-pound 1 (caffeine) against selected microbes.

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Original Article

Bioequivalence Study of Generic Metformin Hydrochloride in Healthy Nigerian Volunteers

Adebanjo Jonathan Adegbola1, Olugbenga James Awobusuyi1, Babatunde Ayodeji Adeagbo1, Bolanle Stephen Oladokun1, Adegbenga Rotimi Owolabi2 and Julius Olugbenga Soyinka1*

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria; 2Department of Medical Pharmacology and Therapeutics, Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria

Abstract

Background and objectives: Metformin is key in the management of type 2 diabetes mellitus but also represents additional financial burden, particularly with the use of branded products. The availability of generic products permits generic substitution with a much-reduced cost of treatment. However, only generic products that offer similar bioavailability with the innovator should be considered. This study was designed to assess the bioequiva-lence of generic metformin tablets within Nigeria.

Methods: Metformin tablets selected from the Nigerian market were appraised for quality following British and United States Pharmacopoeia guidelines. In vivo bioequivalence study in healthy volunteers was applied for a ge-neric and the innovator brand in an open-label, 2-arm, 2-treatment crossover fashion with a 1-week washout period. Blood samples were collected at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24 h post-dose. Plasma concentrations of met-formin were analysed using a validated high-performance liquid chromatography method, and pharmacokinetic pa-rameters were obtained using the non-compartmental approach. The formulations were considered bioequivalent based on the guidelines by United States Food and Drug Administration, Centre for Drug Evaluation and Research.

Results: Nine generic products met the quality assessment standards, and the in vivo bioequivalence study was carried out in 17 healthy volunteers. The mean values for Cmax, Tmax, AUC0–24, and AUC0–∞ for the innovator brand of metformin were 0.43 ± 0.14 µg/mL, 1.35 ± 0.46 h, 2.03 ± 0.68 µg/mL* h and 2.63 ± 1.11 µg/mL* h respec-tively; for the generic product, the values were 0.44 ± 0.13 µg/mL, 1.41 ± 0.59 h, 2.04 ± 0.68 µg/mL* h and 2.85 ± 1.37 µg/mL*h. The 90% confidence intervals for the test formulation/reference formulation ratio for Log Cmax, Log AUC0–10 hr and AUC0–∞ were within the bioequivalence limits of 80% to 125% (95.8–106.8, 94.8–105.5 and 96.3–108.4 respectively).

Conclusions: The bioavailability of the test product was not inferior to innovator metformin.

Introduction

Metformin is an oral antidiabetic drug (OAD) belonging to the biguanide class. Other biguanides are phenformin and buformin, but the former was withdrawn from market due to reported links with serious cases of lactic acidosis.1,2 Metformin, however, re-mains the drug of choice in the management of type 2 diabetes mellitus, particularly in patients whose renal functions have not been compromised. According to the United Kingdom Prospective Diabetes Study (UKPDS), metformin is superior to other OADs in lowering both the macrovascular- and microvascular-related complications that characterize the disease progression in diabetic patients.3,4 Recently, some researchers have spoken out against the claims by UKPDS, citing methodological shortcomings.5 Despite

Keywords: Metformin; Bioequivalence; Antidiabetic; Pharmacokinetics.Abbreviations: API, active pharmaceutical ingredient; AUC, area under plasma concen-tration-time curve; BCS, Biopharmaceutical Classification System; BE, bioequivalence; BMI, body mass index; BP, British Pharmacopoiea; CDER, Centre for Drug Evalua-tion Regulatory; Cmax, maximum concentration; EDTA, ethylene diamine tetraacetic acid; ER, extended release; GMR, geometric mean ratio; HPLC, high performance liquid chromatography; IR, immediate release; LOD, limit of detection; LOQ, limit of quantita-tion; OAD, oral antidiabetic drug; SNPs, single nucleotide polymorphisms; TDM, thera-peutic drug monitoring; Tmax, time to reach maximum concentration; UKPDS, United Kingdom prospective diabetes study; USP, United State Pharmacopoiea.Received: March 16, 2017; Revised: July 15, 2017; Accepted: July 31, 2017*Correspondence to: Julius O. Soyinka, Department of Pharmaceutical Chem-istry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria. Tel: +2348035822785, E-mail: [email protected] to cite this article: Adegbola AJ, Awobusuyi OJ, Adeagbo BA, Oladokun BS, Owolabi AR, Soyinka JO. Bioequivalence Study of Generic Metformin Hydrochlo-ride in Healthy Nigerian Volunteers. J Explor Res Pharmacol 2017;2(3):78–84. doi: 10.14218/JERP.2017.00010.



Adegbola AJ. et al: Metformin bioequivalence study J Explor Res Pharmacol

this dichotomy, metformin remains a first-line drug in obese and non-obese diabetic patients, alongside lifestyle adjustment.6 It is considered superior to sulphonylurea because it causes no weight gain and it is rarely associated with hypoglycaemia.7,8 Moreover, it is safer than the thiazolinedidiones because it offers a cardio-protective effect instead of cardiotoxicity.9,10

Metformin, 1,1-dimethylbiguanide (Fig. 1), has a low molecu-lar weight (129.1 g/mol), good solubility (about 300 mg/mL) in polar solvents, resulting in solution with pH range of 1.2–6.8 at 25 °C; however, its lipophilicity and permeability are unacceptably low.11 Metformin is provided as 500, 850 and 1,000 mg tablets, either as immediate release (IR) and extended release (ER) for-mulations. Glucophage® (a descriptive name to describe its role as a ‘glucose-eater’) is an innovator product that stands out in terms of quality and efficacy over time, but is relatively unafford-able for some patients. In general, the high-price associated with some branded products may predispose patients to opt for generic products, often registered by the drug regulatory body. In Nige-ria, many generic products are in circulation, and they are often preferred by the populace because of the prevailing poor socio-economic status. This trend has helped to curtail rising in pharma-ceutical expenditure, especially in low- to middle-income coun-tries.12 However, generic substitution should not be based solely on the initial cost of treatment but on the overall cost effectiveness of pharmacological treatment.12 As a result, a standard has been set for generic substitution. Interchangeability is permitted when the generic product demonstrates bioequivalence (BE) and therapeutic equivalence with the innovator.

BE of a generic product could be determined by either in vivo or in vitro studies. In vivo BE studies are frequently used to establish therapeutic equivalence, but this approach is usually expensive and more rigorous and may require clinical trial or study expertise.13 In vitro dissolution profiles are proxies for establishing BE when the drug meets the criteria prescribed for a Biopharmaceutics Classifi-cation System (BCS) biowaiver.14 The BCS considers three major factors–dissolution, solubility and intestinal permeability–which influence the rate and extent of drug absorption from IR solid oral dosage forms.15 Metformin is highly soluble in water with poor permeability, and as such it is classified as a BCS class 3. It may enjoy a biowaiver if dissolution of 85% or more of the labelled amount of the active pharmaceutical ingredient (API) in both the generic and the innovator products is attainable within 15 min in standard dissolution media at pH 1.2, 4.5 and 6.8.14

An in vitro dissolution study on four generic products of met-formin showed that none of the four brands of metformin tested met this requirement because the innovator product and two others did not achieve 85% dissolution in 15 min.16 In a similar study conducted by Olusola et al.17 in 2012 on eight generic products of metformin, only three met the criteria for BCS biowaiver after a physiochemical equivalence testing. Thus, using an in vitro dis-

solution profile as a surrogate for in vivo BE is still debatable as in vivo-in vitro correlation has not been established for metformin in most cases.16 Developing countries will benefit from generic prod-ucts, unfortunately the resources for testing drug quality is limited. Thus, this study aimed to assess the bioavailability of generic for-mulations of metformin versus that of the innovator product.

Materials and methods

Materials

Metformin HCl was obtained from AK Scientific chemicals (United States), high-performance liquid chromatography (HPLC) grade acetonitrile and methanol were obtained from Scharlau® Chemicals (Spain). Cimetidine and potassium hydrogen phosphate were purchased from Sigma-Aldrich® Chemical Company (Ger-many). The innovator product of metformin (coded as A) and 13 other generic products of metformin tablets (coded as B, C, D, E, F, G, H, I, J, K, L, M and N) were purchased from retail pharma-cies in Ile Ife, Ilesa and Ibadan South-West, Nigeria. All were IR tablets and the products’ manufacturers and their batch numbers are as follows: Merck Sante, France(50009, manufactured in July 2012); Hovid BDH, Malaysia (03-536 BD, manufactured in March 2013); Jiangsu Ruinian Pharm, China (111208, manufactured in December 2011); Fredun Pharmacutical, India (FT 362, manu-factured in July 2012); NGC Plc, Nigeria (F0802, manufactured in June 2013); Medopharm, India (2G31, manufactured in July 2012); Drugfield Pharm., Nigeria (580302, manufactured in March 2011); Vitaphos Lab Ltd, Nigeria (V054, manufactured in August 2012); Rajat Pharmchem, India (RA 2001, manufactured in June 2012); Juhel Nig. Ltd, Nigeria (0015, manufactured in October 2012); Henan Topfond Ltd, China (120810740, manufactured in August 2012); Vapicare Pharm. India (FVU1201, manufactured in April 2012); Vapicare Pharm., India (EF21002, manufactured in October 2012); and Watson Global Pharm., Nigeria (20120801, manufactured in 2012).

Chemical assay and dissolution testing

Assay and in vitro BE comparison of the innovator and generic products of metformin were carried out as prescribed by the Brit-ish Pharmacopeia (BP) 2013.18 The potency of metformin in each product was established using ultraviolet (UV) spectrophotometric techniques by measuring the absorbance of the stated solution of metformin at 232 nm, with A1 cm1% taken as 798. The dissolution system complied with the requirements in the Monographs of the United State Pharmacopoeia for the dissolution test for tablets.19 Potassium dihydrogen phosphate buffer (0.68% w/v) was adjusted with 1 M sodium hydroxide to pH 6.8. Samples for UV analysis were withdrawn at predetermined intervals over a period of 1 h and the dissolution profile of concentration versus time was plotted.

In vivo BE study design

Ethical approval for the study was given by the Institute of Pub-lic Health, Obafemi Awolowo University, Ile-Ife, Nigeria. Written informed consent was obtained from each voluntary subject be-fore commencement of drug administration and sample collection. Healthy volunteers (n = 22) between the ages of 18 and 28 years-old, with body weight ranging from 45 to 75 kg, were recruited

Fig. 1. Chemical structure of metformin.


Adegbola AJ. et al: Metformin bioequivalence studyJ Explor Res Pharmacol

for the study. Each subject underwent a physical examination and medical history-taking, both conducted by a physician. After an overnight fast, the subjects were given a single oral dose of 500 mg metformin HCl tablet. Two products, consisting of a test product and a reference product, were administered to the subjects in a crossover fashion.

