potential of cocoa pod husk pectin-based modified release...

Research ArticlePotential of Cocoa Pod Husk Pectin-Based Modified ReleaseCapsules as a Carrier for Chronodelivery of Hydrocortisone inSprague-Dawley Rats

Ofosua Adi-Dako 12 Kwabena Ofori-Kwakye 1 Seth Kwabena Amponsah 3

Isaac Boamah4 Noble Kuntworbe1 and Esther Eshun Oppong5

1Department of Pharmaceutics Faculty of Pharmacy and Pharmaceutical Sciences College of Health SciencesKwame Nkrumah University of Science and Technology (KNUST) Kumasi Ghana

2Department of Pharmaceutics and Microbiology University of Ghana School of Pharmacy Accra Ghana3Department of Pharmacology and Toxicology University of Ghana School of Pharmacy Accra Ghana4Department of Medical Microbiology School of Biomedical amp Allied Health Sciences University of Ghana Accra Ghana5Department of Pharmaceutics School of Pharmacy Central University Miotso Ghana

Correspondence should be addressed to Kwabena Ofori-Kwakye koforikwakyeyahoocom

Received 10 April 2018 Revised 4 September 2018 Accepted 19 September 2018 Published 8 October 2018

Academic Editor Sophia Antimisiaris

Copyright copy 2018 Ofosua Adi-Dako et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The potential of cocoa pod husk (CPH) pectin-based modified release (MR) capsules as a carrier for chronodelivery ofhydrocortisone in Sprague-Dawley rats was assessed Extemporaneously formulated CPH pectin-based hydrocortisone (10mg)capsules crosslinked with calcium chloride (Formulation A) or zinc (Formulation B) and a commercial immediate releasehydrocortisone formulation were administered orally to Sprague-Dawley rats and the pharmacokinetic parameters were evaluatedusing noncompartmental analysis Formulation A had a 2 h lag phase followed by an increase in serum drug concentration inthe treated rats Peak concentrations (Cmax) of 21799 plusmn 1993 ngml and 20844 plusmn 2661 ngml were achieved after 6 plusmn 023 h and6 plusmn 035 h (Tmax) respectively for capsules A and B The immediate release formulation had a peak concentration of 15322 plusmn0313 ngml within 1 plusmn 02 hThe relative bioavailability of the CPH pectin-based capsules A and B was 213 and 274 respectivelyFormulations A and B had half-lives more than three times that of the immediate release formulation The MR capsules exhibiteda higher exposure greater bioavailability and versatility in release of cortisol than the commercial immediate release formulationAdditionally the MR capsules exhibited an extended drug release with overnight cortisol rise and early morning cortisol peak andhold promise in the management of adrenal insufficiency

1 Introduction

Drug delivery could be optimized through the design man-ufacturing methods employed and the biopharmaceuticalconsiderations made during formulation development [1]The formulation of novel or optimized drug delivery plat-forms intended to improve human health has been the focusof research in recent years [2]The symptoms of some diurnaldiseases follow a rhythmic pattern and require drug deliveryas per the rhythms However when there is constant drugrelease in such disease states there is the potential for side

effects as the undesired drug released tends to affect thebodyrsquos metabolic system There is therefore the need for thedrug delivery patterns of such formulations to be optimizedto suit unique therapeutic needs

Cortisol is an essential metabolic hormone secreted bythe adrenal cortex in a circadian fashion The highest levelsof cortisol are observed in the morning whiles the lowestlevels are detected around midnight [3 4] A disruption ofthe diurnal rhythm of cortisol causes fatigue depressionill health and insulin resistance Patients suffering fromadrenal insufficiency have low levels of secreted cortisol

HindawiJournal of Drug DeliveryVolume 2018 Article ID 9825363 8 pageshttpsdoiorg10115520189825363

2 Journal of Drug Delivery

Table 1 Composition of CPH pectin-based hydrocortisone capsule formulations

Capsuleformulation

Hydrocortisone(mg)

CPH pectin(mg)

Calciumchloride(mg)

Zinc acetate(mg)

HPMC(mg)

Dicalciumphosphate

(mg)

Starch(mg)

A 10 50 50 - 25 65 -B 10 50 - 50 - - 90

The disease progresses in a gradual and subtle manner withfatal consequences [5 6] Aside adrenal insufficiency otherdisease conditions that display circadian variation in theirpathophysiology include hypertension asthma and gastriculcer Such disease conditions require drug delivery to mimicthis rhythmic pattern [7]

Drug tolerance and side effects are associated with drugsthat are released at a constant rate in diurnal disease states[8ndash10] The administration of immediate release hydrocor-tisone (drug form of cortisol) in the treatment of adrenalinsufficiency has proven to be ineffective due to inabilityof drug to mimic the rhythm of endogenous cortisol Toaddress the deficiencies associated with the use of immediaterelease hydrocortisone preparations in adrenal insufficiencyvarious modified release hydrocortisone formulations whichare intended to replicate the physiological cortisol rhythmhave been produced with varying degrees of success [11ndash14]

There is however the need to develop modified releasehydrocortisone with optimized drug delivery to achieveenhanced and optimal blood drug concentration-time pro-files [2 15] Preclinical studies using animal models providecritical information about the biopharmaceutical and phar-macokinetic properties of such formulations [16]These stud-ies serve as predictors of possible effects of these formulationsin humans [17]

