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roids of sulfasalazine with co-administration of probiotics in experimental rats. ... Keywords: Sulfasalazine, spheroids, delayed release tablets, dissolution, ...

367 Current Drug Delivery, 2018, 15, 367-387

RESEARCH ARTICLE ISSN: 1567-2018 eISSN: 1875-5704

In-vitro and In-vivo Pharmacokinetic Evaluation of Guar Gum-Eudragit® S100 Based Colon-targeted Spheroids of Sulfasalazine Co-administered with Probiotics

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School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab-144 411, India Abstract: Background: Polysaccharide based delivery systems have been successfully used to target drugs to colon. In some recent reports, the superiority of concomitant administration of probiotics with such systems has been established. However, the pharmacokinetics of such symbiotic therapy remain unexplored hitherto.

Received: March 28, 2017 Revised: July 13, 2017 Accepted: August 29, 2017

DOI: 10.2174/1567201815666171207165059

Methods: This study deciphers the pharmacokinetic parameters of guar gum based colon targeted spheroids of sulfasalazine with co-administration of probiotics in experimental rats. Thirty rats were divided into five groups using Latin square design. These were subjected to treatment with delayed release formulation, uncoated spheroids, coated spheroid and coated spheroids along with probiotics. Results: In case of delayed release formulation, negligible presence of sulfasalazine in plasma was observed in first 2h, followed by significant increase in sulfasalazine concentration after 3h. Higher plasma concentrations of sulfasalazine were detected for uncoated spheroids with and without probiotics. Negligible release of drug upto 5h and delayed Tmax in case of guar-gum coated sulfasalazine spheroids with or without probiotics clearly indicated successful formulation of colon targeted spheroids. Further, for coated spheroids (both with and without probiotics), the value of Tmax is found to be significantly higher than those with the other treatments. Conclusion: Colon targeted spheroids were therefore, found to reduce absorption of drug which, in turn, is expected to reduce the side effects as only local action in colon is required for treatment of colitis. This is the first report on pharmacokinetic study of a colon targeted delivery system co-administered with probiotics.

Keywords: Sulfasalazine, spheroids, delayed release tablets, dissolution, probiotics, pharmacokinetics. 1. INTRODUCTION

Ulcerative colitis (UC) is a chronic inflammation of colon which is characterized by bloody diarrhoea with absence of parasites in the stool culture. The etiological factors include, damage of patients’ immune system [1], abnormal immunological response of colonic mucosa against colonic microflora [2], environmental factors [3], genetic factors [4, 5], and oxidative damage [6]. Commonly used treatments include corticosteroids and immunomodulators like azathioprine (AZA), 6-mercaptopurine (6-MP), infliximab and cyclosporine [7, 8]. These drugs are reported to cause limiting side effects on long term usage. Sulfasalazine (SFZ) is the cornerstone drug for the treatment of UC and is reported to be superior to corticosteroids, AZA, 6-MP and cyclosporine in terms of its safety and efficacy. Side effects associated *Address correspondence to this author at Department of Pharmaceutical Sciences, Lovely Professional University, Phagwara, 144411, India; Tel: +919888720835; Fax: +91 1824501900; E-mail: [email protected] 1875-5704/18 $58.00+.00

with the use of Sulfasalazine include fever, arthralgia, rashes, Heinz body anaemia and agranulocytosis which are attributed to its metabolite, sulfapyridine [8, 9]. SFZ is poorly absorbed from colon, hence, formulation of colon targeted delivery system of SFZ is expected to reduce its side effects [10].