Study inclusion criteria included strict adherence to the follow-ing parameters: healthy adults; 18–45 years of age; non-smokers; not pregnant; and body mass index (BMI) between 18–32 kg/m2. Medically healthy was determined according to medical history and findings of physical examination. All study participants were required to provide voluntary written informed consent and show willingness and ability to fast overnight. Exclusion criteria in-cluded: history of renal impairment; pregnancy or lactation; recent significant blood donation; recent participation in similar studies (within 28 days); evidence of alcoholism or drug abuse, especially of drugs that could cause hypoglycaemic effect; and history of hy-persensitivity to biguanides.

Study treatment

The voluntary subjects were invited to the study centre at about 7:30 am on the study day. The subjects were told to observe over-night fasting prior to that day. The study was implemented as a single dose, two-period and two-treatment with a test and a ref-erence product in crossover design. The test product was chosen based on its equivalence with the reference product as determined by the quality assessment using assay and dissolution profiling of the two products. During the first period, half of the subjects received 500 mg metformin as the reference product, which was given as a single oral dose, while the remaining half received oral-ly 500 mg metformin as the test product. Venous blood samples were collected at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24 h following drug administration. A 1-week washout period could ensure that the level of metformin in plasma had fallen far below the limit of quantitation. Then, subjects who received the test product in period 1 were treated with the reference product and vice-versa. Venous blood samples of the subjects were collected into ethylenediami-netetraacetic acid bottles at predetermined time intervals over 24 h. The samples were centrifuged to obtain plasma and the plasma was stored at −20 °C until analysis in our Therapeutic Drug Monitoring (TDM) laboratory.

Quantification of metformin in human plasma

The analytical procedure involved modification of an extraction and HPLC method previously reported in the literature.20 A 1 mg/mL stock solution of metformin and cimetidine were prepared by dissolving 25 mg of each in a 25 mL volumetric flask using metha-nol, and the solutions were stored at 4 °C. The chromatographic system consisted of an Agilent 1,100 series liquid chromatography system (Agilent Technologies, United States) fitted with a quater-nary pump and a diode array UV detector (DAD; at 190–900 nm). Chromatographic separation was achieved at 25 °C on a reverse-phase Agilent Zorbax (C18) column (5 µm × 4.6 mm) while the mobile phase was acetonitrile-potassium hydrogen phosphate buffer (0.01 M, adjusted to pH 6.67), 55:45, applied at a flow rate of 1.2 mL/min. Sample was injected through a Rheodyne model 7725 valve (United States) fitted with a 20 µL loop.

The eluents were monitored with UV detection at 234 nm λmax, while chromatograms were recorded with HP Chemstation soft-ware. To 100 µL of plasma in a 2-mL Eppendorf tube was added

50 µL of 20 µg/mL cimetidine solution (internal standard), 100 µL of 8M NaOH and 1.25 mL of 1-butanol/n-hexane (50:50, v/v), followed by shaking for 2 min. After centrifugation at 10,800 g for 5 min, the whole organic layer was separated and transferred into another tube. Metformin was back-extracted with 100 µL of 1% acetic acid. The mixture was vortex-mixed and centrifuged for 1 min. The organic phase was removed, and a 20 µL volume of aqueous phase was injected into the chromatograph. The peak area ratio for each sample was generated from the peak response of metformin and cimetidine using UV detection at 234 nm.

The assay was validated according to FDA draft guidance (CDER, 2013) to reflect acceptable linearity, sensitivity, specific-ity, accuracy and precision. Calibration curves were constructed within 0.05 µg/mL to 5.0 µg/mL, based on the relationship be-tween the peak area ratios and the standard solutions of metform-in. A 100 µL aliquot of drug-free plasma samples were spiked with 50 µL of internal standard solution (20 µg/mL cimetidine) and standard solutions (between the range of 0.05–5.0 µg/mL) of metformin.

For each sample, the above-stated extraction procedure was car-ried out and the supernatant (20 µL) was injected into an HPLC column. A plot of peak area ratios versus concentrations of the standard solutions was made. The limit of detection (LOD) and limit of quantification (LOQ) were generated based on regression analysis of the calibration curve. Intra-day precision and accuracy were determined by analysis of five replicates of each QC level at 0.05 µg/mL, 0.5 µg/mL and 5.0 µg/mL concentrations. Inter-day precision was measured by analysis of duplicates of each QC con-centration on three different days.

Pharmacokinetic and statistical analysis

The plot of plasma concentration (C) against time (t) data of met-formin was carried out using Microsoft Excel 2010. The data were analysed to obtain pharmacokinetic parameters using the non-compartmental model by means of the KINETICA Pharma-cokinetic Software (United States). The results were recorded as mean ± standard deviation (SD). AUC0–10 h was computed using the linear method. The trapezoidal rule was applied when Cn > Cn−1. t0 was defined as C0. The AUCT was estimated as the sum of AUC0–10 h. The AUC10 h–∞. ke was the elimination rate con-stant and was obtained as the slope of linear regression of the ln transformed plasma concentration–time curve in the elimination phase. Half-life (t½) was computed from t½ = ln(2)/ke, clearance (CL) from Dose/AUCT and volume of distribution (Vd) from Vd = CL/ke = (t½ * CL)/0.693.

Comparisons of the pharmacokinetic parameters for the two products for determining BE were made using t-test and ANOVA by means of the SPSS 16 Software. After transforming BA param-eters (Cmax, AUC0–10 h, AUCT) to the logarithm scale, the data from both arms were compared by the 90% confidence intervals (CIs) using the ratio of geometric means. The test product was consid-ered to be BE compared with the reference sample if the 90% CIs for AUC and Cmax were within the predetermined BE range of 80% to 125% (CDER, 2014).

Results

The innovator product and 13 generic brands of metformin were selected for preliminary quality appraisal screening by weight uni-formity test, quantitative analysis and dissolution profiling. Twelve



generic brands passed the weight uniformity test, while ten brands passed both the assay and dissolution tests, following the stand-ard requirement stipulated by BP 2013 as they contained between 95–105% of the API of metformin and not less than 70% of API dissolved within 45 min during in vitro dissolution (USP, 2007).

Overall, nine generic products met all the standards stipulated in the official guidelines. The results for the assay and dissolution test are presented in Table 1 and Figure 2. Also, the quantity of met-formin that went into solution within 15 min is presented in Table 1, in order to show if the products rapidly dissolved to attain 85 %

Table 1. Product description and quality assessment of metformin tablets distributed within Nigeria

Product Weight Uniformity Test UV-Assay (% Content)Dissolution (% Released)

After 15 Min After 45 Min

A Pass 96.1 70.6 ± 0.20 85.3 ± 0.15

B Pass 100.0 86.7 ± 0.27 91.6 ± 0.67

C Pass 96.1 82.9 ± 0.21 86.4 ± 0.14

D Pass 93.9 44.6 ± 3.12 56.25 ± 0.10

E Pass 98.6 78.8 ± 0.20 84.2 ± 0.35

F Pass 100.2 76.5 ± 0.19 87.5 ± 0.16

G Pass 92.9 81.0 ± 0.36 86.6 ± 0.29

H Pass 98.5 58.0 ± 1.46 84.0 ± 0.11

I Pass 99.8 55.5 ± 0.78 88.4 ± 0.46

J Fail 95.2 67.5 ± 0.50 86.2 + 0.16

K Pass 100.2 2.5 ± 0.17 2.8 ± 0.04

L Pass 95.6 87.7 ± 0.22 89.1 ± 0.12

M Pass 95.3 84.6 ± 0.23 87.6 ± 0.06

N Pass 101.4 89.0 ± 0.06 89.6 ± 0.38

Fig. 2. Dissolution profile of metformin tablets in the Nigerian market.



total release within 15 min with the aim of determining whether the products meet criteria for biowaiver. Though similar dissolution profiles were observed for all the products, except products C and K, only three generic products rapidly released ≥85% metformin API. This led to the extension of this project to in vivo study.

For lack of resources, only product B was considered for in vivo comparative study. Seventeen healthy volunteers, including six fe-males and eleven males, completed the in vivo study (Table 2). Treatment with both generic and innovator products was well tol-erated. Metformin was quantifiable in all the subjects from 30 min to 10 h post-dose sampling points. The bioanalytical procedure for metformin analysis was validated based on FDA/CDER guide-lines. The LOD and LOQ were 10.2 and 30.9 ng/mL respectively. Accuracy, recovery and intra-day and inter-day precision are pre-sented in Table 3. The average plasma concentrations-time profile of metformin for the innovator (reference) and the generic (test) products is depicted in Figure 3. Derived pharmacokinetic parame-ters, 90% CI and geometric mean ratio (GMR) of the test/reference products for logarithm-transformed BE parameters (Cmax, AUC0–10 hr and AUC0–∞) are presented in Tables 4 and 5.

Discussion

Out of 14 brands of metformin tablets in the Nigerian market, 10 products were found to be fit according to weight uniformity test, UV-spectrophotometric quantitative analysis and dissolution test. Eight of those products were generic and they were found to dem-onstrate pharmaceutical equivalence with the innovator brand by releasing 75% or more within 45 min using a basket rotating at 100 rpm. The products could have enjoyed in vivo biowaiver but some, including the innovator brand, were not sufficiently released in pH 6.8 phosphate buffer during the dissolution test to meet the specification of 85% or more release within 15 min.

Metformin is highly hydrophilic, with poor permeability and

it belongs to class III of BCS. To establish in vitro BE-in vivo BE correlation, metformin products should release ≥85% of API within 15 min.15 The reason for this is that if the products rapidly dissolved under all physiological conditions, one will expect such products to behave like oral solutions in vivo. Only three generic products released ≥85% of its API within 15 min. This pattern was similar to that of previously reported dissolution studies on met-formin tablets in Nigeria, where the products were noted not to be rapidly dissolving in any of the three media having pH 2.0, 4.5 and 6.8.16,17 The feasibility of generic substitutions of OAD with the same amount and quality of API in the management of diabetes relies on the fact that the products are therapeutically equivalent and are able to offer glycaemic control of <7.0% HbA1C and pre-prandial capillary plasma glucose of 80–130 mg/dL.21

As in vitro BE-in vivo BE correlation is still debatable for met-formin, further in vivo BE study was launched in accordance with the ICH guidelines on Good Clinical Practice and Guidance for Industry Bioavailability and Bioequivalence Studies.22,23 The test product was selected based on the prequalification dissolution test and assay of metformin tablets, while the innovator brand of 500 mg metformin IR tablet served as the reference product. The healthy subjects treated with the test and reference products in a crossover fashion seemed to tolerate the treatment very well, as no adverse events were recorded. We started with 22 healthy volun-teers, but we had to exclude 5 subjects because they contradicted the rule for this study either by taking other drugs during the study period, taking a meal unduly, or not being available at the time required for pharmacokinetic sampling.

In this BE study, metformin quantification from human plasma was achieved by adapting a simple, sensitive and selective HPLC method by Amini et al.20 This method was slightly modified and validated in our TDM laboratory. Samples were pre-treated by bas-ification with sodium hydroxide, extraction with 50%v/v butanol-hexane, and back-extracted with 1% acetic acid. Cimetidine was used in lieu of ranitidine. Though the LOD and LOQ in our study were slightly above the corresponding data generated by Amini’s group, 10.2 and 30.9 ng/mL respectively compared with 5 and 15.6 ng/mL,20 quantification of plasma levels of metformin at 0–10 h after a single oral dose of 500 mg metformin tablet was adequately realizable.