Cocoa pod husks (CPH) which are the empty pod shellsof the cocoa (eobroma cacao) fruit have been shown tohave suitable physicochemical properties as a pharmaceuticalexcipient [18] An in vitro evaluation of the CPHpectin-basedformulation as a carrier for chronodelivery of hydrocortisonedemonstrated its potential use in adrenal insufficiency [14]Findings from this earlier in vitro study showed that formu-lated CPH pectin-based systems containing hydrocortisonecan achieve a burst release of the drug at circadian timing afterapproximately 6 hour lag phase [14] The burst release of thedrug at circadian timingwas attributed to enhanced solubilityand degradation of pectin in simulated colonic mediumThecurrent study sought to design a simple formulation of a CPHpectin-based oral multiparticulate matrix system which issuitable for chronodelivery of hydrocortisone An evaluationof the pharmacokinetic profiles of the CPH pectin-basedhydrocortisone systems compared with an immediate releasecommercial hydrocortisone formulation was undertaken inSprague-Dawley rats

2 Materials and Methods

21 Materials Micronized hydrocortisone powder sim50120583m(Sigma Aldrich USA) commercial 10mg hydrocortisone

tablets (Auden McKenzie UK) calcium chloride (FischerScientific UK) dicalcium phosphate (SujianModern Chemi-cal China) hydroxypropyl methylcellulose [Methocel 44779HPMC (viscosity 40-60 cps 2 aqueous solution)] (AlphaAesar USA) microcrystalline cellulose (Blanver Farmo-quimica Brazil) maize starch (Vasa Pharmachem India)zinc acetate (Central Drug House India) dexamethasoneinjection (Lab Sanderson SA Chile) gelatin capsule shells(Capsule Connection USA) ether (Merck Germany) andfreeze dried CPH pectin extracted from minced fresh cocoapod husks by hot aqueous extraction [18] were used for thestudy

22 Preparation of Modified Release CPH Pectin-BasedHydrocortisone Capsules Modified release pectin-basedhydrocortisone capsules containing sim10mg hydrocortisonewere extemporaneously prepared (Table 1) The stipulatedamounts of the powders except CPH pectin were weighedand mixed by geometric dilution CPH pectin was dispersedin sufficient amount of hot water to form a viscous dispersionand added gradually to the mixed powders to form a wetmass The wet powder mass was passed through a 20sieve and the granules dried at 40∘C for 15 h in an ovenThe moisture content of the granules was determined byweighing 1 g of the granules into each of three Petri dishesand dried in a hot air oven at 105∘C to constant weightThe moisture content was calculated as the ratio of theweight of moisture loss to weight of sample expressed as apercentage [18] The particle size and surface characteristicsof the granules were evaluated with a Jeol Scanning ElectronMicroscope (JSM-6390LV Japan) by placing samples ondouble-sided carbon adhesive tapes on specimen stubs andcoated with platinum in the autofine coater The sampleswere then scanned and viewed

The capsules containing sim200mg granules each wereprepared manually by the traditional punch method Thegranules were poured onto a powder weighing paper andthe base of capsule size ldquo0rdquo was held vertically and the openend repeatedly pushed or punched into the powder until thecapsule was filled The cap was replaced to close the capsuleEach filled capsule was weighed using an empty capsule as acounterweight and granules were added or removed until thecorrect weights were obtained [19 20]

23 Weight Analysis of Capsules Ten capsules were individ-ually weighed The contents of each capsule were removedand the weights of the empty shells recorded separately Thedifference between both weights was calculated and the netweight of the contents was determined for each capsule [21]

Journal of Drug Delivery 3

24 Content Analysis of Capsules The hydrocortisone con-tent of the capsules was determined using a validatedReversed-Phased High-Performance Liquid Chromatogra-phy (RP-HPLC) method [14 22] with minor modificationsAnalysis was done using a JT Baker ODS C18 5 120583m46 times 150mm column injection volume 20 120583L flow rate1mlmin wavelength of detection 254 nm and temperatureambient The contents of ten capsules of each formulationwere removed and finely powdered separately A quantityof powder equivalent to 25mg hydrocortisone was accu-rately weighed The powders were transferred into 10ml ofmethanol in 25ml volumetric flasks sonicated and madeup to 25ml volume with methanol The solutions obtainedwere filtered using Whatman filter paper No 41 and thefiltrates were analyzed by RP-HPLC The peak areas of thesamples were determined and the amount of hydrocor-tisone extrapolated from the linear regression calibrationcurves

25 In Vitro Drug Release Studies The release of hydrocor-tisone from the CPH pectin-based capsules was monitoredusing a USP Dissolution Tester Apparatus II (Lid-8 Dis-solution Tester Vanguard Pharmaceuticals Machinery IncUSA) The dissolution medium was 900ml phosphate bufferpH68 the paddle speedwas 50 rpm and the test temperaturewas 37 plusmn 05∘C At specified time intervals 5ml aliquots weredrawn for analysis and replaced with the same volume ofdissolution mediumThe hydrocortisone levels were detectedwith a Spectroquant Pharo 300 UV Spectrophotometer(Merck Darmstadt Germany) Three replicate determina-tions were made for each capsule formulation

