See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/281375201 Anti-Nociceptive Synergism of Pregabalin and Xylopic acid Co-administration in Paclitaxel-induced Neuropathy: Isobolographic Analysis Article  in  Pharmacognosy Journal · June 2015 DOI: 10.5530/pj.2015.6.8 CITATIONS 6 READS 259 5 authors, including: Some of the authors of this publication are also working on these related projects: Anti-nociceptive properties of medicinal plants View project Misc Projects View project Elvis Ofori Ameyaw University of Cape Coast 88 PUBLICATIONS   497 CITATIONS    SEE PROFILE Eric Woode University of Health and Allied Sciences 155 PUBLICATIONS   1,499 CITATIONS    SEE PROFILE Samuel Kyei University of Cape Coast 84 PUBLICATIONS   451 CITATIONS    SEE PROFILE Robert Peter Biney University of Cape Coast 39 PUBLICATIONS   117 CITATIONS    SEE PROFILE All content following this page was uploaded by Samuel Kyei on 31 August 2015. 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Synergism of Pregabalin and Xylopic acid Co-administration in Paclitaxel-induced Neuropathy: Isobolographic Analysis ABSTRACT Background: Paclitaxel-induced neuropathy still remains a clinical problem for patients undergoing chemotherapy. Objective: The objective of the study was to determine the interaction between xylopic acid and pregab- alinco-administration as well as their toxicity in paclitaxel-induced neuropa- thy. Materials and Methods: Neuropathic pain was induced in rats with 2 mg/kg of paclitaxel on four alternate days (days 0, 2, 4 and 6). Equi effective doses of xylopic acid and pregabalin that produced 50% anti-nociception (ED50) were determined from their log-dose response curves in the cold allodynia and thermal pain tests. Xylopic acid and pregabalin were again ad- ministered to rats in a fixed ratio combination (1:1) of their ED50’s in order to determine the experimental ED50 (Zexp) of the co-administered compounds. Isobolograms were constructed to compare the Zexp to a theoretical ED50 (Zadd). Blood samples from the various treatment groups of rats were col- lected for toxicological assessment of the co-administered compounds. Results: The Zexp lay below the Zadd on the isobologram of the cold allodnia test. The co-administration exhibited additivity in the thermal pain test. The co-administration did not produce significant (p>0.05) toxicity in rats. The co-administration may be beneficial in paclitaxel-induced neuropathy. Key words: Isobolograms, Neuropathic pain, Paclitaxel, xylopic acid, Toxicity. SUMMARY •  Paclitaxel-induced neuropathy is still a clinical problem. •  Pregabalin and Xylopic acid co-administration produced anti-nociceptive syn- ergism against this pain type. •  The co-administration was fairly safe in animals. PICTORIAL ABSTRACT Abbreviations used: XA: Xylopic acid, PG: Pregabalin, Zadd: Theoritical ED50, Zexp: Experimental ED50. Correspondence: Dr. Elvis Ofori Ameyaw, Department of Biomedical and Forensic Sciences, School of Biological Sciences, College of Agriculture and Natural Sciences, University of Cape Coast, Cape Coast, Ghana. Phone no: +233279128631 Email: elvisameyaw@gmail.com DOI : 10.5530/pj.2015.6.8 Pharmacogn. J. A multifaceted peer reviewed journal in the field of Pharmacognosy and Natural Products www.phcogfirst.com/phcogj Original Article INTRODUCTION Pain has been associated with a wide range of diseases such as cancer and arthritis. Pain can sometimes be the disease itself seen in conditions of neuropathy.1 Recently, it is reported that more than 1.5 billion people worldwide suffer from chronic pain. Again, approximately 3-4.5% of the global population suffers from neuropathic pain, with incidence rate in- creasing in complementary to age.2 Neuropathic pain arising from the cancer chemotherapeutic agent, paclitaxel still remains a clinical prob- lem for patients undergoing chemotherapy.3 Paclitaxel is one of the most effective and