All the findings for pharmacokinetic parameters in this study are in concordance with the other data reported previously. This study found 4.31 ± 2.2 h as the half-life, 221.86 ± 86.5 L/h as the clearance, and 1,190.33 ± 421.75 L as the volume of distribution respectively after a single oral dose of 500 mg of reference prod-uct. The corresponding data for a single oral dose of test product are 5.67 ± 1.74 h, 214.29 ± 96.91 L/h and 1,273.35 ± 468.87 L. The within-subject variability during the two treatments among the Nigerian healthy volunteers was low, as the average clearance and the average volume of distribution of metformin determined for both products were not statistically significantly different from each other.

However, wide variations in both clearance and volume of dis-tribution do exist between subjects in this study. Similar findings

Table 2. Demographic features of the study participants

Demographic Features

Participants, total 22

Participants, completed the study 17

Gender, female/male 6/11

Mean ± SDa

Age (Years) 27.4 ± 5.54

Height (m) 1.61 ± 0.08

Weight (Kg) 55.5 ± 11.8

BMI (kg/m2) 23.5 ± 3.81

ameans ± the standard deviation of Demographic features.

Table 3. Validation of bioanalytical HPLC- UV detection method for metformin

Concentration, µg/mL Accuracy, % (n = 6) Recovery, % (n = 6)Precision, CV %

Intra-day Inter-day

0.05 78.0 98.0 ± 1.82 14.9 12.5

0.5 103.2 83.9 ± 0.83 3.0 8.7

5.0 97.8 96.6 ±2.16 3.4 18.0



have been reported for metformin pharmacokinetics in another population.24 The reason put forward to explain these variations is inter-subject variability in the oral bioavailability (F) of met-formin and inter-subject variation in the ratio of the renal clearance of metformin to creatinine clearance which is independent of F. Another strong rationale is the single nucleotide polymorphisms in metformin transporters-organic cation transporters in either healthy subjects and diabetic or obese individuals.24–27

Other pharmacokinetic parameters, such as maximum plasma concentration (Cmax), time to reach the maximum concentration (Tmax) and plasma exposure (AUC), were considered for the deter-mination of BE of the two products. There was no significant dif-ference for Cmax, Tmax and AUC, as shown in Table 3. After loga-rithm transformation of BE parameters, the GMR and 90% CI was within the BE acceptable range, from 80% to 125%.

Future research directions/recommendations

As a result of the great importance of generic drugs in healthcare, it is imperative that their pharmaceutical quality and in vivo perfor-mance be reliably assessed before they could be used interchange-

ably with the innovator product in the marketplace. It must be demonstrated that the safety and efficacy of the generic drugs are comparable to those of the innovator drugs.

Conclusions

This BE study found that the 500 mg of the test product is equiva-lent to 500 mg of the reference product of metformin. The out-come of the in vitro study correlates well with the in vivo study and both formulations met the regulatory standards for assuming BE in healthy volunteers.

Acknowledgments

The authors appreciate the facilitators and program coordinators of the bioequivalence online course organized by Karolinska In-stitute. The commitment of the subjects who participated in the in vivo study and the technical input of Babajide Shenkoya and Toyin in drug analysis are gratefully recognized.




Designing the research (JOS, AJA, BAA), recruiting volunteers (JOS, AJA, BAA, OJA, BSO, ARO), certifying medical fitness of volunteers (ARO), analysing samples (JOS, AJA, BAA, OJA, BSO), preparing the manuscript (JOS, AJA, BAA, OJA, BSO,

Fig. 3. Plasma concentration versus time profile of metformin following oral administration of an innovator and a generic product.

Table 4. Derived pharmacokinetic parameters of metformin after oral administration of 500 mg tablet of innovator and a generic product

Parameter Reference A (Mean ± SD) Test B (Mean ± SD) p-value*

Cmax, µg/mL 0.43 ± 0.14 0.44 ± 0.13 0.8133

Tmax, µg/mL 1.35 ± 0.46 1.41 ± 0.59 0.5795

AUCL, µg/mL * h 2.03 ± 0.68 2.04 ± 0.68 0.9718

AUCT, µg/mL * h 2.63 ± 1.11 2.85 ± 1.37 0.4434

t½, h 4.31 ± 2.2 5.67 ± 1.74 0.0003

CL, L/h 221.86 ± 86.5 214.29 ± 96.91 0.6126

Vd, L 1,190.33 ± 421.75 1,273.35 ± 468.87 0.4426

VSS, L 1,335.17 ± 441. 00 1,371.33 ± 382.01 0.7110

*Threshold of significance was set at <0.05.

Table 5. Derived pharmacokinetic parameters of the test/reference products for logarithm-transformed BE parameters

Parameter 90% CI GMR Test/Reference

Log Cmax 95.8–106.8 101.3

Log AUC0–10 hr 94.8–105.5 100.2

Log AUC0–∞ 96.3–108.4 102.3

Abbreviations: CI, confidence interval; GMR, geometric mean ratio.



ARO).

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Review Article

Introduction

Psychotic disorders, such as schizophrenia, are among the top ten causes of disability worldwide,1 and are associated with a spec-trum of neurocognitive deficits, which may be present from the very early stages and worsen with the onset of frank psychosis.2 Typically, the onset of psychosis occurs in late adolescence or early adulthood,3,4 with 70% of these individuals experiencing a second episode within 5–8 years.3,5 Although studies have consist-ently identified a number of environmental as well as genetic risk

factors that contribute to the risk architecture of psychosis,4,6–8 mechanistic understanding as to how they may increase the risk of psychosis is unclear. Such understanding is critical to the iden-tification of novel therapeutic targets as well as of biomarkers that may predict the risk of disease before the actual onset of illness, of relapse following onset, or indeed of biomarkers of response to treatment.

In this review, we explore the current understanding of the neu-robiological underpinnings of psychosis, focusing on knowledge gained from one prominent risk factor in particular. Following this, we propose a novel approach that may help shed mechanistic insight on aspects of the presentation of psychosis. For the pur-poses of the present review, the risk factor of interest is cannabis use, and its activity within the related endocannabinoid system in man.

Cannabis, the endocannabinoid (eCB) system, and psychosis

Recreationally, cannabis is one of the most widely used illicit drugs in the world.9 However, its use is also recognised as one of the most preventable risk factors for the onset and relapse of psy-chotic disorders.10–13 A recent meta-analysis revealed that the risk of onset of psychosis amongst cannabis users is 2–4 times higher than in non-users, depending on degree of exposure.14 A separate

Investigating the Role of the Endocannabinoid System in Early Psychosis

Aisling O’Neill1 and Sagnik Bhattacharyya1*

1Department of Psychosis Studies, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London, UK

Abstract

Accumulating evidence suggests that dysfunction within the endocannabinoid (eCB) system may play a role in psychosis. However, little is understood about how this may be related to the neurocognitive abnormalities and symptoms of psychosis. In this paper, we summarize some of the evidence supporting the role of eCB system in psychosis, as well as the current understanding of the neurocognitive underpinnings of psychosis. We particularly focus on neuroimaging evidence pertaining to alteration in the functional integration between different brain re-gions in patients with psychosis, and then relate this to evidence from neuroimaging studies of the effects of can-nabis and its main ingredients, such as delta-9-tetrahydrocannainol and cannabidiol. Specifically, we explore this in the context of the hypothesis that psychosis is a disorder of dysconnectivity between different brain regions, focusing particularly on three large scale functional networks (the default mode, central executive, and salience networks), alterations in which have been implicated in psychosis, and we discuss the gaps in this research thus far. Finally, we propose that an approach to investigating the role of the eCB system in psychosis may be to employ a pharmacological cannabinoid challenge paradigm to examine how experimental perturbation of the eCB system may be related to abnormalities in the brain networks implicated in psychosis. We discuss challenges associated with this approach, and suggest safe and practical options to overcome the main issues involved with such an experimental approach. Studies employing such an approach have the potential of offering insight into the neu-rocognitive mechanisms underlying psychosis, and identifying novel therapeutic targets.

Keywords: Psychosis; Endocannabinoid system; Connectivity; Functional neuroim-aging; Cannabidiol.Abbreviations: eCB, endocannabinoid; Δ9-THC, delta-9-tetrahydrocannabiniol; 2-AG, 2-arachidonoylglycerol; CBD, cannabidiol; FC, functional connectivity; fMRI, functional magnetic resonance imaging; DMN, default mode network; SN, salience network; CEN, central executive network; rs-fMRI, resting-state functional MRI; MRC, Medical Research Council; NIHR, National Institute for Health Research; NHS, National Health Service.Received: March 14, 2017; Revised: May 22, 2017; Accepted: May 29, 2017*Correspondence to: Sagnik Bhattacharyya, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, 16 De Crespigny Park, SE5 8AF, UK. Tel: +44 20 7848 0955, Fax: +44 20 7848 0976, E-mail: [email protected] to cite this article: O’Neill A, Bhattacharyya S. Investigating the Role of the Endocannabinoid System in Early Psychosis. J Explor Res Pharmacol 2017;2(3):85–92. doi: 10.14218/JERP.2017.00009.



O’Neill A. et al: Endocannabinoid system and psychosisJ Explor Res Pharmacol

meta-analytic review found that the prognosis for individuals with psychosis who continued to use cannabis following onset of illness was significantly worse compared to those who discontinued use after onset, in terms of outcomes, relapse rates, hospital admis-sions, and positive symptoms.15

Of particular concern has been the heavy use of high-potency strains of cannabis, especially given evidence that the availability of high-potency cannabis has been on the rise over the last two decades.16–18 In the context of cannabis use, potency is determined by the level of delta-9-tetrahydrocannabiniol (Δ9-THC), the main psychoactive ingredient present in the extract of the cannabis plant. Indeed, the frequent use of high-potency strains of cannabis, which contain high levels of Δ9-THC, has been associated with signifi-cantly greater risk of onset and of relapse in psychosis, compared to use of less potent forms or to less frequent use.12,16,19

Δ9-THC, the primary psychotropic constituent of cannabis, binds to the endogenous cannabinoid receptors, which are distrib-uted throughout what is known as the “endocannabinoid system” (eCB system).20,21 The eCB system mainly constitutes endogenous cannabinoid receptors (CB1 and CB2 receptors) and their ligands (including anandamide, and 2-arachidonoylglycerol, or 2-AG) dis-tributed throughout the central and peripheral nervous systems in the mammalian brain.22 Expression of the CB1 type endocannabi-noid receptor is particularly high in the hippocampus, cerebellum, basal ganglia, and neocortex23—regions involved in a number of cognitive processes of particular interest in the context of psycho-sis, such as learning, memory, and attention processing.24–26 Ex-pression of the CB2 receptor is predominantly observed in immune cells, from where it is thought to exert an effect on immune func-tions.21 CB2 receptor expression has also been observed in central nervous system neurons, albeit at much lower levels than the CB1 type.21 Various animal studies have proposed links between CB2 receptor function and anxiety,27 emesis,28 schizophrenia-related behaviours,29 alcohol preference,30 and impulsive behaviours.31 However, these functions – and the cellular mechanisms through which CB2 receptors exert these functions—are still disputed.32