26 Experimental Animals Eighteen male Sprague-Dawleyrats (200-250 g) were obtained from the Animal House ofthe Centre for Plant Medicine Research (CPMR) Mampong-Akuapem Ghana The animals were housed in stainless steelwire mesh cages and supplied with water and fed with chow(Agricare Co Ltd Accra) ad libitum Each rat was made tooccupy a minimum space of 2 cubic feet (61 cm times 31 cm times31 cm) with soft wood shavings as bedding for their comfortThe animals were acclimatized for 7 days and maintainedunder standard laboratory conditions (temperature 25plusmn 1∘Crelative humidity 60-70 and 12 hour light-dark cycle)The animals were randomly grouped into six rats per group(Groups 1 2 and 3) The study was conducted in accordancewith the guidelines of the Institutional Research Committeeof CPMR responsible for animal care and use as well as theNational Institute of Health Guidelines for the Care and Useof Laboratory Animals [23]

27 Administration of Hydrocortisone Formulations Endoge-nous cortisol of the experimental rats was suppressed with50120583gkg of dexamethasone before the administration of thehydrocortisone formulations [24] A commercial immediaterelease hydrocortisone tablet (40mgkg) was orally admin-istered to Group 1 rats The modified release CPH pectin-based capsule Formulations A and B were administeredorally (40mgkg) to Groups 2 and 3 rats respectively Alladministrations were done at 2100 h GMT

28 Blood Sampling and Serum Cortisol DeterminationBlood sampling of rats in Group 1 was at 30min intervals(based on half-life of immediate release formulation) from2100 h to 2230 h GMT and at 0130 h GMT Blood samplingof Group 2 andGroup 3 rats was at 2 h intervals (based on for-mulation type delayed release) from 2100 h to 500 h GMTParallel blood sampling approach and a sampling time spanof 8 h were used to reduce stress among the rats Stress amonganimals could lead to an increase in levels of endogenouscortisol which invariably affect assay of administered cortisol(hydrocortisone) Animals were mildly anesthetized withether vapor and blood samples collected via cardiac punctureinto BD-serum separator vacutainer tubes and immediatelycentrifuged to obtain serum All serum samples were storedat 4∘C and analyzed for cortisol levels within 24 h The serumconcentration of cortisol was measured using a competitiveimmunoassay (Rat cortisol Elisa kit Cusabio South Africa)with an analytical sensitivity of 0049 ngml The intra-assayand interassay precision coefficient of variation were lt 8and lt 10 respectively

29 Data Analysis Results were expressed as mean (withstandard error) and percentages The in vivo pharmacoki-netic parameters of the hydrocortisone formulations in the3 groups were estimated with noncompartmental analy-sis using GraphPad Prism 70 and Excel 2013 Peak drugconcentration (Cmax) and time to achieve this (Tmax) weredetermined from concentration-time profile curves The areaunder the drug concentration-time curve (AUC) was calcu-lated by the linear trapezoidal rule AUC was determinedtill the last sampling point and was also extrapolated toinfinity (AUC0997888rarrinfin) The elimination rate constant (Ke) wasdetermined by linear regression of the linear terminal part ofthe log serum cortisol concentration-time curve The elim-ination half-life (t12) was calculated from the formula t12= 0693Keminus1 Comparison of pharmacokinetic parameters ofthe hydrocortisone formulations was by one-way analysis ofvariance (ANOVA) followed by Tukeyrsquos post hoc multiplecomparison test Statistical significance was set at p lt 005

3 Results

The CPH pectin-based hydrocortisone granules were irregu-larly shapedwith rough surfaces (Figure 1)Themean particlesize and moisture content of the granules were FormulationA (1153 plusmn 019mm 002) and Formulation B (1858 plusmn025mm 009) respectivelyThe twomodified release CPHpectin-based hydrocortisone capsule formulations preparedshowed satisfactory physicochemical properties The averageweight and hydrocortisone content of Formulation A were20076 plusmn 045mg and 9599 plusmn 034 respectively and thoseof Formulation B were 20099 plusmn 059mg and 10329 plusmn 025respectively The capsule weight deviations were less than75 In vitro drug release of the modified release capsules(Formulations A and B) and the commercial immediaterelease hydrocortisone tablet showed biphasic release ofhydrocortisone (Figure 2) Formulation A a matrix of CPHpectin calcium chloride dicalcium phosphate and HPMCcontaining hydrocortisone showed negligible drug release


(a) (b)

Figure 1 Scanning electron micrographs of (a) CPH pectincalciumHPMC hydrocortisone granules [Formulation A] (mag x 200) and (b)CPH pectinzinc hydrocortisone granules [Formulation B] (mag x 50)

Formulation AFormulation BCommercial tablet

0

20

40

60

80

100

Am

ount

of d

rug

rele

ased

()

30 60 90 120 1500Time (min)

Figure 2 Dissolution profiles of the two CPH pectin-based hydro-cortisone modified release capsule formulations and a commercialconventional 10mghydrocortisone tablet in phosphate buffer pH68(mean plusmn SEM n = 3)

within the first 30minutes afterwhich a continuous release ofhydrocortisone was observed Approximately 28 and 79of FormulationAwas releasedwithin 1 h and 2 h respectivelyThe release profile for Formulation B a matrix of CPHpectin zinc and hydrocortisone was similar to FormulationA with negligible release within the first 30min followed byincreased drug release In all approximately 20 and 64 ofFormulation B was released within 1 h and 2 h respectivelyEven thoughFormulationB appeared to show a slower releaseof hydrocortisone than Formulation A the difference was notsignificant (pgt 005) The first phase of drug release is due toinitial wetting-swelling of pectin and HPMC (FormulationA) or pectin (Formulation B) followed by diffusion of thedrug through the gel matrix The commercial hydrocortisoneproduct exhibited accelerated drug release (71) within thefirst 15min followed by continuous decrease in release Theinitial phase of drug release could be attributed to diffusionof drug molecules from the tablet in direct contact withthe aqueous medium The second phase of continuous butprolonged decrease in drug release may be due to the poorsolubility of hydrocortisone in aqueous medium