commonly used anticancer drugs for the treatment of solid tumours. Paclitaxel produces neuropathy by destroying the nerves via its binding to β-tubulin of microtubules. This impairs axoplasmic transport leading to a progressive dying-back axonopathy, hence the neuropathy. Evidence of swollen and vacuolated mitochondria in peripheral sensory axons, both C-fibers and myelinated axons has been reported.4 Its use is limited by Myelossupression and peripheral neurotoxicity.3 Clinically, the sensory neurotoxicity produced by paclitaxel include allodynia, numbness, tingling, and burning pain which often begin symmetrical- ly in the feet and sometimes appear simultaneously in both hands and feet. The neuropathic pain sometimes becomes a chronic problem even after cessation of paclitaxel administration.5 Despite this problem con- fronted by patients, there is currently no valid drug to prevent or treat the paclitaxel-induced pain.6 Pregabalin is the only drug currently ap- proved in the USA for the treatment of spinal neuropathic pain, a type of pain condition affecting 40% of spinal cord injury patients.7 There is the likelihood for food-drug interaction occurring between xylopic acid and pregabalin especially in Africa. Patients prescribed with pregabalin for their disease conditions may ingest xylopic acid which is present in the fruits of Xylopia aethiopica. Xylopia aethiopica is consumed as a delicacy in most parts of the world.8 This presents important drug-food inter- action because xylopic acid is the highest occurring compound in the fruits of X. aethiopica.9 Xylopic acid has demonstrated antinociceptive property in several models of pain. The relief of pain by xylopic acid has been attributed to its action on the opioidergic, adrenergic, bradykinin and prostaglandin nociceptive pathways.10-12 Pregabalin, an antiepileptic drug has demonstrated analgesic property in paclitaxel-induced neuro- pathic pain in pre-clinical, clinical research and therapy.13 Ameyaw Elvis Ofori1*, Woode Eric3, Kyei Samuel2, Biny Peter Robert3 and Boampong Johnson Nyarko1 1Department of Biomedical and Forensic Sciences, School of Biological Sciences, College of Agriculture and Natural Sciences, University of Cape Coast, Cape Coast, Ghana. 2Department of Optometry, School of Physical Sciences, College of Agriculture and Natural Sciences, University of Cape Coast, Cape Coast, Ghana. 3Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. AMEYAW ELVIS and KYEI SAMUEL: Isobolographic analysis of pregabalin and xylopic acid co-administrarion 364� Pharmacognosy Journal, Vol 7, Issue 6, Nov-Dec, 2015 To improve the analgesic potency and efficacy of xylopic acid and pre- gabalin against paclitaxel-induced neuropathy in rats, isobolographic study was performed on these two agents. Because the effects of co-ad- ministered drugs are often highly variable, such variability necessitates the use of statistical robust methodology. Isobolographic study is a tool that provides unambiguous terminologies of drug interactions and a sta- tistical framework for the analysis of drug combinations or co-adminis- tration. Drug combination regimens have been utilised in areas of HIV- AIDS, cancer and tuberculosis with the aim of achieving a therapeutic efficacy greater than that achieved with monotherapy with some success. Other benefits include decreased toxicity—antagonistic interaction, the delay or prevention of drug resistance development, and the favourable effects of synergistic drug interactions. Synergism is especially impor- tant in clinical situations with drugs because it allows the use of smaller amounts of the constituent drugs. An adverse effect may also syner- gise, presenting a phenomenon of special importance in clinical situa- tions which often leads to reduction of doses of the individual drugs for therapy. The detection of synergism may also be useful in illuminating mechanism of drug action