Consistent with the known CB1 receptor distribution, acute ad-ministration of Δ9-THC in healthy individuals has been shown to induce transient psychotic symptoms,33,34 and to cause impairments in aspects of memory and learning,33,35 abnormalities in inhibitory control processing and attentional salience processing,34,36,37 as well as alter the normal activity of the neural substrates underly-ing all of these processes.38,39 Evidence that modulation of these brain regions and cognitive processes by acute administration of Δ9-THC resembles aspects of the neural abnormalities and psy-chopathology that are also observed in schizophrenia further sup-port a role for alterations in the eCB system in the pathophysiology of the disorder, and highlight it as an important target for further research.20

Independent of its role in modulating the psychoactive and psy-chotomimetic effects of cannabis, the eCB system has also been implicated in schizophrenia in other ways.40 One potential con-tributing element to the overall role of the eCB system in schizo-phrenia may be the relationship between eCB dysfunction and ab-normal dopamine levels.41 Normal dopamine activity is involved in a number of cognitive processes, such as motivational salience, decision making, and attention and cognitive control, which are altered in schizophrenia.42–44 Striatal dopamine hyperactivity, in particular, is one of the most consistent findings in the pathophysi-ology of psychosis.45

Although causality is unclear, as the influence of one on the other may in fact be bidirectional, irregularities in both the availa-bility of this neurotransmitter and in the activity of the eCB system are very likely related. This may be inferred from the dysregulated

neural levels of anandamide—an endogenous cannabinoid—ob-served in hyperdopaminergic rat models of schizophrenia,46 and increases in dopamine in the nucleus accumbens of healthy rats following acute administration of anandamide.47 Additionally, an accumulating body of evidence suggests that acute and chronic cannabis use in humans may affect dopamine release and synthesis differentially, as reviewed by Sami et al.48

eCB system alterations have also been implicated in the patho-physiology of schizophrenia through the findings of post-mortem studies, which have identified exaggerated CB1 receptor binding in the dorsolateral prefrontal cortex as well as abnormal levels of anandamide across the brain in individuals with schizophre-nia.49,50 Indeed, dysregulated levels of anandamide have also been observed in animals following repeated Δ9-THC administration,51 and in the cerebrospinal fluid of first-episode psychosis patients with co-morbid high frequency cannabis use, as compared to first-episode patients with low frequency cannabis use, and healthy controls,52 further linking alterations in components of the eCB system to the psychosis-like effects of Δ9-THC.

These findings from studies of both the effects of cannabis use on behaviour and brain activity, and abnormal eCB function in psychosis warrant a systematic investigation of the eCB system in the context of psychosis, and indicate that an optimal approach would be an experimental medicine paradigm, in conjunction with cannabinoid administration. However, ethical and safety consid-erations preclude studies involving the administration of cannabis or Δ9-THC to individuals with psychosis.

Cannabidiol (CBD)

The use of CBD, a non-psychotropic component of the cannabis plant, is a potential approach that could overcome the ethical issues surrounding pharmacological challenge studies involving adminis-tration of cannabis or Δ9-THC in patients with psychosis. There is evidence to suggest that short-term CBD administration (4 weeks) may result in an increase in peripheral anandamide levels in pa-tients with schizophrenia, associated with a reduction in psychotic symptoms53; and may also counteract the psychotic symptoms, cognitive impairments, and associated brain activation abnormali-ties induced by Δ9-THC administration in healthy volunteers.54,55

Though the mechanism of action underlying the effects of CBD is still unclear, a range of molecular mechanisms have been suggested as acting either individually or in conjunction with oth-ers to produce the aforementioned notable effects.53,56 One theory relates to the potential action of CBD as a high-potency antagonist of CB1 receptor agonists,57 which—in opposition to the partial agonist activity of Δ9-THC at CB1 receptors—may result in the contrasting effects induced by the two exogenous cannabinoids. Another prominent argument concerns the ability of CBD to en-hance anandamide signalling via the inhibition of anandamide uptake and intracellular degradation.56,58 As noted earlier, this in-crease in anandamide levels has been associated with decreases in psychotic symptoms, and though the exact nature of this relation-ship remains inconclusive, it is likely that this ability to increase anandamide signalling is related to the antipsychotic properties of CBD.53

Nonetheless, consistent with this evidence overall, in recent years there has been considerable interest in a potential role for CBD as an antipsychotic treatment.59 Additionally, CBD has also been found to display neuroprotective properties, and has demon-strated a low side effect profile, and tolerability in doses of up to 1,500 mg.60,61 All of these factors combine to make CBD an ideal tool for the safe perturbation of the eCB system in a pharmacologi-


O’Neill A. et al: Endocannabinoid system and psychosis J Explor Res Pharmacol

cal challenge paradigm, and will allow the further investigation of abnormalities of the eCB system in clinical populations.

Psychosis as a disorder of dysconnectivity

Although abnormal patterns of brain activity in specific brain re-gions are well documented in psychosis and have been associated with aspects of the illness,62 these localised abnormalities have thus far failed to provide a comprehensive account of the neuro-cognitive mechanisms underlying the multiple, complex pheno-typic features of the disorder.63,64 There is increasing recognition that attributing even very specific symptoms, such as auditory hal-lucinations, to dysfunction in just one localised area of the brain is overly simplistic, and that such an approach is far less plausible for the broad range of positive and negative psychotic symptoms and ubiquitous cognitive deficits that characterize psychotic disorders, such as schizophrenia.64

This is further complicated by the fact that these symptoms are not only heterogeneous in their presentation across individu-als with psychosis, but can also change in prominence across time within the same individual. This limitation has prompted a more integrated approach, focusing instead on the interaction over time of brain regions that have been traditionally functionally segre-gated. The ensuing research drove the development of what is known as the dysconnectivity theory of schizophrenia,65 which describes the psychopathology of the disorder as resulting from an underlying dysconnection syndrome.65–67 The term functional connectivity (FC) describes the temporal relationship between ac-tivation measured in different brain regions, either at rest or dur-ing a task,68 and is inferred from the correlation between regional fluctuations in the blood-oxygen-level-dependent signal of differ-ent brain regions, measured using functional magnetic resonance imaging (fMRI).

A number of studies have investigated FC alterations in the con-text of psychosis, which are summarized below.

Results of resting state studies

A popular approach in neuroimaging literature has been to de-scribe brain regions as being organized into functional neural net-works, three of which are of particular interest in the context of FC research across neuropsychiatric diagnoses.64 These networks are the Default Mode Network (DMN), the Salience Network (SN), and the Central Executive Network (CEN). Resting-state func-tional MRI (rs-fMRI) techniques have proven particularly useful in the study of such networks in psychosis. They provide a means of representing intrinsic brain function and connectivity between brain regions under resting as opposed to activation conditions, which involve external stimuli or induced reactions.69,70

The DMN is largely active at rest, and as such, is engaged by a range of internally directed thought processes, including self-ref-erential thought, aspects of autobiographical memory, and future simulations—all processes that are notably disrupted in schizo-phrenia.71 Anatomically, the core nodes of the DMN include the posterior cingulate cortex and the precuneus, the medial prefron-tal cortex, and the angular gyrus.72 Additional regions involved in DMN processes include the dorsal medial subsystem, and the medial temporal subsystem.72

Normally, DMN and CEN activity are thought to be anti-cor-related.73 That is, DMN activity is reduced during externally ori-ented task states, at the same time as CEN activity is increased,

and vice versa for the internally oriented/resting state. As such, the CEN is thought to be responsible for higher level cognitive func-tions (e.g. attentional control, and executive task performance), and is rooted in the dorsolateral prefrontal cortex and the posterior parietal cortices.74

Critical to the appropriate engagement and disengagement of CEN and DMN activity, the SN is believed to moderate this “switching” between networks through the attribution of salience to external or internal stimuli.64 In salience literature, the attribu-tion of salience refers to the assignment of importance to external stimuli or internal mental events, critical in the processing of an individual’s experiences.75 The anterior insula (a core region in the SN) is thought to moderate the shift between activity in the DMN (internally directed processes) and CEN (externally directed processes) by increasing cognitive and task control system activ-ity, whilst suppressing DMN activity when a salient event is de-tected.75,76 In contrast, in individuals with schizophrenia, abnormal levels of dopamine in the SN are thought to result in aberrant an-terior insula activity (with a particularly high expression of do-pamine D1 receptors in the anterior insula), which in turn results in the misattribution of salience to external/internal stimuli, and consequently the dysfunctional switching between DMN and CEN engagement.64, 77–79

The functional consequences of this sequence include greater connectivity between the DMN and CEN, greater connectivity within the DMN, and decreased anterior insula activity occurring at rest, as well as a failure to suppress DMN activity during ex-ternally driven tasks.77,80 Decreased FC between the SN and both the DMN and CEN at rest has also been reported in patients with schizophrenia,77,81 as well as an overall reduction in the strength of negative FC between task-positive and task-negative networks during both rest and task in patients diagnosed with schizophrenia, as well as with other psychotic disorders.73,80

It is thought that the potential symptomatic consequences of these connectivity abnormalities range from hallucinations and deficits in emotional processing (resulting in part from misattribu-tion of salience) to deficits in self-referential thinking (resulting in part from the over-engagement of the DMN). As such, this triple network model is thought to provide the most unified account to date of the mechanisms underlying the spectrum of different psy-chosis symptom domains—from deficits of self to the classic posi-tive, negative and cognitive domains.82

Apart from the anterior insula, an additional core node of the SN is the dorsal anterior cingulate cortex, though its broader func-tions also rely on input from the amygdala, ventral striatum, and the substantia nigra/ventral tegmental area75—with a high expres-sion of both dopamine and CB1 receptors observed in the dorsal and ventral striatum, and in the substantia nigra.21,83

If eCB dysfunction were to have a role in the pathophysiology of psychosis, one would expect it to modulate components of the three networks described here, in a manner consistent with altera-tions observed in those with psychosis. However, this has yet to be examined. Indeed, few studies have investigated the effects of Δ9-THC on FC during cognitive tasks or at rest. The limited available evidence suggests that Δ9-THC can induce a reduction in connec-tivity between the SN and the CEN, increase connectivity between the DMN and CEN, and increase connectivity within the DMN, during salience processing in healthy individuals37,84—reflective of those disturbances described in the triple network model. This, coupled with the high distribution of CB1 receptors within the SN and the propensity for acute Δ9-THC to impair performance on salience processing tasks in healthy individuals,85 would suggest a plausible if as yet undefined role for eCB dysfunction in the triple network model of schizophrenia.