Commercial tabletFormulation AFormulation B

2 4 6 8 100Time (h)

0

5

10

15

20

25

Con

cent

ratio

n of

dru

g (n

gm

l)

Figure 3 In vivo concentration-timeprofiles of themodified releaseCPHpectin-based hydrocortisone (10mg) capsule formulations anda commercial conventional 10mg hydrocortisone tablet in Sprague-Dawley rats (mean plusmn SEM n = 3)

The cortisol concentration-time data (absorption pro-files) obtained alternatively from 3 rats in each group(average) for Formulations A and B and the commercialconventional hydrocortisone tablet is shown in Figure 3 Asa result of the abrupt end of the concentration-time curvefor Formulations A and B extrapolations were made toobtain postabsorption-elimination phases so as to be ableto calculate AUC0997888rarrinfin t12 and Rel F () As a result nostatistical comparisons are made for these 3 pharmacokineticparameters among the 3 groups Formulation A had a 2 h lagphase followed by an increase in serum drug concentrationThe lag time observed for Formulation A could be attributedto the decreased solubility of the pectincalciumHPMC drugmatrix in the gastric medium

The pharmacokinetic parameters Cmax Tmax AUC0997888rarrinfinand t12 of the drug formulations are summarized in Table 2

The administration of the oral immediate release hydro-cortisone product resulted in a maximum concentration(Cmax) being obtained within 1 plusmn 02 h With regard to themodified release Formulations A and B the time (Tmax) toreach Cmax was achieved after 6 plusmn 023 h and 6 plusmn 035 h


Table 2 Pharmacokinetic parameters of the two CPH pectin-based hydrocortisone capsule formulations compared with a commercialimmediate release hydrocortisone tablet

Pharmacokineticparameters

Commercialhydrocortisone tablet Formulation A Formulation B

Cmax (ngml) 1532 plusmn 031 2180 plusmn 199lowast 2084 plusmn 266lowastTmax (h) 1 plusmn 020 6 plusmn 023lowastlowast 6 plusmn 035lowastlowastt12 (h) 192 plusmn 148 726 plusmn 312120593 1046 plusmn 443120593

AUC0997888rarrinfin(nghrml) 4278 plusmn 4207 91147 plusmn 2013120593 117463 plusmn 4845120593

Rel F () 100 213120593 274120593

lowastplt005 lowastlowastplt0001120593 results obtained after extrapolations from concentration-time curves

respectively Post hoc analysis showed that the Tmax for For-mulations A and B were statistically different from the com-mercial immediate release formulation (plt0001) Post hocanalysis also showed that there was a significant differencein Cmax between Formulation A and the commercial productand between Formulation B and this immediate release prod-uct (plt005) However there was no significant difference inCmax between Formulations A and B (pgt005)

4 Discussion

The treatment of adrenal insufficiency with conventionalhydrocortisone dosage forms is challenging and generallyleads to suboptimal drug serum levels Studies involvingoral modified release formulations of hydrocortisone havebeen shown tomimic a circadian-based physiological cortisolrelease profile than the conventional release hydrocortisoneformulations [11 25] The overnight rise and morning peakof cortisol is a crucial aspect of the replication of endogenouscortisol circadian rhythm when hydrocortisone is used

In the current study the potential of CPH pectin-basedcapsule system in providing an extended release of hydrocor-tisone during the nocturnal cortisol gradual rise andmorningpeakwas evaluated in Sprague-Dawley rats TwoCPHpectin-based oral multiparticulate matrix capsules were formulatedto achieve suitable modified release of hydrocortisone in vitroand in vivo CPH pectin was modified to reduce its solubilityin the upper gastrointestinal tract with combinations ofHPMC calcium and zinc to achieve colon specific release ofhydrocortisone and ultimately the desired release profile invivo [26ndash28]

Content analysis of theCPHpectin-based hydrocortisonecapsules yielded results in the acceptance criteria of 90-110 [29] hence the formulations contained the requisiteamount of hydrocortisone The capsules were also within thestipulated pharmacopoeial limits of capsule weight variationCommercial immediate release hydrocortisone preparationsare routinely administered to attain acceptable therapeuticlevels of cortisol in adrenal insufficiency using a thrice dailyregimen of 10mg hydrocortisone In the current study a com-mercial immediate release 10mg hydrocortisone tablet wasused as a standard conventional hydrocortisone preparation

The CPH pectin-based modified release formulationswere prepared as multiparticulate encapsulated granules

in gelatin capsules in view of their potential benefits incolonic drug delivery Multiparticulate systems are associatedwith increased bioavailability decreased systemic toxicitydecreased risk of dose dumping reduced local irritation anda potentially longer residence time in the ascending colon ascompared to single unit systems [30]