and in the development of new theories.14,15 It is therefore possible to upsurge the analgesic efficacy and potency of xylopic acid and pregabalin and correspondingly decrease their side ef- fects via combination therapy. METHODS AND MATERIALS Extraction of xylopic acid (15𝛽-Acetoxy(-)-kaur-16-en-19-oic acid) Xylopic acid was extracted according to the process described else- where.9,10 Briefly, 360 g of dried fruit of Xylopia aethiopica was macer- ated with 5 L of petroleum ether (40–60oC). This was and allowed to stand for three days. The petroleum ether was drained and concentrated with rotary evaporator at a temperature of 50oC. Crude xylopic acid formed after three days was washed with petroleum ether at 40–60oC and purified with 96% ethanol. The yield of the xylopic acid was 1.41%, melting point of 261-262oC. The purity of the extracted xylopic acid as determined with High Performance Liquid Chromatography was 95% as described previously.9 Animals and husbandry Sprague-Dawley rats (150–200 g) of both sexes were housed in the ani- mal facility of the Department of Biomedical and Forensic Sciences. The animals were housed in groups of six in stainless steel cages (34×47×18 cm) with soft wood shavings as bedding. They were fed with normal commercial pellet diet (AGRICARE, Kumasi, Ghana), given water ad libitum and maintained under standard laboratory conditions. All pro- cedures and techniques used in these studies were in accordance with the National Institute of Health Guidelines for the Care and Use of Labora- tory Animals. All protocols used were approved by the Departmental Ethics Committee. Drugs and reagents Pregabalin (Lyrica®) was purchased from Pfizer Pharmaceuticals, Ar- zneimittelwerk Godecke, Freiburg, Germany, cremophor from Sigma- Aldrich Inc., St. Louis, MO, USA and paclitaxel (Intaxel®) from Fresinius Kabi Oncology, Badi, India. Paclitaxel administration Baseline measurements of thermal and cold stimuli were performed after allowing the rats to acclimatise to the behavioural testing environment. Neuropathic pain was induced in the rats by intraperitoneal (i.p.) injec- tion of paclitaxel (2 mg/kg) dissolved in saline on four alternate days (days 0, 2, 4 and 6) as described.16 On day 16 post- paclitaxel administra- tion, the various groups of animals were treated with vehicle (cremo- phore solution) xylopic acid (10-100 mg/kg in cremophore) and prega- balin (10-100 mg/kg) for five days. Cold allodynia The analgesic effect of orally co-administered xylopic acid and prega- balin on cold allodynia was assessed by immersing the rat’s hind paw into cold water (4.5°C). The latency for a rat to withdrawal its paw was measured with a digital timer.17 Just one hind paw was assessed during each immersion at a time with a cut-off time of 20 s. For each animal, two recordings were made for each hind paw, and the withdrawal responses were reported as the mean of both hind paw values. The treatment was continued with vehicle, xylopic acid and pregabalin for 5 days. Thermal Hyperalgesia The tail immersion test was done by immersing the distal portion of the tail (3-4 cm) of the various treated groups of rats in hot water maintained at 52°C until the tail was withdrawn. The duration of immersion was recorded and a cut-off time of 10 s was used.18 The treatment was contin- ued with vehicle, xylopic acid and pregabalin for 5 days. Isobolographic analysis of analgesic property and toxicological assessment of xylopic acid and pregabalin co-administration The potencies of xylopic acid and pregabalin estimated previously in the cold allodynia and thermal hyperalgesia tests were used for the isobolo- graphic studies. The estimated potencies (ED50’s) of xylopic acid and pre- gabalin in both tests were also used to compute the theoretical potency (Zadd) as follows; Zadd = f (ED50) of pregabalin + (1-f) ED50 of xylopic acid Where f is the fraction of the each component