Results of cognitive activation studies

In the context of characterising overall functional dysconnectivity in schizophrenia, studies that employ cognitive tasks are essential for a more complete understanding of the nature of disturbances observed during psychosis. Learning and memory impairments are particularly well-documented phenomena in the neurocogni-tive profile of individuals with schizophrenia, both in the context of comorbid cannabis use and its absence,86,87 and are similarly observed in healthy individuals following both acute Δ9-THC administration and chronic cannabis use.39,88 Across all illness stages of schizophrenia, but particularly during the first episode of psychosis, memory tasks involving verbal learning and encod-ing have been found to display significant impairments, compared to healthy individuals.26 Similarly, verbal learning, memory, and attention appear to be the most consistently impaired cognitive do-mains in studies of acute and chronic cannabis use.89

Encoding refers to the mental storage of information for later retrieval or recollection from short-term or long-term memory, and is crucial for learning.86 The brain regions largely involved in ver-bal learning and encoding include, but are not limited to, the me-dial temporal lobe (formation of new memories) and the prefron-tal cortex (essential for executive control functions and salience processing).90–92 Both regions display significant abnormalities in activation and connectivity during encoding and recall across illness stages in schizophrenia.86, 91–96 Specifically, reports of connectivity-related abnormalities have included decreases in FC between the DMN and some regions involved in executive control, and decreased connectivity within the DMN during encoding and recall tasks.94–96 Connectivity within the DMN and the regions in-volved in executive control was found to correlate positively with task performance,93 indicating a failure to recruit crucial neural resources that is linked to level of cognitive impairment.

Neural abnormalities in corresponding regions have been ob-served during encoding and recall tasks in healthy individuals ad-ministered Δ9-THC, including decreases in insular activity during encoding,97 increases in parahippocampal activity while learning during repeated trials of encoding, and a change in ventrostriatal activation during repeated trials of cued word recall condition.98 While the effect of Δ9-THC on the FC between these regions dur-ing encoding and recall has not previously been explored, these studies do highlight the importance of such investigations, and of further research of the eCB system overall.

Effect of eCB system perturbation on neurocognitive sub-strates implicated in psychosis

As outlined earlier, while the justification for investigating the role of eCB dysfunction in psychosis is clearly there—focusing particularly on the relationship between experimentally induced perturbations of the eCB system and the function of neural sub-strates implicated in psychosis, as well as symptoms and cogni-tive changes characteristic of psychosis – this has yet to be carried out systematically. Additionally, as discussed previously, though its safety and pharmacological profile make CBD an ideal tool for safe perturbation of the eCB system in clinical populations, to our knowledge, no study as yet has investigated the effects of such per-turbation on the neurocognitive substrates implicated in psychosis, in psychosis patients directly. However, given the clear parallels between neurocognitive abnormalities observed in psychosis and those induced by Δ9-THC, results of studies investigating the op-posing effects of CBD and Δ9-THC in healthy individuals are also

highly informative.In healthy individuals, the neural effects of CBD compared

to those of Δ9-THC are relatively consistent, generally showing a direct and opposite effect on brain activation and connectivity during cognitive tasks, including salience processing, emotional processing, learning, and short-term memory.34,37,55,99,100 The re-sults of both human and animal studies exploring the behavioural effects of CBD in comparison to Δ9-THC on these same cognitive processes that are also strongly affected in schizophrenia, specifi-cally learning and short-term memory, have thus far been less con-sistent.54,55,101,102 This variability may be related to a number of factors, such as the heterogeneity of study designs (including vary-ing CBD dosage) and modest sample sizes, together with limited overall research on the topic. In particular, differences in cognitive activations tasks employed in previous studies may have contrib-uted to inconsistency in results. Not only have previous studies employed cognitive tasks that engage different cognitive domains, they also commonly vary in degree of difficulty,20 hindering cross-study comparability.

Future research directions

As outlined above, despite the importance of such investigations for understanding the neurobiological underpinnings of psycho-sis, there is a clear lack of studies that have investigated the re-lationship between dynamic perturbation of the eCB system and functional brain abnormalities, or indeed FC between the DMN, SN and CEN in patients with psychosis. We posit that the optimal approach to address this gap would be for studies to investigate the effects of acute and/or short-term perturbation of the eCB sys-tem in patients with psychosis. Exploration of the role of the eCB system in the neurobiology of psychosis is most ideal in the early stages of psychosis, as such studies will be able to overcome issues relating to the longer term effects of illness course and antipsy-chotic treatment on aspects of cognition.2,103.

Indeed, previous research has also shown functional dyscon-nectivity to become increasingly widespread from the early to the latter stages of schizophrenia.104. Changes in FC have also been ob-served after relatively short-term antipsychotic use (12 weeks).105 Utilising a paradigm that focuses both on resting state abnormali-ties as well as the cognitive domains that are notably impaired in early psychosis, such as verbal memory, will be particularly useful in informing a comprehensive understanding of the role of the eCB system in large scale network dysconnectivity in psychosis.

Conclusions

Overall, such an approach may help connect multiple theoretical strands in schizophrenia research and rationally integrate a role for the eCB system into the relatively well-established dysconnectiv-ity theory, focusing on the dysconnectivity of large-scale networks in psychosis. This may help formulate a comprehensive frame-work for the neurocognitive abnormalities underlying psychosis.

Acknowledgments

This work was supported by grants to Dr Sagnik Bhattacharyya from the Medical Research Council (MRC), UK (MR/J012149/1; MC_PC_14105 v.2). Sagnik Bhattacharyya has also received support from the National Institute for Health Research (NIHR)



(NIHR Clinician Scientist Award; NIHR CS-11-001) and from the NIHR Mental Health Biomedical Research Centre at South Lon-don and Maudsley National Health Service (NHS) Foundation Trust and King’s College London. Aisling O’Neill was supported by the NIHR Collaboration for Leadership in Applied Health Re-search and Care South London at King’s College Hospital NHS Foundation Trust. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.




Both of the authors contributed in a substantial way to the study, and approved the manuscript content. Both authors were involved in the design, analysis and interpretation of findings. O’Neill A wrote the first draft of the manuscript, and both authors contrib-uted to its critical revision and gave final approval of the version for publication.

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Review Article

Introduction

Cannabis

Cannabis in both its pure and altered forms has been beneficial for human use since antiquity.1 Members of the genus Cannabis mostly produce dioecious annual herbs.2,3 The exact number of Cannabis species is a point of great debate, as according to differ-ent researchers there are variable number of species.4–10 The spe-cies that are most pertinent include, Cannabis sativa, Cannabis ruderalis and Cannabis indica. However, among these, the highly polymorphic species Cannabis sativa L. is considered as the most active, based on studies targeting its morphology, anatomy, phyto-

chemistry and genetics.11 The morphological diversity of this plant is phenomenal, and it has tremendous potential as foodstuff and fuel (edible food/oil from its achene), fibre (stem) and pharmaceu-ticals. It also has unrivalled biochemical riches with regard to its considerable balance of active and biologically significant com-pounds and their potential medical uses.12

History

The history of C. sativa use dates back to over 10,000 years, sup-porting its recognition as one of the oldest domestic plants known to humanity.7,13 It originated from Central Asia and is one of the oldest known psychotropic drugs. C. sativa was cultivated and consumed long before civilization; therefore, uncovering the ori-gin of its use by humans is a difficult task. Archaeological discov-eries have shown that it has been recognized and acknowledged since the Neolithic era in China, (around 4000 BC).13 However the psychoactive potential of this plant was recognized by western medicine quite latter, with the year of 1839 seeing the first of its real description of actions.14

China’s Emperor Shen Nung wrote in his 2737 BC compen-dium the first description of the properties and medicinal uses of C. sativa.14 Subsequently, it was cultivated for its fibre, fuel, seeds and medicinal purposes.11 A distinguished surgeon in ancient Chi-na, Hua Tuo (115–205 AD), reportedly used cannabis as an anaes-thetic. The analgesic and anaesthetic tendencies of C. sativa were also revealed in the biography of Chinese physician Hoa-tho, who practiced around 220 AD.15 C. sativa then spread to the rest of the world, to ancient Egypt, prehistoric Europe, ancient Greece and Rome, Persia and Arabia, India, South America, Europe and North

Cannabis: A Prehistoric Remedy for the Deficits of Existing and Emerging Anticancer Therapies

Bakht Nasir1, Humaira Fatima1, Madiha Ahmed1, Abdul-Rehman Phull2 and Ihsan-ul-Haq1*

1Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan; 2Department of Biological Sci-ences, College of Natural Sciences, Kongju National University, 56 Gongju Daehak-Ro Gongju-Si, Chungnam 32588, Republic of Korea

Abstract

Cannabis has been used medicinally for centuries and numerous species of this genus are undoubtedly amongst the primeval plant remedies known to humans. Cannabis sativa in particular is the most reported species, due to its substantial therapeutic implications that are owed to the presence of chemically and pharmacologically diverse cannabinoids. These compounds have long been used for the palliative treatment of cancer. Recent ad-vancements in receptor pharmacology research have led to the identification of cannabinoids as effective antitu-mor agents. This property is accredited for their ability to induce apoptosis, suppress proliferative cell signalling pathways and promote cell growth inhibition. Evolving lines of evidence suggest that cannabinoid analogues, as well as their receptor agonists, may offer a novel strategy to treat various forms of cancer. This review summarizes the historical perspective of C. sativa, its potential mechanism of action, and pharmacokinetic and pharmacody-namic aspects of cannabinoids, with special emphasis on their anticancer potentials.

Keywords: Cannabis sativa; Cannabinoids; Psychoactive agents; Cancer therapy.Abbreviations: ACF, aberrant crypt foci; CB1, cannabinoids receptor type 1; CB2, cannabinoids receptor type 2; CBC, cannabichromene; CBD, cannabidiol; CBE, can-nabielsoin; CBG, cannabigerol; CBL, cannabicyclol; CBN, cannabinol; CBND, can-nabinodiol; CBT, cannabitriol; COX1, cyclooxygenase 1; COX2, cyclooxygenase 2; CRC, colorectal carcinoma; EGFR, epidermal growth factor receptor; ERK, extracel-lular signal-regulated kinase; HCC, hepatocellular carcinoma; HER2, human epider-mal growth factor receptor 2; iNOS, inducible nitric oxide synthase; MCL, mantle cell lymphoma; MMTV, mouse mammary tumour virus; p21ras, K-ras oncogene product; PKA, protein kinase A; PPARγ, peroxisome proliferator activated receptor gamma; THC, tetrahydrocannabinol; TNF, tumour necrosis factor; TRP, transient receptor po-tential; VEGF, vascular endothelial growth factor.Received: March 22, 2017; Revised: June 21, 2017; Accepted: June 22, 2017*Correspondence to: Ihsan-ul-Haq, Department of Pharmacy, Faculty of Biologi-cal Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan. Tel: +92-51-90644143, E-mail: [email protected]; [email protected] to cite this article: Nasir B, Fatima H, Ahmed M, Phull A-R, Ihsan-ul-Haq. Can-nabis: A Prehistoric Remedy for the Deficits of Existing and Emerging Anticancer Ther-apies. J Explor Res Pharmacol 2017;2(3):93–104. doi: 10.14218/JERP.2017.00012.