In past, a number of colon-targeted delivery systems have been reported that include time, pressure and osmotically controlled systems, pH and prodrug based and colonic microflora activated delivery systems [11]. Among them, microflora activated polysaccharide (e.g. guar gum, pectin, amylose, inulin etc.) based delivery systems have proven to be quite successful [11]. Different microflora activated polysaccharide based colon targeted drug delivery systems along with their merits and limitations have been extensively discussed in our previous reports [11, 12]. There are many formulations that have been developed in past such as compression coated matrix tablets, microspheres, beads, pellets, have been designed so far for colon targeting using polysaccha© 2018 Bentham Science Publishers



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Abhinav Sharma, Bimlesh Kumar, Sachin Kumar Singh*, Monica Gulati, Yogyata Vaidya, Manik, Harish Rathee, Deepak Ghai, Adil Hussain Malik, Ankit Kumar Yadav, Peddi Maharshi, Palak Bawa, Sarvi Yadav Rajesh, Parth Sharma, Narendra Kumar Pandey and Souvik Mohanta



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rides as well as pH dependent polymers (Table 1). Polysaccharides used so far include guar gum, Xanthan gum, Gellan gum, Chitosan, Pectin, Amylose, ethyl cellulose hydroxy propyl methyl cellulose, Karaya gum and pH dependent polymers include Eudragit® S100, Eudragit® FS30D, Eudragit® L100, Kollicoat SR30D etc. The major challenge associated with site specific delivery of drugs to colon includes traversing through the entire gut in the intact form, exposure to varying pH and anaerobic nature of GIT, heterogeneity of fermentation activity in different regions, and transient occurrence of mobility [12-14]. Another challenge associated with colonic delivery includes diseased state of the patients. It has been reported that during ulcerative colitis/colon cancer the pH of GIT/intestine shifts towards higher pH and becomes alkaline [56]. Hence, the pH dependent polymers that are used as coat or, core fail to protect the formulation intact before it reaches to colon. Further, it has also been reported that during ulcerative colitis/Crohn’s disease/colon cancer, the microflora of colon gets disturbed and their count gets drastically decreased [57]. The drugs used for the treatment of such diseases also cause damage to the microflora [57]. These microflora are required to degrade the polysaccharides used as coat or in core for colon targeting. During the diseased state, reduction in bacterial count and damage to the bacterial population by the drugs used to treat disease prevent the degradation of polysaccharide coat. This, in turn, leads to compromise in the release of drug at the colonic site.


The data published by various researchers so far generally depicts the pharmacokinetic profile of drug from various formulations in healthy subjects, wherein the physiology and bacterial picture of GIT, remains undisturbed. During the diseased state, physiology and microfloral picture of GIT gets altered leading to failure of release of drugs from such systems.

2.4. Micromeritic Characterization of Prepared Blends

It has been reported that SFZ reduces the colonic count of anaerobic bacteria that adversely affects physiology of colon [58]. Due to this, chances of increase in opportunistic infections are there that may further aggravate the situation [58]. Administration of probiotics in UC has already been reported to be effective in the restoration of colonic microflora [9]. Moreover, there are several reports that indicate a positive effect of synbiotics on the symptoms of IBD [59]. Hence, the strategy of co-administration of synbiotics in polysaccharide assisted colon targeted delivery systems could provide additional benefits besides facilitating their targeted release in colon. In the previous two studies significant role of synbiotics on the therapeutic efficacy of polysaccharide based colon targeted SFZ spheroids and Mesalamine microspheres in the rat model of UC has been reported by the same research group [9, 34]. However, in both the studies, role of coadministration of probiotics was focused towards achieving better pharmacodynamic profile by improving microbial count and site specificity rather than proving it pharmacokinetically. Present study is focused on the role of synbiotics on pharmacokinetics of guar gum-based colon-targeted SFZ spheroids. The study aims at proving site specific release of drug in rats. This is the first report on pharmacokinetic study of a colon targeted delivery system co-administered with probiotics.