in the mixture.15 To obtain the combination potency of co-administered xylopic acid (XA) and pregabalin (PG), the two agents were orally administered to paclitaxel-induced neuropathic pain suffering rats daily at doses of their respective ED50’s and in fixed ratio combinations of fractions of their re- spective ED50 values (equieffctive doses) of 1/2, 1/4, 1/8, 1/16. The treatment regime continued for 5 days in the cold allodynia and thermal hyperal- gesia tests. The experimental potency (Zexp) of the co-administered test agents (XA+PG) was determined by least square method of regression. To determine the drug-drug interaction in the living system, isobo- logram (a cartesian plot of pairs of doses that, in combination, yield a specified level of effect) was constructed by connecting the theoretical ED50 of xylopic acid plotted on the abscissa and pregabalin plotted on the ordinate to obtain the line of additivity. The ED50 (experimental) was compared to the theoretical additive ED50 using a‘t’-test. Synergistic an- algesic effect is obtained when the effect of a drug combination is higher and statistically different (ED50 significantly lower) than the theoretically calculated equieffective dose of a drug combination with the same pro- portions. If the ED50’s are not statistically different, the effect of the com- bination is additive which implies each constituent contributes with its own potency to the total effect. The interaction index was also computed as Zexp/Zadd. If the value is close to one, the interaction is additive. Values lower than one indicates the magnitude of synergistic interactions and values higher than 1 represent antagonistic interactions. Blood samples were also collected for haematology and biochemical assay for signs of toxicity on the fifth day post-treatment. The haematological parameters were determined with an automatic analyzer (Sysmex XT-2000lCELL- DYN 1700, Abbot Diagnostics Division, Abbot Laboratories, Abbot Park, Illinois, USA). Haematological parameters estimated included haemoglobin (Hb), erythrocyte count (RBC), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count AMEYAW ELVIS and KYEI SAMUEL: Isobolographic analysis of pregabalin and xylopic acid co-administrarion Pharmacognosy Journal, Vol 7, Issue 6, Nov-Dec, 2015� 365 Figure 1: Dose-response curves of (a) xylopic acid and (b) pregabalin in the tail immersion test Data is presented as mean ± S.E.M Figure 2: Dose-response curves of xylopic acid and pregabalin in the cold allodynia test Data is presented as mean ± S.E.M (PLT), and total white blood cells (WBCs). The serum biochemistry pa- rameters studied were alanine aminotransferase (ALT), aspartate amino- transferase (AST), alkaline phosphatase (ALP), gamma glutamyl trans- ferase (GGT), bilirubin, blood urea nitrogen (BUN), creatinine, total protein (protein) and albumin. Statistical analysis The data were analysed with GraphPad Prism Version 5 (GraphPad Soft- ware, San Diego, CA, USA). The results are presented as mean±S.E.M. The difference between the means of co-treated groups and negative control group was analysed with One-way analysis of variance followed by Tukey’s post hoc test. Doses for 50% of the maximal effect (ED50) for each drug were determined using an iterative computer least squares method, with the following nonlinear regression (three-parameter logistic) equation: Where, X is the logarithm of dose and Y is the response. Y starts at a (the bottom) and goes to b (the top) with a sigmoid shape. P<0.05 was considered statistically significant. The fitted midpoints (ED50s) of the curves, i.e., Zexp and Zadd were compared statistically using t-test.19 Isobo- lographic calculations were performed with the program Pharm Tools Pro (version 1.27, the Mc Cary Group Inc.). Results are presented as mean ± S.E.M. RESULTS Estimation of the potencies of xylopic acid and pregabalin in the cold allodynia and