Nasir B. et al: Cannabis as an anticancer therapyJ Explor Res Pharmacol

America.16

Habitat

C. sativa is a species well-adapted to diverse climates, from plains to altitudes of 10,000 feet. It probably originated from Central Asia and is now distributed widely, enjoying a global reputation. The major pool of cannabis supply is from China, Russia, India, Paki-stan and Iran, but it is also cultivated in other parts of the world.

Forms/preparation

The strong narcotic obtained from the resin of stem, leaves, flow-er and fruit is predominantly available in three different forms known by their Indian names, ‘bhang, ganja and charas’. These preparations vary according to their potency, extraction and ad-ministration. Bhang is much weaker than charas or ganja and is actually constituted of a seeded blend of C. sativa flowers, as well as its stem and leaves. Ganja is essentially derived from seedless unfertilized female flowering tops, while charas (hash-ish in Arabic) is procured through hand rolling or sieving and screening of cannabis trichomes.17 All preparations are presented in Figure 1.

Phytochemistry

The chemical makeup of C. sativa is highly complex, due to the wide array of chemical constituents and their possible interac-tions with each other. These compounds are from diverse chemical classes (e.g., flavonoids, steroids, hydrocarbons, mono and sesquit-erpenes, nitrogenous compounds and amino acids).18 More than 500 constituents have been identified in C. sativa.18–23 C21 ter-penophenolic cannabinoids are the most common and extensively studied class of cannabis constituents. The term cannabinoids rep-resents a group of compounds found distinctively in C. sativa.24 The introduction of synthetic cannabinoids and discovery of en-docannabinoids (endogenous cannabinoids receptor ligands which are chemically different from those isolated from cannabis) has

prompted utilization of the word “phytocannabinoids” to describe these compounds.25 The number of natural compounds identified in C. sativa is continuously increasing; for example, there were 423 in 1980,26 483 in 199523 and more than 525 in 2008.18–23 De-scribed below is a brief overview of the phytochemical aspects of C. sativa, with special focus on the psychoactive components (e.g., cannabinoids).

Cannabinoids

Cannabinoids, along with their analogues and transformation products, are the characteristic carbon 21 group of compounds found in C. sativa.18 The known cannabinoids can be classified into a few main structural types, while the variations amongst them are fairly basic (e.g., presence or absence of a carboxyl group on the phenolic ring, with one of the hydroxyl moieties of the basic structure been replaced by a methoxy group or a methyl, propyl or butyl side chain replacing the pentyl one). These compounds can be categorized into various sub-classes (Fig. 2).

Among these, the first compound to be isolated from resin obtained from marijuana was cannabigerol (CBG).27 Besides, another group of compounds named as cannabidiol (CBD), dis-covered in 1940, has trans-absolute configuration and most prob-ably negative optical rotation. Similarly, cannabicyclol (CBL) are compounds first considered to have structural similarity with trans-tetrahydrocannabinol (-THC) type compounds28 but later were confirmed to represent a different class, on the basis of results obtained via nuclear magnetic resonance and X-ray analysis.29–31 A few miscellaneous types of cannabinoids have also been report-ed, and these include terpenoids, flavonoids, carbohydrates, fatty acids, hydrocarbons, simple alcohols, aldehydes, ketones, acids, esters and lactones.32 Structures of the various cannabinoids are presented in Figure 3.

Pharmacology of cannabinoids

Cannabinoid receptors

The G protein-coupled receptors associated with the endocannabi-

Fig. 1. Cannabis products. First row, left to right: Indian, Lebanese, Turkish and Pakistani hashish. Second row, left to right: Swiss hashish, Zairean marijuana, Swiss marijuana, Moroccan hash oil. Adopted from: https://www.icmag.com/ic/.


Nasir B. et al: Cannabis as an anticancer therapy J Explor Res Pharmacol

noid signalling system mediate actions of cannabinoids, namely the cannabinoids receptor type 1 (CB1) and type 2 (CB2). Activa-tion of any of the aforementioned receptors leads to diminished cy-clic adenosine monophosphate levels intracellularly, coupled with activation of phosphoinositide 3-kinase and mitogen-activated protein kinase pathways.33

The central and peripheral arms of the nervous system are known to possess the maximum concentration of CB1 receptors, with basal ganglia, cortex, olfactory lobes, hippocampus, spinal cord and cerebellum showing highest receptor densities. The pres-ence of CB1 receptors at these areas accounts for the pharmaco-logical effects of cannabinoids on movement, cognition, memory and emotions. Nociceptive transmission is mediated by the CB1

receptors located in the spinal cord, predominantly in its peri-aq-ueductal grey matter and dorsal horn. These receptors are very few in the brainstem, justifying the fact that respiratory depression is absent after administration of cannabinoids.

CB2 receptors are found peripherally and they are in close connection with cells in the immune system, particularly the macrophages and spleen, wherein they contribute to regulation of the immune system through meditated release of cytokines. These receptors are not allied with psychoactive effects. Another phytocannabinoid, CBD employs anti-inflammatory effects via stimulation of transient receptor potential vanillin (TRPV) chan-nel proteins and inhibition of cyclooxygenase enzymes 1 and 2 respectively.34

Fig. 2. Various classes of cannabinoids isolated from C. sativa.



Pharmacokinetics

The two most common routes for the intake of natural as well as synthetic cannabinoids include the inhalation and oral routes, although other routes are also available. A number of factors in-fluence the concentration of THC present in its natural prepara-tion, including plant variety, type of preparation (hash oil > hash > sinsemilla [seedless plant] > smoked or ingested leaves and flow-ers) and the technique of cultivation.35 All the cannabinoids are absorbed swiftly when administered through the inhalation route, taking 15 min to achieve their maximum brain concentration.

Absorption seems to be variable depending upon the route of administration. After oral administration, THC undergoes first-pass hepatic metabolism, which leads to generation of its psy-choactive metabolite (i.e. 11-hydroxyΔ9-THC). Cannabinoids can cross the placenta, enter the foetal circulation and may pass into breast milk.34 Cannabinoids are also greatly lipid soluble, so they accumulate in fatty tissues and are released slowly into the circu-lating blood. Because of this accumulation, elimination from the body is very slow and may take several days.

Cannabinoids are metabolised primarily in the liver by the CYP2 C subfamily, and a large inter-individual difference exists in the metabolizing capabilities of cannabinoids. Therefore, the rate of metabolism is slowed down in case of liver disease. 11-hydroxy-(Δ9-THC), which is the chief metabolite and more potent than Δ9-THC, may be responsible for some of the characteristic effects of cannabis. THC is predominantly eliminated in the faeces and to a less extent in the urine.25,34

Cannabinoids as medicine/anticancer agents

C. sativa has been known for its euphoric and psychoactive effects, but limited research has been done on its possible pharmaceuti-cal application. Studies exploring the pharmacological potential of this plant were provoked by the isolation of its main psychoactive component (i.e. THC).36 Thus far, cannabinoids have been used efficiently in the treatment of nausea, vomiting, lack of appetite and pain,37 representing the side effects most usually manifested in cancer patients during chemotherapy. Cannabinoid use in on-cology is somehow underrated, since studies reporting the growth inhibitory action of various cannabinoids on different cancer cell types have grown in number.

The first study that reported the antitumor effects of cannabi-noids was done in 1975.38 In vitro and in vivo inhibition (mice) of Lewis lung adenocarcinoma cell growth was demonstrated by the administration of Δ9-THC, Δ8-THC and CBN. Since then, the anti-angiogenic, antimetastatic, antiproliferative and pro-apoptotic effects of cannabinoids have been shown in various cancer types (e.g., lung, glioma, skin, thyroid, lymphoma, skin, pancreas, uter-us, breast and prostate carcinoma) using both in vitro and in vivo models.39–43

A promising area of study for the therapeutic applications of cannabinoids has been their antitumor activity against a variety of aggressive cancers.44 Table 138–41,45–59 shows the types of tu-mours that are sensitive to growth inhibition induced by cannabi-noids. Other in vitro studies have also suggested that THC and other naturally occurring cannabinoids have antineoplastic effects

Fig. 3. Structures of various cannabinoids isolated from C. sativa and their derivatives.

Table 1. Cannabinoids action against various tumours

Type Study model Action [Reference]

Breast carcinoma In vitro Cell cycle arrest [47,55,56]

Colorectal carcinoma In vivo (mouse); in vitro Apoptosis; reduced cell proliferation [45,50–52]

Glioma In vivo (mouse, rat); in vitro Decreased tumour size; apoptosis [39,40,49,57]

Lung carcinoma In vivo (mouse); in vitro Decreased tumour size; inhibition of cell growth [38]

Lymphoma In vivo (mouse); in vitro Decreased tumour size; apoptosis [53]

Neuroblastoma In vitro Apoptosis [48,49]

Skin carcinoma In vivo (mouse); in vitro Decreased tumour size; apoptosis [41]

Prostate carcinoma In vitro Apoptosis [46,47,54]

Uterus carcinoma In vitro Inhibition of cell growth [58,59]



against breast cancer, gliomas, lung carcinomas, lymphomas, neu-roblastoma, colorectal carcinoma (CRC), skin carcinomas, pros-tate carcinoma and uterine carcinoma.45,60 Antineoplastic effects of naturally occurring cannabinoids have also been shown through various in vivo studies involving mice having xenografts of lung and skin carcinomas, lymphomas and gliomas.39,60

Mechanism of anticancer action

The roles of cannabinoid receptors in mediating the anticancer action of cannabinoids have been advocated by expression stud-ies and by the inhibitory action of cannabinoid antagonists. It has also been found that CB2 plays a more important role than CB1 in bringing about the anticancer activity of cannabinoids.46 Also, the concentration of cannabinoid receptors on tumour cells has been found to be much higher than on the corresponding normal tissue

in different cancer types. For instance, CB2 expression in human epidermal growth factor receptor-2 (HER2)-positive breast cancer is significantly higher (91%) than in HER2-negative breast cancers (35–72%) and normal breast tissue (5%).61,62

Furthermore, cannabinoids may cause selective growth inhibi-tion in tumour cells while sparing normal tissue.63,64 For example, cannabinoid exposure causes ceramide-induced cell death in glioma cells, whereas the same cannabinoid-mediated mechanism is re-sponsible for the protection of astrocytes from oxidative stress.64 The antitumor effects of cannabinoids can be mediated by their ac-tion on either CB146,47,65,66 or CB2 receptors or both,40,41,67 and on TRPV1 receptors in the case of endocannabinoid anandamide.48,68,69 The association between expression of CB1 and/or CB2 receptors and prognosis has also been reported for several tumour types.70

Despite the data presented by numerous authors, the exact mechanism of the antitumor action of these molecules has not been fully characterized. Furthermore, the mechanisms of action

Table 2. Mechanism of action and intracellular signals involved in the anticancer action of cannabinoids60,71