The flow rate of the powders was determined as the ratio of mass (g) to time (s) using glass funnel with an orifice diameter of 10 mm (n = 3). The procedure was followed as per our previously reported study for PPK-solid dispersion with minor modifications using fixed funnel and free-standing cone method [60]. On a flat horizontal surface, a graph paper was placed and a funnel was clamped above a graph paper by maintaining about 7 cm gap between paper and tip of funnel. Accurately weighed powders were poured through the funnel until the apex of the cone, thus formed, just reached the tip of the funnel. Average diameters of the base of the powder cones were determined and tangent of the angle of repose calculated using Eq. (1) [60]:

2.1. Materials All the materials and equipments used for formulation of guar gum-Eudragit® S100 based SFZ spheroids and probiotics were same as reported in Prudhviraj et al., 2015 [9]. 2.2. Preparation of Guar Gum Based Colon Targeted Spheroids Blend Spheroids blend was prepared by mixing SFZ, guar gum, microcrystalline cellulose PH 101 and talc. In brief “For a batch of 30g, spheroids containing 40% Sulfasalazine (12g), 40% guar gum (12g), 18% microcrystalline cellulose PH 101 (5.4g) and 2% talc (0.6g) by weight ratios were mixed together in a “V” cone blender (Swastika Pvt. Ltd. India) at 60 rpm for 30 min.

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2.3. Preparation of Delayed Release Sulfasalazine Tablet Blend The blend of drug and excipient was prepared by mixing SFZ, Microcrystalline cellulose pH102 (MCC PH 102), sodium starch glycolate and talc were mixed together in a “V” cone blender (Swastika Pvt. Ltd. India) at 60 rpm for 30 min. For the batch of 100 tablets, SFZ (1000mg), microcrystalline cellulose pH102 (650mg), sodium starch glycolate (300mg), and talc (50mg) were used.

The powder blends prepared for spheroids and tablets were further subjected to micromeritic characterization for true, bulk, and tapped density, flow rate, angle of repose, Carr's compressibility index. 2.4.1. Flow Rate and Angle of Repose

tan  = 2h/D

Eq. (1)

Here, h = Height of the heap of powder; D = Diameter of the base of the heap of powder 2.4.2. True Density The study was carried out as reported by Beg et al., (2016) [61]. True density for blend of S-SNEDDS prepared by using different carriers was determined by liquid displacement method. Ethanol was used as the immiscible solvent. The study was carried out in triplicate and mean data

Colon Targeted Spheroids of Sulfasalazine

Table 1.

Current Drug Delivery, 2018, Vol. 15, No. 3


List of various formulations prepared for oral colon targeted delivery. Delivery System (Formulation)




Eudragit® S100 or L100 in matrix bases comprised of xanthan gum

Tablets (Matrix design)

Wet granulation technique

Drug loss during transit in stomach and small intestine is minimal and the tablets can act as potential colon specific drug delivery device.



Eudragit® S100 and Eudragit® L100


Wet granulation

Tablet disintegration occurred in the ileocecal region in three subjects and the ascending colon in five subjects



5 fluoro uracil

FHV 60, FHV 70 and FHV 80 of guar gum

Matrix Tablets

Direct compression

Only 2.38% got released in stomach and small intestine and the rest in colon




Guar gum

Tablets (immediate and colon release)

Wet granulation

Drug was not released in the stomach and small intestine but in the colon



5- Fluorouracil

Guar gum

Tablets (immediate and colon release)

Wet granulation

Colon-targeted tablets showed delayed t , delayed absorption time (t ), decreased C and Cmax decreased absorption rate constant when compared to the immediate release tablets.













Dilitiazem hydrochloride







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S. N.

Chitosan and Kollicoat SR30D (1:2.5,1:3.5,1:5 w/w)


Wet granulation

In-vivo pharmacokinetic study shows delayed T max, decreased C max and prolonged MRT and there is a targeted delivery


Pectin and HPMC


Direct compression

4:1 ratio of HPMC and pectin gave the best results since the tablets maintained their integrity for 6 h dissolution test and targeted the colon.


Chitosan acetate :ethylcellulose (87.5:12.5)


Direct compression

The dosage provided a lag time rendering the tablets to reach the simulated colonic fluids and drug release was 90% over 12 h.