thermal hyperalgesia tests The potencies of xylopic acid and pregabalin were 8.97 ± 0.96 mg/kg and 8.1 ± 0.85 mg/kg in the thermal pain tests respectively (Figure 1). In the cold allodynia test, the ED50 of xylopic acid and pregabalin were 20.64 ± 0.99 mg/kg and 11.65 ± 095 mg/kg respectively (Figure 2). The fractions of the ED50 of xylopic acid and pregabalin used for the isobolographic studies are shown on Table 1. Isobolographic assessment of the analgesic effect of xylopic acid and pregabalin co-administration in the cold allodynia and thermal hyperalgesia tests The co-administration of XA and PG resulted in significant analgesic ef- fect compared to the vehicle treated animals. The theoretical ED50’s in the cold allodynia and thermal hyperalgesia tests were 16.15 ± 1.2 and 8.54 ± 1.4 respectively. The experimental ED50 (Zexp) for the mixture (XA+PG) in the cold allodynia and thermal pain tests were 7.25 ± 0.23 and 8.16 ± 1.6 respectively. The Zexp (open circle) lay significantly below the line of additivity and the Zadd (closed circles) on the isobologram of the cold allodynia but not the thermal hyperalgesia test (Figure 3a and 3b) indi- cating synergism of analgesic effect (P<0.001) in the cold allodynia test but not the thermal pain test. The degree of interaction calculated as the interaction indices were 0.45 and 0.96 for the cold and thermal tests re- spectively (Table 2). Effect of xylopic acid and pregabalin co-administration on haematological parameters and heart, liver and kidney enzymes The vehicle treated group and XA+PG co-treated groups had reduced but not significant white blood cells concentration compared to sham control groups (Table 3). The liver and bile enzymes, AST, ALT, GGT and ALP from the animals co-administered with various concentrations of XA and PA combination were not statistically different from the nega- tive control groups. Albumin and total protein concentrations were also not different from the negative control group. The ALT, ALP and AST concentrations of the vehicle treated group were slightly elevated but not statistically significant from that of the sham group (Table 3). The kidney Table 1: The ED50’s ± S.E.M. and fractions of Xylopic acid and Pregabalin used for determining the Zexp in the cold allodynia and thermal hyperalgesia tests Doses used (mg/kg) Tests Xylopic acid Pregabalin Thermalpain (Tail immersion test) 8.97 ± 0.96 8.1 ± 0.85 4.49 4.05 2.24 2.025 1.12 1.013 0.56 0.506 Cold allodynia 20.64 ± 0.99 11.65 ± 0.95 10.32 5.825 5.16 2.913 2.85 1.456 1.29 0.728 ( ) ( )( )501 10 LogED X a b a Y � + � = + AMEYAW ELVIS and KYEI SAMUEL: Isobolographic analysis of pregabalin and xylopic acid co-administrarion 366� Pharmacognosy Journal, Vol 7, Issue 6, Nov-Dec, 2015 Figure 3a: Isobologram for the combination of xylopic acid and pregabalin in (a) cold allodynia test in rats. Filled circles (●) are the theoretical ED50’s ± S.E.M. and open circles (o), the experimental ED50’s ± S.E.M Figure 3b: Isobologram for the combination of xylopic acid and pregabalin in (b) thermal hyperalgesia tests in rats. Filled circles (●) are the theoretical ED50’s ± S.E.M. and open circles (o), the experimental ED50’s ± S.E.M enzymes and function marker, creatinine and BUN were insignificantly altered in the XA+PG co-treated groups compared to the vehicle treated group (Table 3). The various co-treatments did not produce toxicity on the erythrocytes and their contents (Table 3). DISCUSSION Oral co-administration of pregabalin and xylopic acid produced synergis- tic analgesic properties and no significant toxic effects in rat model of pa- clitaxel-induced neuropathic pain. The potency of co-administered agents, xylopic acid and pregabalin was significantly higher, and lay below the line of additivity and the theoretical potency of the two agents in the cold allo- dynia test. The additivity line represents the level of effect for the