Modulation of systemic absorption of drugs in the colonis an emerging approach in the treatment of diseases thatdisplay circadian variation [31 32]The performance and for-mulation characteristics of themodified release capsules wereintended to delay drug release in the upper gastrointestinaltract after which drug release will be triggered by colonic bac-teria [31] The use of biodegradable polymers such as pectinin the design of modified release capsules holds great promisein targeted release of hydrocortisone in the colonic region

The use of matrix systems in targeted delivery is associ-ated with a rapid initial release of drug which is a challengewhen delayed drug release is desired Pectin is a hydrophilicpolymer with the potential for premature drug release dueto its high solubility in the gastrointestinal tract To over-come this drawback the CPH pectin-based hydrocortisonecapsules were chemically modified with calcium zinc andHPMC to reduce their aqueous solubility [33] Calciumbinding with pectin ensures reduced solubility increased gelstrength and extensive hydration in the gastrointestinal tractwhile inclusion of HPMC strengthens the hybrid polymermatrix [28] Cross-linking of CPH pectin with zinc ionsensures reduced aqueous solubility of pectin [26] Earlierreports suggest that zinc is a better cross-linker than calciumfor pectin [26 34 35] However the inclusion of HPMCin Formulation A appeared to enhance the gel strength ofthe calcium and pectin drug matrix leading to FormulationA (containing calcium and HPMC) and Formulation B(containing zinc and starch) showing similar drug absorptionprofiles

Biphasic release the process whereby drug release isinitially delayed followed by accelerated drug release at alater stage is useful for colon specific drug delivery [36 37]Biphasic release is well-documented in press coated and filmcoated drug delivery systems but is rarely observed withmatrix systems [28] The biphasic release of FormulationsA and B is potentially beneficial in the chronodelivery ofhydrocortisone which may be useful in clinical conditionsinfluenced by diurnal rhythms Colonic delivery of drugs


through biphasic or sigmoidal release mechanisms is usefulfor the maintenance of sustained blood drug levels [38]

In the current study the time for drug to peak (Tmax)was longer for the modified release formulations than thecommercial immediate release formulation This confirmsthe delayed and extended release profiles of FormulationsA and B These modified release capsules may be useful inachieving the cortisol overnight rise and morning peak incortisol deficiency This finding is similar to earlier clinicalreports where a novel formulation ofmodified release hydro-cortisone formulation achieved a physiological nocturnalcortisol rise and morning peak [12] In a related study theadministration of a 10mg modified release hydrocortisoneand 10mg immediate release formulation resulted in a Tmaxof 783 h and 18 h for the modified release and immediaterelease respectively [25] Whitaker et al also reported amean Tmax of 83 hours for DIURF 002 a modified releasehydrocortisone formulation whereas the immediate releasehydrocortisone formulation had a Tmax of 1 hour [10]

The maximum serum concentration (Cmax) was alsohigher for Formulations A and B than the commercialimmediate release product indicating a possible controlledabsorption rate for the modified release formulations Anelimination half-life (t12) of 7261 plusmn 312 h and 10462 plusmn4425 h for Formulations A and B respectively demonstratesthe prolonged effect of both modified release formulationscompared with the immediate release hydrocortisone for-mulation Furthermore Formulation B with the longesthalf-life and Cmax and Tmax ideal for a delayed releasehydrocortisone drug could be a drug of choice if dosingper day needs to be reduced to improve patient complianceThe data obtained showed that the total drug exposure(AUC0997888rarrinfin) was greater for Formulations A and B than thecommercial formulation The relative bioavailability (Rel F)of the CPH pectin-based Formulations A and B was sim213and 274 respectively This demonstrates the ability of themodified release hydrocortisone formulations to provide agreater extent of drug exposure with time in the experimentalanimals compared to the conventional dosage form [39]Despite these results the short sampling time used whichled to an abrupt end of the in vivo pharmacokinetic profileof rats administered with Formulations A and B and theextrapolation of the postabsorption-elimination phases ofrespective concentration-time curves is deemed a limitationof this aspect of the study

Both CPH pectin composites of calcium and HPMC andzinc formulations were robust enough to successfully delaythe release of hydrocortisone in the upper gastrointestinaltract increase the residence time of hydrocortisone in theplasma and increase bioavailability However Formulation Aexhibited a better control of drug release than FormulationB In situ cross-linking of CPH pectin with calcium and zincions played a major role in retarding the release of hydro-cortisone Zinc pectinate composites have been associatedwith slower initial release of drugs fromprevious reports [35]However the extent of initial release of hydrocortisone fromFormulation B can be attributed to the influence of starchEarlier reports indicate the potential rapid release of drugsfrom starch based systems due to extensive swelling and

enzymatic degradation in the gastrointestinal tract [40] Theresults suggest possible accelerated release of cortisol fromFormulations A and B on arrival in the colon after microbialdegradation of pectin [31]

The nocturnal burst release observed with the modifiedrelease formulations is critical for restoring the cortisolpeak associated with the disruption of cortisol rhythm Theextended release profiles of CPH pectin-based formulationsshould aid in meeting levels of overnight rise and earlymorning cortisol peak required in adrenal insufficiency

5 Conclusion

This in vivo investigation of CPH pectin-based hydrocorti-sone multiparticulate capsules has shown that cortisol can bereleased in a circadian fashion in Sprague-Dawley rats and theformulation holds promise in achieving the chronodeliveryof hydrocortisone in adrenal insufficiency in humans Thereis the need however for further clinical investigations inhuman subjects to confirm replicability