Cannabinoids/Cannabimimetics Mechanism of action Intracellular signals

CB1 agonists Inhibition of the mitogen-induced stimulation of G0/G1-S phase of cell cycle

Extracellular signal-regulated kinase (ERK) activation; suppression of p21ras activity; protein kinase A (PKA) inhibition

CB2 agonists Induction of apoptosis ERK activation; sustained ceramide generation through elevated de novo synthesis

Endocannabinoids Inhibition of the mitogen-induced stimulation of G0/G1-S phase of cell cycle

ERK activation; obstruction/inhibition of p21ras activity

CB1/vanilloid receptor ‘hybrid’ agonists

Inhibition of the mitogen-induced stimulation of G0/G1-S phase of cell cycle; induction of apoptosis

PKA suppression; ERK activation; vigorous/strong elevation in Ca2+ level

Fig. 4. Cannabinoid receptors, intracellular signals and mechanisms of the anticancer action of cannabinoids/cannabimimetics. Cannabinoids receptor type 1 (CB1) activation in human breast and prostate cancer cells signal in various intracellular pathways which have a role in control of cell fate. The apo-ptotic action of cannabinoids on glioma cells rely on de novo ceramide generation and extracellular signal-regulated kinase (ERK) activation. Cannabinoid action on cannabinoids receptor type 2 (CB2) in vascular endothelial cells tends to block the angiogenic process and metastasis in mouse models of glioma and skin carcinoma. The figure is a simplified version and crosstalk between different pathways has been omitted. (PKA, protein kinase A; p21ras

, K-ras onco-gene product; VEGF, vascular endothelial growth factor).60,71



of these molecules vary according to the various tumour cell type under examination. Review of various studies has indicated that cannabinoids can act via diverse cellular mechanisms (Fig. 460,71), and the mechanism may involve induction of apoptosis and/or au-tophagy, suppression of cell signalling pathways working in cell proliferation, cell growth inhibition or cell cycle arrest, but it may also include targeting of angiogenesis and cell migration.60,63,71–73

The possible mechanisms and intracellular signals involved in the anticancer action of cannabinoids are summarized in Table 260,71.

Cannabinoids in treatment of cancer

Brain cancer

Clinical management of malignant gliomas is an important research area. Gliomas represent the most common form of brain tumour, are associated with an adverse prognosis and unresponsiveness to treat-ment. Natural and synthetic cannabinoids, as well as those found endogenously, have been reported to affect the cell proliferation rate in cell lines derived from the central nervous system. A study dem-onstrated that THC and WIN-55,212-2 (a mixed CB1/CB2 agonist) stifled the growth of rat glioma C6 cells inoculated intra-cerebrally (rat) or subcutaneously (mice), with the mechanism of action being based in activation of cannabinoid receptors.39,49,74

In another study that aimed to determine the possible in vitro antiproliferative effect of CBD, a non-psychoactive cannabinoid, two glioma cell lines of human origin were used (U87 and U373). A significant decrease in viability and mitochondrial oxidative metabolism was observed in the glioma cells, in a concentration-dependent manner with an apparent IC50 of 25 µM. The antiprolif-erative effect of CBD was also shown to be correlated to induction of apoptosis. The significant antitumor activity of CBD suggested its possible application as an antineoplastic agent.75

Torres et al.76 examined the combined effect of Δ9-THC and temozolomide (TMZ) in the treatment of glioblastoma multiforme. A significant antitumor action in glioma xenografts was observed. Moreover, tumours that are resistant to TMZ treatment were also shown to be responsive to the combination. This formulation also increased the autophagy and the mechanism was confirmed by pharmacological or genetic inhibition of this process, which re-sulted in the prevention of cell death. Likewise, a fair safety pro-file and antiproliferative action of Δ9-THC on tumour cells was reported in its phase 1 clinical trial in 9 patients with recurrent glioblastoma multiforme.77

When comparison of CB2 receptor expression in paraffin-embedded sections from primary brain tumours of paediatric and adult patients was made, their expression was found to be high in most glioblastomas and to correlate with tumour grade. High CB2 immunoreactivity was also observed in some benign paediatric astrocytic tumours, such as subependymal giant cell astrocytoma. Thus, these tumours might be vulnerable to cannabinoid treat-ment.78 Despite a few contradictory reports regarding the mecha-nism of action of cannabinoids, these studies offer exciting new dimensions in the treatment of brain cancers.

Breast cancer

About 30% of newly diagnosed cancers each year are breast cancer cases, and the ErbB2 tyrosine kinase receptor is over expressed in almost one-third of them (Her2 in humans, Neu in rats).79 ErbB2-positive cancer characterizes highly aggressive phenotypes and

such tumours are often described by their reduced responsiveness to standard treatment plans. To determine the potential of cannabi-noids as a new therapeutic choice in the management of ErbB2-positive breast tumours a study was conducted by which their an-titumor potential was examined in a clinically relevant model of ErbB2-driven metastatic breast cancer (the MMTV-neu mouse). A series of human breast tumours were used to analyse the ex-pression of cannabinoid targets. The results obtained showed that Δ9-THC and non-psychotropic CB2 cannabinoid receptor agonist JWH-133 reduced tumour progression to a significant extent and that these belligerent and less responsive tumours could be effec-tively treated with these agents.61

A group of researchers investigated the antitumor potential of numerous plant cannabinoids (i.e. CBD, CBG, CBC, CBD acid, and THC acid), and compared the efficacy and advantage of canna-bis extracts compared to the use of pure cannabinoids. Amongst the tested compounds, CBD was found to be the most powerful growth inhibitor in cancer cells, with IC50 values between 6.0 and 10.6 µM. The researchers concluded that CBD and cannabis extract enriched in this natural cannabinoid may serve as an encouraging choice in non-psychoactive antineoplastic strategy. It inhibited cell growth as well as tumour metastasis, particularly in the case of a highly malignant human breast carcinoma cell line. The mechanism of antineoplastic action of CBD in human breast carcinoma included activation of CB2 and TRPV1 receptors and initiation of oxidative stress, all of which contribute to apoptosis induction.80

In another study, the down-regulation of Id-1 in aggressive hu-man breast cancer cells was reported with CBD and to occur in a dose-dependent fashion.81 Basic helix-loop-helix transcription factors are negatively regulated by Id-1.82 There is strong evidence to suggest that Id-1 controls cellular processes related to tumour progression.83 A study in which Id-1 was reduced showed marked decline in proliferation and invasiveness of breast cancer cells in vitro.84 CBD, therefore, offers a novel nontoxic exogenous choice, effectively decreasing Id-1 expression in metastatic breast cancer cells and rendering the tumour less aggressive.81

In a different study intended to discover a compound that could efficiently co-target different antitumor pathways associated with Δ9-THC and CBD, screening for different analogues was done and around 40 resorcinol derivatives were selected for examina-tion owing to their structural similarity with CBD. The compound O-1663 was found to be the most potent of all the tested deriva-tives in inhibiting breast cancer cell proliferation and invasion in culture and metastasis in vivo. The study suggested the potential use of cannabinoids in the treatment of patients with metastatic breast cancer, and proposed a framework for using these lead com-pounds for the synthesis of novel cannabinoid analogs.85 Likewise, another research group reported that Δ9-THC blocked cell cycle progression and induced apoptosis, thereby reducing human breast cancer cell proliferation. They also reported that these effects were produced via activation of CB2 cannabinoid receptors. The Δ9-THC caused down-regulation of Cdc2, arresting cells particularly in G2-M phase. Normal human mammary epithelial cells, in terms of their proliferation pattern, were less affected by the cannabinoid used, which is quite encouraging; these findings may lead to an in-novative approach for the treatment of breast cancer.86

Colon cancer

CRC, also known as bowel cancer, is regarded as the third most common cancer worldwide both in men and women, with 50,830 deaths and 142,820 new cases estimated to have occurred in 2013.87 A trend of steadily rising treatment costs associated with CRC has



been shown by pharmacoeconomic studies and surveys, which is due to the increasing use of targeted biological therapies.88

CBG is a safe and non-psychotropic cannabinoid and inter-acts with specific targets involved in carcinogenesis. The effect of CBG against colon tumorigenesis was investigated by a group of researchers; mouse models of colon cancer were employed to as-sess the in vivo antineoplastic effect of CBG. The study revealed growth inhibition of CRC cells by CBG, occurring largely through a pro-apoptotic mechanism. The in vivo growth and development of colon carcinogenesis was also retarded. The inhibitory role of CBG in tumoural cell growth has a close association to reactive oxygen species overproduction. Notably, the action against CRC cells was rather selective. CBG was hypothesized to be a worthy anti-CRC curative and preventive therapeutic agent, keeping in line with the safety profile of Cannabis-derived cannabinoids.45

In another study, CBD was investigated for its possible chemo-preventive effect in an experimental model of colon cancer and its likely mechanism of action evaluated in CRC cell lines. The azoxymethane (AOM)-induced colon cancer mouse model was used to study the effect. The effects of AOM treatment were aber-rant crypt foci (ACF), tumour and polyp formation, phospho-Akt, iNOS and COX-2 up-regulation, and caspase-3 down-regulation. CBD impeded AOM-induced phospho-Akt and caspase-3 changes and reduced ACF, polyps and tumours, while in CRC cell lines it prevented oxidative damage of DNA, diminished cell proliferation in a CB1, PPARγ and TRPV1 antagonists sensitive manner, it also elevated endocannabinoid levels. It was concluded from the find-ings that CBD exerts an in vivo chemopreventive effect and retards cell proliferation via numerous mechanisms worthy of clinical consideration in colon cancer prevention.50

A study conducted on CRC investigated related expression and the underlying molecular mechanism of apoptotic activity associ-ated with CB1 and CB2 up-regulation. The receptor expression was studied in cell lines of colon cancer (DLD-1 and HT29) as well as human cancer specimens. CB1 expression was found to be high in normal human colonic epithelium, while CB2 expression was sig-nificantly high in tumour tissue. Activation of these receptors, es-pecially CB2 triggered apoptosis and elevated ceramide level in the cell lines investigated. Pharmacologic inhibition of new ceramide synthesis inhibited apoptosis. Ceramide upsurge and consequent apoptosis initiated by cannabinoid receptor activation was abrogated by the knockdown of TNF-α mRNA. The study’s authors concluded that apoptosis was induced through ceramide de novo synthesis in colon cancer cells via activation of either the CB1 or CB2 receptor. TNF-α was determined to primarily serve as a link between activa-tion of cannabinoid receptors and ceramide production.51

In another study, CBD-rich C. sativa extract was tested for its possible effect on healthy colonic and CRC cell proliferation (us-ing DLD-1 and HCT116 cell lines), while the in vivo effect was estimated using experimental models of colon cancer. The results showed that proliferation in tumour cells was reduced by CBD, but it did not show a similar response in normal cells. AOM-induced polyps, preneoplastic lesions and tumour progression in a xeno-graft model of colon cancer was reduced by CBD in vivo. It was, thus, concluded that CB1 and CB2 receptor activation by CBD was responsible for attenuation of colon carcinogenesis and inhibi-tion of colorectal cancer cell proliferation.52