Guar gum


Direct compression

Guar gum has a great potential for colon specific drug delivery and use of 4% w/v of rat caecal contents in PBS obtained after seven days of enzymatic induction can provide best conditions for in-vitro evaluation of guar gum


Xanthan gum: guar gum (10:20)

Rapidly disintegrating core tablets

Direct compression

The tablets containing XG:GG(10:20) showed an increased cumulative percent drug release of 67.2% in presence of 2% cecal content and 80.34% in presence of 4% cecal content after 19 h of incubation


Hydroxyl propyl cellulose and phenyl propanol amine hydrochloride

Enteric coated timed release press coated tablets

Wet granulation

The tablets successfully reached colon and proved ETP as an effective tool for oral site specific delivery including targeting of the colon



Guar gum

Matrix, multilayer and compression coated tablets

Wet granulation, Wet The compression coated tablets with 350 and 435 granulation and mg of guar gum coat is the best suited for colon targeting since it shows minimal release in the direct compression first 5 h and then 45% drug in colonic fluids.




Guar gum, xanthan gum and xanthan gum


Direct compression

Coated tablets showed 30-40% drug release in 8 h and their further in-vitro studies in dissolution media with rat caecal contents showed 54.4860.42% of drug release




Guar gum and hydroxyl propyl methyl cellulose


Direct compression

Tablets remained intact in stomach and small intestine but partial and complete release of a tracer compound used occurred in the colon and it was concluded that guar gum along with HPMC can be successfully used as a carrier for drug delivery to the colon.


Table (1) contd….

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Delivery System (Formulation)





5 Fluorouracil

Guar gum and shellac

Matrix tablets

Wet granulation

Matrix tablets coated with 10% shellac solution and containing probiotics can be successfully used for colon targeting since it ensures maximum drug delivery at colon




Guar gum


Emulsification method

In-vitro= 91 % cumulative release and in-vivo 79% release




Calcium pectinate


Ionotropic gelation

Microspheres prepared at 2.5% w/v CaCl2 concentration can modulate drug release by both time and pH control and 100% drug release in colon is achieved with in less than 24 hours






Emulsion polymerisation technique

In-vitro studies showed that pectin-metronidazole pro drug didn’t show any release at acidic pH and in-vivo results also shown the same results so the PT-ME pro drug is far better than the only pectin microspheres









Bovine serum albumin






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Gellan gum, karaya gum and povidone


Emulsion polymerisation technique

This combination is found to be effective for microspheres and the gastric retention was aided with titanium dioxide since it extended the drug release up to 12 h


Guar gum and xanthan gum


Emulsion polymerisation technique

Results suggested that the concomitant administration of synbiotics along with mesalamine can be used as a convenient method for achieving efficient and cost effective targeting of the drug to colon


Calcium-pectinate with polyethyleneminein the cross linking solution


Ionotropic gelation method

Optimised Ca-pectinate beads can be hardened with PEI and can encapsulate the drug easily with achieving colon specific delivery.


Calcium pectinate


Extrusion of BSA loaded pectin solution into properly agitated calcium chloride solution

Calcium pectinate beads are extraordinary as carriers for oral delivery of proteins meant for colonic delivery.


Calcium pectinate


Inotropic gelation

Calcium pectinate gel is a promising carrier for controlled release drug delivery of poorly soluble drugs


Nutriose: ethyl cellulose blends



The release of the drug was supressed in upper GIT simulated conditions but shown significant release in a time controlled manner as it came in contact with the faecal samples of the IBD patients



Guar gum and Eudragit® FS30D

Pellets (ph and enzymatic delivery system)

Fluidised bed coater

In-vitro results showed that triggered release is controlled by ph and enzymatic dependent coating and in-vivo results showed that coating with this two polymers increased the plasma drug concentration peak time and absorption lag time.




Eudragit® S100 and Eudragit® L100


Extrusion and spheronisation

Tablets coated with 1:4 (EL100:ES100) gave a drug release of