Data Availability

The in vitro and in vivo pharmacokinetics data used tosupport the findings of this study are included within thearticle and are also available from the corresponding authorby request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support given toOfosua Adi-Dako by the Universities Science Humanitiesand Engineering Partnerships in Africa (USHEPiA) theUniversity of Ghana Office of Research and Innovation(ORID) and Professor Kelly Chibale of University of CapeTown South Africa for the study

References

[1] G S Banker J Siepmann andCRhodesModernParmaceuticsCRC Press 2002

[2] R Langer ldquoNewmethods of drug deliveryrdquo Science vol 249 no4976 pp 1527ndash1533 1990

[3] C de Weerth R H Zijl and J K Buitelaar ldquoDevelopment ofcortisol circadian rhythm in infancyrdquo Early Human Develop-ment vol 73 no 1-2 pp 39ndash52 2003

[4] M Debono and R J Ross ldquoWhat is the best approach totailoring hydrocortisone dose to meet patient needs in 2012rdquoClinical Endocrinology vol 78 no 5 pp 659ndash664 2013

[5] S Chan and M Debono ldquoReplication of cortisol circadianrhythm new advances in hydrocortisone replacement therapyrdquoerapeutic Advances in Endocrinology and Metabolism vol 1no 3 pp 129ndash138 2010

[6] C J Elder and P Dimitri ldquoHydrocortisone for adrenal insuffi-ciencyrdquo ADC - Education and Practice Edition vol 100 no 5pp 272ndash276 2015


[7] E M Alevritis F A Sarubbi R M Jordan and A N PeirisldquoInfectious Causes of Adrenal Insufficiencyrdquo Southern MedicalJournal vol 96 no 9 pp 888ndash890 2003

[8] S-Y Lin and Y Kawashima ldquoCurrent status and approaches todeveloping press-coated chronodelivery drug systemsrdquo Journalof Controlled Release vol 157 no 3 pp 331ndash353 2012

[9] M M Patel ldquoColon A gateway for chronotherapeutic drugdelivery systemsrdquo Expert Opinion on Drug Delivery vol 12 no9 pp 1389ndash1395 2015

[10] M J Whitaker M Debono H Huatan D P Merke W Arltand R J Ross ldquoAn oral multiparticulate modified-releasehydrocortisone replacement therapy that provides physiologi-cal cortisol exposurerdquo Clinical Endocrinology vol 80 no 4 pp554ndash561 2014

[11] G Johannsson R Bergthorsdottir A G Nilsson H LennernasT Hedner and S Skrtic ldquoImproving glucocorticoid replace-ment therapy using a novel modified-release hydrocortisonetablet a pharmacokinetic studyrdquo European Journal of Endo-crinology vol 161 no 1 pp 119ndash130 2009

[12] S Verma C Vanryzin N Sinaii et al ldquoA pharmacokineticand pharmacodynamic study of delayed- and extended-releasehydrocortisone (ChronocortTM) vs conventional hydrocor-tisone (CortefTM) in the treatment of congenital adrenalhyperplasiardquo Clinical Endocrinology vol 72 no 4 pp 441ndash4472010

[13] G Johannsson H Lennernas C Marelli K Rockich and SSkrtic ldquoAchieving a physiological cortisol profile with once-daily dual-release hydrocortisone A pharmacokinetic studyrdquoEuropean Journal of Endocrinology vol 175 no 1 pp 85ndash932016

[14] O Adi-Dako K Ofori-Kwakye M E Boakye-Gyasi S OppongBekoe and S Okyem ldquoIn Vitro Evaluation of Cocoa PodHusk Pectin as a Carrier for Chronodelivery of HydrocortisoneIntended for Adrenal Insufficiencyrdquo Journal of Drug Deliveryvol 2017 Article ID 8284025 10 pages 2017

[15] M J Rathbone J Hadgraft andM S RobertsModified-releaseDrug Delivery Technology CRC Press 2002

[16] P Li and L Zhao ldquoDeveloping early formulations Practice andperspectiverdquo International Journal of Pharmaceutics vol 341no 1-2 pp 1ndash19 2007

[17] A K Shah and S A Agnihotri ldquoRecent advances and novelstrategies in pre-clinical formulation development An over-viewrdquo Journal of Controlled Release vol 156 no 3 pp 281ndash2962011

[18] O Adi-Dako K Ofori-Kwakye S Frimpong Manso M EBoakye-Gyasi C Sasu and M Pobee ldquoPhysicochemical andAntimicrobial Properties of Cocoa Pod Husk Pectin Intendedas a Versatile Pharmaceutical Excipient and NutraceuticalrdquoJournal of Pharmaceutics vol 2016 Article ID 7608693 12pages 2016

[19] E C Bucholtz L M French J P Lavoie and C J GaebeleinldquoQuality control analysis of student-generated pharmaceuticalcapsulesrdquo Journal of Chemical Education vol 87 no 10 pp 1108-1109 2010

[20] E M Rudnic and J B Schwartz ldquoOral solid dosage formsrdquo inRemington e Science and Practice of Pharmacy A GennaroEd 20th edition 2000