Liver cancer

A study conducted to investigate the effects of cannabinoids on growth of hepatocellular carcinoma (HCC) showed that Δ9-THC and JWH-015 (a CB2 receptor agonist) via stimulation of CB2 re-

ceptors reduced the viability of HuH-7 (HCC cells) and HepG2 (human HCC cell line). The investigators found that Δ9-THC and JWH-015 promoted human HCC death via induction of autophagy, both in cell culture and xenografted mice experimental systems. Those study results might well help in designing novel therapeutic strategies for the management of HCC.89

Lung cancer

Survival rate in lung cancer patients is low, leading to a demand for design of new approaches that will allow for better management of the disease. Cannabinoid-based antitumor therapies represent new strategies and many studies have reported their antiprolif-erative potential. The first study to show that oral administration of Δ9-THC can retard the growth of Lewis lung adenocarcinoma also indicated that the mechanism of these effects involved DNA synthesis inhibition.60 Another study showed that Δ9-THC caused inhibition of epidermal growth factor–induced phosphorylation of ERK1/2, c-Jun-NH2-kinase1/2, and Akt in the A549 human lung cancer cell line. Moreover, when studied in vivo, it resulted in sup-pression of subcutaneous tumour growth and metastasis in severe-ly immunodeficient mice.90

Lymphoma

Numerous studies have reported on the antitumor potential of cannabinoids in various lymphoma tumours. Lymphoma tumours EL-4, LSA and P815, when exposed in vitro to Δ9-THC, showed increased apoptosis and significantly reduced cell viability; moreo-ver, the Δ9-THC administration to EL-4 tumour–bearing mice re-duced tumour load significantly, potentiated apoptosis in tumour cells and, likewise, the survival of tumour-bearing mice.91 In an-other study, cannabinoid receptor ligands (anandamide and WIN-55,212-2) were used to treat mantle cell lymphoma (MCL), with a resultant decrease in cell viability.92 Cannabinoid receptor–medi-ated apoptosis was shown to be induced by (R)-methanandamide and WIN-55,212-2 in MCL. These studies propose that CB1 and CB2 receptors might be targeted by cannabinoids and/or their ago-nists, taking the field one step further in the quest towards finding new therapeutic strategies for the treatment of lymphoma.93

Another research group reported expression of CB2 receptors after examination of numerous human leukaemia and lymphoma cell lines, including Jurkat, Molt-4 and Sup-T1. These cell lines showed susceptibility to apoptosis induced by Δ9-THC, HU-210 (a synthetic cannabinoid), anandamide (endogenous cannabinoid) and CB2 receptor agonist JWH-015. Δ9-THC also induced apo-ptosis and reduced cell viability in primary acute lymphoblastic leukaemia cell culture. The results suggest that CB2 receptors may represent possible targets for apoptosis induction and that selec-tive CB2 agonists bearing minimal or no psychotropic effects may serve as novel anticancer agents.53

Pancreatic cancer

Pancreatic cancer is classified as one of the most fatal cancer types. Cannabinoid exposure has been shown to cause apoptosis of pancreatic tumour cells, and this effect was found to rely on CB2 receptor and ceramide-dependent up-regulation of the stress regulation protein p8 and ATF-4 and TRB3 stress–related genes.93 However, to identify the true potential of cannabinoid-based thera-py for the management of pancreatic cancer, detailed studies are a



prerequisite to determine the mechanism of cell death induced by these compounds.

Prostate cancer

Prostate cancer is amongst the most prevalent cancers diagnosed in men, and it has been quite a challenge to develop novel therapeutic strategies for its management. A study aimed at developing new targets for the treatment of prostate cancer showed significantly higher expression levels of CB1 and CB2 in cells derived from adenocarcinoma of human prostate tissue (CA-human papilloma virus-10), as well as some other human prostate cells (LNCaP, CWR22RN1, DUI45 and PC3) as compared to cells derived from normal human prostate tissue (PZ-HPV-7). A dose-and time-reliant cell growth inhibition was observed in LNCaP cells when treated with WIN-55,212-2, the effect being prevented by CB1 and CB2 receptor antagonists (SR 141716 and SR 144528, respectively). The results suggested the possible development of cannabinoid re-ceptor agonists for the treatment of prostate cancer.94

Another study revealed the expression of cannabinoid recep-tors in prostate tissue and PC-3 cells (human prostate cancer cell line). However, the induction of apoptosis by Δ9-THC treatment was found to occur through a receptor-independent mechanism.54

It was also found that the endogenous cannabinoid anandamide possesses antiproliferative and apoptotic effects in human prostate cancer cell lines (LNCaP, DU145 and PC3); the effects of which being mediated by epidermal growth factor receptor (EGFR) down-regulation and ceramide accumulation.46 Various cannabi-noid analogues and various cannabinoid receptor ligands are listed in Table 3,46,53,61,75,85,89,92–95 along with their pharmacological ac-tions. Structures of the cannabinoid analogues and synthetic can-nabinoids are presented in Figure 5.

Cannabinoids in cancer palliation

Cannabinoids were used for the palliative treatment of cancer long before its medicinal use in oncology was recognized.39 Rather than treating the underlying cause of cancer, they were primarily used for the relief of symptoms associated with cancer.96 The pallia-tive use of cannabinoids in cancer include its uses as an antiemet-ic,97–100 analgesic101–103 and appetite stimulant.104

Limitations and possibilities

The possible use of THC in oncology might be accompanied by

Fig. 5. Structures of various cannabinoid analogues.

Table 3. Cannabinoid analogy and cannabinoid receptor ligands

S. No Compound Chemistry Pharmacology [Reference]

1 Anandamide Endogenous cannabinoid MCL, Prostate cancer [46,92]

2 HU-210 Synthetic cannabinoid Leukaemia, lymphoma [53]

3 HU-331 Synthetic cannabinoid (quninone synthesized from CBD) colon carcinoma [95]

3 JWH-015 Synthetic CB2 receptor agonist Leukaemia, lymphoma, HCC [89,53]

4 JWH-133 Synthetic CB2 receptor agonist ErbB2-positive breast tumours [61]

5 Methanandamide Synthetic endocannabinoid analogy Lymphoma [93]

6 O-1663 Synthetic cannabinoid Breast cancer [85]

7 WIN-55-212-2 Synthetic cannabinoid (full CB1 agonist) Glioma, MCL, prostate cancer [75,92,94]



certain limitations, especially its side effects at the central nervous system level, which include hallucinations, somnolence, dyspho-ria, abnormalities in thoughts and perception, and depersonaliza-tion.105 On the contrary, direct psychotropic proprieties are not usually reported in cases of most non-THC plant cannabinoids, particularly for CBD.106,107 CBD even blocks the conversion of THC to the more psychoactive 11-hydroxy-THC, thus mitigating its psychoactivity.108,109 Furthermore, it has been reported that the response to marijuana, whether neurophysiological or behavioural, is not affected by the systematic variations in its constituents (i.e. CBD and CBC).110 Numerous such observations and studies have strengthened the concept of promoting non-psychoactive cannabi-noids as potential anticancer agents. Their development, therefore, will demand more advanced studies focusing on the underlying anticancer mechanisms as well as the pros and cons of their pos-sible use in treating different cancer types.

A perplexing situation has arisen from reports of the cancer cell growth-stimulating properties of cannabinoids at low doses in vitro.90 The role of proteins is of great importance and in some in-stances their dual roles make them a double-edged sword. Studies have shown that there are numerous proteins capable of existing in more than one subcellular location. Moreover, additional activities have been reported for identical proteins found in different loca-tions within the cell. Such differential localization profiles may be illustrative of a new mechanism through which cells can exploit a partial sum of genomic information to elicit complex behavioural and biological phenotypes.

Cancer prognosis can be negatively affected by dysregulation of translocation. Proteins exhibiting multiple, independent functions (other than those already identified) are designated as “moonlight-ing proteins”. Apart from change in cellular localization, the func-tions of such moonlighting proteins can vary based on changes in redox state of cell, oligomeric state of the protein, and temperature or variations in cellular concentration of a substrate, ligand, co-factor or product.111 The target proteins of different cancers have been identified as moonlighting proteins owing to their ability to accomplish mechanistically discrete functions.

Proteins with dual characteristics relevant to anticancer action of cannabinoids include the following: ERK, c-Jun, and Akt in lung cancer; ATF4 and TBR3 in pancreatic cancer; and EGFR in prostate cancer.112–115 Thus, the critical review and rationalization of understanding related to protein functions in cancer pathways is of primary importance. Notwithstanding the fact that the observed effects of cannabinoids are multifaceted, complicated and contra-dictory in some instances, formidable evidence exists to advocate that cannabinoids might present useful alternatives in our pursuit for new chemotherapeutic agents.42,43,80,93,116,117

Future research direction

Development of new anticancer therapies is the need of the hour; however, their introduction to and potential application in clinic should be made with great caution and systematically. The anti-cancer value of this ancient remedy can only be fully exploited if future research is directed towards drawing a realistic correlation between the significant in vitro findings and results obtained from clinical trials run in parallel.

A decent safety profile and palliative effects of cannabinoids in cancer patients make them useful candidates for clinical trials. The anticancer mechanism of bioactive principles from C. sativa can be better understood if further research is performed on molecular level explaining the cellular pathways involved in the anticancer effects of

cannabinoids. Moreover, a deep understanding of the multiple func-tions of some proteins involved in these pathways can also help in optimizing the use of cannabinoids as potential anticancer agents.

Conclusions

C. sativa has been known for its psychoactive activity attributed to the exclusive presence of cannabinoids. Identification of can-nabinoid receptors has triggered researchers to validate the under-explored pharmacological prospects of this prehistoric remedy. These efforts have provided momentous evidence of the promise of cannabinoids in the quest for the treatment of numerous neo-plastic brutalities involving brain, breast, colon, liver, pancreas, lung, blood and prostate, among others. Psychoactive effects as-sociated with cannabis utilization pose serious hurdles against its therapeutic application, so that extensive research is required in standardization of its pharmacokinetic parameters. In order to identify the real potential of cannabinoid-based therapeutics and their anticancer mechanism for the management of various types of cancer, detailed clinical studies are still a prerequisite to ascer-taining their safe utility as anticancer agents.

Acknowledgments

The authors are grateful to the Pakistan Scientific and Technological Information Center (PASTIC) and Higher Education Commission of Pakistan (HEC) for providing free access to its database and the relevant literature. We would also like to acknowledge Mr. Ibrahim Shah, PhD scholar, University of Reading, UK, for his insights and comments on an earlier version of the manuscript. No funds of any sort or magnitude were availed from any organization or institute.




Compiling the data (BN, HF, MA), designing and writing the man-uscript (BN, HF, MA), providing intellectual insights in under-standing the anticancer cellular pathways and overall explanation of the pathways (ARP), providing guidance at every step of writ-ing and compilation of the manuscript (IUH), revising and refining all the data provided in the paper (IUH).

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https://www.ncbi.nlm.nih.gov/gene/1386




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