individual agents and the closeness of the experimental potency of the mixture to this line in the thermal pain test demonstrates additivity.20 Both compounds were more potent in the thermal pain test compared to the cold allodynia test. On the contrary, the magnitude of potentiation, with reference to the interaction indices was greater in the cold allodynia test than the thermal pain test. The difference in efficacy of the co-administered agents in the two tests may be due to the different pathophysiology associated with the two pain types. It is obvious that the efficacy of the two agents as well as the co-administration vary with respect to type of noxious stimulus. Paclitaxel, after its administration accumulates in the dorsal root gan- glia and the brain at very low concentrations. Accumulation has also been reported in the sciatic nerve and spinal cord at intermediate con- centrations.21 Morphological changes such as swollen and vacuolated mitochondria as well as reduced respiration and energy production in axonal mitochondria that results from paclitaxel administration initiates apoptosis pathways by altering intracellular calcium levels.4,22 Therefore, the neuropathy in this study after low dose paclitaxel administration was due to atypical (swollen and vacuolated) mitochondria in peripheral sensory axons, both C-fibers and myelinated axons as well as the loss of intraepidermal nerve fibres. Both agents may have acted centrally to produce analgesic synergism. This is supported by the tail immersion test. The tail immersion thermal pain test investigates pain primarily of spinal origin.23 Pregabalin is a well-established anticonvulsant and anal- gesic agent that has shown efficacy and dose dependent effects either as monotherapy or in combination with analgesics in relieving pain and re- lated symptoms. It is noteworthy that pregabalin has received Food and Drug Association (FDA) approval for the treatment of diabetic neuropa- thy and post-herpetic neuralgia. Pregabalin produces its analgesic effect through high-affinity alpha2-delta-1 subunit of voltage-gated calcium channels. Both xylopic acid and pregabalin are calcium channel blockers Table 2: Theoretical (Zadd) and experimental (Zexp) ED50 ± S.E.M. values of Xylopic acid and Pregabalin co-administration in the cold allodynia and thermal hyperalgesia tests Tests ED50’s (XA+PG: 1:1) Thermal pain (Tail immersion test) Cold allodynia Zadd (mg/kg) 8.54 ± 1.4 16.15 ± 1.2 Zexp (mg/kg) 8.16 ± 1.6 7.25 ± 0.23*** Interaction index 0.96 0.45 Values are presented as Mean ± S.E.M. ***P<0.001compared to their respective Zadds AMEYAW ELVIS and KYEI SAMUEL: Isobolographic analysis of pregabalin and xylopic acid co-administrarion Pharmacognosy Journal, Vol 7, Issue 6, Nov-Dec, 2015� 367 with the ability to stabilise injured nerve membrane.24,25 This mechanism may underline the analgesic effect of the co-administered agents in the thermal pain test because additivity is at best observed when two agents activate the same pathway.15 Xylopic acid produces analgesic effect in neuropathic pain by acting on NMDA and adrenergic nociceptive pathways.12 Any of these nocicep- tive pathways might have been modulated by xylopic acid in addition to the inhibition of Ca2+ channel-mediated neurotransmitter release, activation of excitatory amino acid transporters, modulation of potas- sium channels and inhibition of pathways involving inflammatory me- diators by pregabalin to produce the observed synergism in the cold al- lodynia test.24 Although xylopic acid and pregabalin co-administration produced analgesic synergism and additivity, the combination may not be without toxic effects, especially on prolong usage. Paclitaxel slightly reduced the red blood cells and platelets count and these effects were reversed by the co-administered agents. Paclitaxel as well as the co- administered agents may have the potential to induce hepato-biliary