[21] British Pharmacopoeia British Pharmacopoeia CommissionLondon the Department of Health Social Services and PublicSafetyvol 1 pp 719-720 2013

[22] O Adi-Dako S Oppong Bekoe K Ofori-Kwakye E Appiahand P Peprah ldquoNovel HPLC Analysis of Hydrocortisone in

Conventional andControlled-Release Pharmaceutical Prepara-tionsrdquo Journal of Pharmaceutics vol 2017 Article ID 9495732 8pages 2017

[23] N R Council Guide for the care and use of laboratory animalsInstitute of Laboratory Animal Resources Commission onLife Sciences National Academy of Sciences Washington DCUSA 1996

[24] M A Cole P J Kim B A Kalman and R L Spencer ldquoDexam-ethasone suppression of corticosteroid secretion Evaluation ofthe site of action by receptor measures and functional studiesrdquoPsychoneuroendocrinology vol 25 no 2 pp 151ndash167 2000

[25] M Debono C Ghobadi A Rostami-Hodjegan et al ldquoModi-fied-release hydrocortisone to provide circadian cortisol pro-filesrdquo e Journal of Clinical Endocrinology amp Metabolism vol94 no 5 pp 1548ndash1554 2009

[26] O Chambin G Dupuis D Champion A Voilley and YPourcelot ldquoColon-specific drug delivery Influence of solutionreticulation properties upon pectin beads performancerdquo Inter-national Journal of Pharmaceutics vol 321 no 1-2 pp 86ndash932006

[27] S Das K Y Ng and P C Ho ldquoDesign of a pectin-basedmicro-particle formulation using zinc ions as the cross-linking agentand glutaraldehyde as the hardening agent for colonic-specificdelivery of resveratrol in vitro and in vivo evaluationsrdquo Journalof Drug Targeting vol 19 no 6 pp 446ndash457 2011

[28] B Wu Z Chen X Wei N Sun Y Lu and W Wu ldquoBiphasicrelease of indomethacin from HPMCpectincalcium matrixtablet I Characterization and mechanistic studyrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 67 no 3pp 707ndash714 2007

[29] United States Pharmacopeia USP 29ndashNF 24 Rockville MDUSP 2005

[30] L F Asghar and S Chandran ldquoMultiparticulate formulationapproach to colon specific drug delivery current perspectivesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 9 no 3 pp327ndash338 2006

[31] M K Chourasia and S K Jain ldquoPolysaccharides for colontargeted drug deliveryrdquoDrug Delivery vol 11 no 2 pp 129ndash1482004

[32] P-Y Yeh M M Berenson W S Samowitz P Kopeckovaand J Kopecek ldquoSite-specific drug delivery and penetrationenhancement in the gastrointestinal tractrdquo Journal of ControlledRelease vol 36 no 1-2 pp 109ndash124 1995

[33] L S Liu M L Fishman and K B Hicks ldquoPectin in controlleddrug delivery - A reviewrdquoCellulose vol 14 no 1 pp 15ndash24 2007

[34] S Das A Chaudhury and K-Y Ng ldquoPreparation and eval-uation of zinc-pectin-chitosan composite particles for drugdelivery to the colon Role of chitosan in modifying in vitro andin vivo drug releaserdquo International Journal of Pharmaceuticsvol 406 no 1-2 pp 11ndash20 2011

[35] I El-Gibaly ldquoOral delayed-release system based on Zn-pectinate gel (ZPG) microparticles as an alternative carrier tocalciumpectinate beads for colonic drug deliveryrdquo InternationalJournal of Pharmaceutics vol 232 no 1-2 pp 199ndash211 2002

[36] E Dinte E Bodoki S Leucuta andCAdela Iuga ldquoCompatibil-ity studies between drugs and excipients in the preformulationphase of buccal mucoadhesive systemsrdquo Farmacia vol 61 no4 pp 703ndash712 2013

[37] S S Menon B V Basavaraj S Bharath R Deveswaran andVMadhavan ldquoFormulation and evaluation of ibuprofen tabletsusing orange peel pectin as binding agentrdquo Der PharmaciaLettre vol 3 no 4 pp 241ndash247 2011


[38] G S MacLeod J T Fell and J H Collett ldquoAn in vitro investi-gation into the potential for bimodal drug release frompectinchitosanHPMC-coated tabletsrdquo International Journal ofPharmaceutics vol 188 no 1 pp 11ndash18 1999

[39] H H Gadalla G M Soliman F A Mohammed and A M El-Sayed ldquoDevelopment and in vitroin vivo evaluation of Zn-pec-tinate microparticles reinforced with chitosan for the colonicdelivery of progesteronerdquoDrug Delivery vol 23 no 7 pp 2541ndash2554 2016

[40] L Tuovinen S Peltonen M Liikola et al ldquoDrug release fromstarch-acetatemicroparticles and films with and without incor-porated alpha-amylaserdquo Biomaterials vol 25 no 18 pp 4355ndash4362 2004

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Table 1 Composition of CPH pectin-based hydrocortisone capsule formulations

Capsuleformulation

Hydrocortisone(mg)

CPH pectin(mg)

Calciumchloride(mg)

Zinc acetate(mg)

HPMC(mg)

Dicalciumphosphate

(mg)

Starch(mg)