diseases on prolong usage. Generally, a slight elevation of ALP and total bilirubin was observed for paclitaxel and the relative high dose combination treatments. The liver enzymes were not significantly af- fected by the co-administration. It is also likely that the long term use of the co-administered agents will have deleterious effect on the kidneys. Blood urea nitrogen and serum creatinine were slightly but not significantly elevated in the paclitaxel and co-administered agents treated groups. This may indicate the dimin- ished excretory efficiency of the kidneys of these animals.26 This implies the co-administration of xylopic acid and pregabalin in neuropathic pain states associated with paclitaxel cancer chemotherapy is fairly safe. How- ever, further studies to determine the long term safety of the co-admin- istration is needed to exclude toxicity from cumulative doses. CONCLUSION Co-administration of xylopic acid and pregabalin may be beneficial in some types of painful neuropathy associated with paclitaxel administra- tion. The co-administration is also fairly safe. ACKNOWLEDGEMENTS The authors express their sincere gratitude to Mr. Kwame Kumi Asare, Daniels Konja and Rex Annane Frimpong. Department of Biomedical and Forensic Sciences, University of Cape Coast. CONFLICT OF INTEREST The authors declare no conflict of interest. REFERENCES 1.  Hansson P. Neuropathic pain: clinical characteristics and diagnostic workup. Eu- ropean Journal of Pain 2002; 6(SA): 47–50. 2.  Global Industry Analysts, Inc. Report, January 10, 2011. http://www.prweb.com/ pdfdownload/8052240.pdf. 3.  Rowinsky EK, Eisenhauer EA, Chaudhry V, Arbuck SG, Donehower RC. Clinical toxicities encountered with paclitaxel (Taxol). 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Table 3: Effect of Xylopic acid and Pregablin co-administration on blood biochemistry and haematological parameters Parameter Sham NC Doses of XA and PG (mg/kg) XA+PG XA+PG/2 XA+PG/4 XA+PG/8 XA+P/16 BUN (mg %) 38.4 ± 0.5 39.1 ± 0.2 39.3 ± 0.6 38.9 ± 0.2 39.2 ± 0.5 38.8 ± 0.4 38.5 ± 0.5 Creatinine (mg/dl) 29.4 ± 1.1 30.5 ± 2.5 31.4 ± 0.7 30.7 ± 1.5 29.5 ± 1.8 30.5 ± 1.1 30.2 ± 1.8 Total bilirubin (mg/dl) 0.96 ± 0.1 1.1 ± 0.2 1.1 ± 0.9 1.1 ± 0.1 1.1 ± 0.2 1.0 ± 0.2 1.0 ± 0.5 ALP (IU/l) 0.7 ± 0.8 0.8 ± 0.1 0.9 ± 0.4 0.9 ± 0.2 0.8 ± 0.1 0.9 ± 0.3 0.8 ± 0.1 ALT(IU/l) 195 ± 3.4 193 ± 1.8 200 ± 2.5 163 ± 9.7 187 ± 4.3 189 ± 1.2 191 ± 2.4 AST/(IU/l) 59.5 ± 1.2 60.5 ± 0.5 68.2 ± 0.2 66.5 ± 1.4 59.8 ± 0.1 59.9 ± 1.5 59.7 ± 1.2 GGT(IU/l) 150 ± 1.3 153 ± 1.4 152 ± 1.2 150 ± 1.8 145 ± 0.5 144 ± 0.5 150 ± 1.3 Protein (g/dl) 11.4 ± 1.4 12.4 ± 0.3 12.1 ± 1.5 10.1 ± 1.3 7.2 ± 0.5 9.4 ± 1.5 10.5 ± 0.2 Albumin (g/l) 75.4 ± 0.5 77.5 ± 0.8 74.2 ± 0.4 75.3 ± 1.5 71.2 ± 1.6 77.4 ± 0.9 74.3 ± 0.8 WBC (×103/µL) 40.2 ± 0.8 42.3 ± 0.4 39.5 ± 08 42.1 ± 0.4 43.2 ± 1.2 42.1 ± 0.9 40.3 ± 0.5 Platelets (×103/µl) 13.2 ± 0.9 11.9 ± 0.9 11.1 ± 0.4 12.2 ± 0.1 13.5 ± 1.1 14.95 ± 0. 14.95 ± 0. RBC (×106/µL) 301 ± 1.3 298 ± 1.1 305 ± 0.9 308 ± 1.5 298 ± 1.1 304 ± 0.8 309 ± 01.3 HCT (%) 8.5 ± 2.5 8.9 ± 1.3 8.7 ± 2.3 9.2 ± 0.7 8.1 ± 1.2 8.5 ± 0.65 8.8 ± 1.3 Hb (%) 34.1 ± 1.2 32.2 ± 1.5 30.1 ± 1.5 32.8 ± 0.8 35.5 ± 0.9 30.9 ± 1.3 33.8 ± 1.2 MCH (pg) 12.2 ± 0.4 12.7 ± 0.5 13.7 ± 1.1 10.3 ± 0.9 12.5 ± 0.2 12.4 ± 0.9 10.9 ± 1.2 MCV (µm3) 30.4 ± 0.5 28.4 ± 1.2 26.4 ± 0.8 28.4 ± 1.2 25.6 ± 0.5 29.4 ± 0.9 29.1 ± 1.4 MCHC (%) 54.8 ± 1.2 53.4 ± 0.9 50.2 ± 0.8 54.3 ± 0.2 51.8 ± 1.2 56.1 ± 0.9 56.1 ± 1.4 NC=Negative Control. AMEYAW ELVIS and KYEI SAMUEL: Isobolographic analysis of pregabalin and xylopic acid co-administrarion 368� Pharmacognosy Journal, Vol 7, Issue 6, Nov-Dec, 2015 ABOUT AUTHORS Dr Elvis Ofori Ameyaw: Senior Lecturer of Department of Biomedical and Forensic Sciences, School of Biological Sciences, College of Agriculture and Natural Sciences, University of Cape Coast, Cape Coast, Ghana. 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