A 10 50 50 - 25 65 -B 10 50 - 50 - - 90

The disease progresses in a gradual and subtle manner withfatal consequences [5 6] Aside adrenal insufficiency otherdisease conditions that display circadian variation in theirpathophysiology include hypertension asthma and gastriculcer Such disease conditions require drug delivery to mimicthis rhythmic pattern [7]

Drug tolerance and side effects are associated with drugsthat are released at a constant rate in diurnal disease states[8ndash10] The administration of immediate release hydrocor-tisone (drug form of cortisol) in the treatment of adrenalinsufficiency has proven to be ineffective due to inabilityof drug to mimic the rhythm of endogenous cortisol Toaddress the deficiencies associated with the use of immediaterelease hydrocortisone preparations in adrenal insufficiencyvarious modified release hydrocortisone formulations whichare intended to replicate the physiological cortisol rhythmhave been produced with varying degrees of success [11ndash14]

There is however the need to develop modified releasehydrocortisone with optimized drug delivery to achieveenhanced and optimal blood drug concentration-time pro-files [2 15] Preclinical studies using animal models providecritical information about the biopharmaceutical and phar-macokinetic properties of such formulations [16]These stud-ies serve as predictors of possible effects of these formulationsin humans [17]

Cocoa pod husks (CPH) which are the empty pod shellsof the cocoa (eobroma cacao) fruit have been shown tohave suitable physicochemical properties as a pharmaceuticalexcipient [18] An in vitro evaluation of the CPHpectin-basedformulation as a carrier for chronodelivery of hydrocortisonedemonstrated its potential use in adrenal insufficiency [14]Findings from this earlier in vitro study showed that formu-lated CPH pectin-based systems containing hydrocortisonecan achieve a burst release of the drug at circadian timing afterapproximately 6 hour lag phase [14] The burst release of thedrug at circadian timingwas attributed to enhanced solubilityand degradation of pectin in simulated colonic mediumThecurrent study sought to design a simple formulation of a CPHpectin-based oral multiparticulate matrix system which issuitable for chronodelivery of hydrocortisone An evaluationof the pharmacokinetic profiles of the CPH pectin-basedhydrocortisone systems compared with an immediate releasecommercial hydrocortisone formulation was undertaken inSprague-Dawley rats

2 Materials and Methods

21 Materials Micronized hydrocortisone powder sim50120583m(Sigma Aldrich USA) commercial 10mg hydrocortisone

tablets (Auden McKenzie UK) calcium chloride (FischerScientific UK) dicalcium phosphate (SujianModern Chemi-cal China) hydroxypropyl methylcellulose [Methocel 44779HPMC (viscosity 40-60 cps 2 aqueous solution)] (AlphaAesar USA) microcrystalline cellulose (Blanver Farmo-quimica Brazil) maize starch (Vasa Pharmachem India)zinc acetate (Central Drug House India) dexamethasoneinjection (Lab Sanderson SA Chile) gelatin capsule shells(Capsule Connection USA) ether (Merck Germany) andfreeze dried CPH pectin extracted from minced fresh cocoapod husks by hot aqueous extraction [18] were used for thestudy

22 Preparation of Modified Release CPH Pectin-BasedHydrocortisone Capsules Modified release pectin-basedhydrocortisone capsules containing sim10mg hydrocortisonewere extemporaneously prepared (Table 1) The stipulatedamounts of the powders except CPH pectin were weighedand mixed by geometric dilution CPH pectin was dispersedin sufficient amount of hot water to form a viscous dispersionand added gradually to the mixed powders to form a wetmass The wet powder mass was passed through a 20sieve and the granules dried at 40∘C for 15 h in an ovenThe moisture content of the granules was determined byweighing 1 g of the granules into each of three Petri dishesand dried in a hot air oven at 105∘C to constant weightThe moisture content was calculated as the ratio of theweight of moisture loss to weight of sample expressed as apercentage [18] The particle size and surface characteristicsof the granules were evaluated with a Jeol Scanning ElectronMicroscope (JSM-6390LV Japan) by placing samples ondouble-sided carbon adhesive tapes on specimen stubs andcoated with platinum in the autofine coater The sampleswere then scanned and viewed

The capsules containing sim200mg granules each wereprepared manually by the traditional punch method Thegranules were poured onto a powder weighing paper andthe base of capsule size ldquo0rdquo was held vertically and the openend repeatedly pushed or punched into the powder until thecapsule was filled The cap was replaced to close the capsuleEach filled capsule was weighed using an empty capsule as acounterweight and granules were added or removed until thecorrect weights were obtained [19 20]

23 Weight Analysis of Capsules Ten capsules were individ-ually weighed The contents of each capsule were removedand the weights of the empty shells recorded separately Thedifference between both weights was calculated and the netweight of the contents was determined for each capsule [21]








3 Results



(a) (b)



0

20

40

60

80

100

Am

ount

of d

rug

rele

ased

()

30 60 90 120 1500Time (min)




2 4 6 8 100Time (h)

0

5

10

15

20

25

Con

cent

ratio

n of

dru

g (n

gm

l)











Rel F () 100 213120593 274120593



4 Discussion
















5 Conclusion


Data Availability




Acknowledgments


References


















































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of









3 Results



(a) (b)



0

20

40

60

80

100

Am

ount

of d

rug

rele

ased

()

30 60 90 120 1500Time (min)




2 4 6 8 100Time (h)

0

5

10

15

20

25

Con

cent

ratio

n of

dru

g (n

gm

l)











Rel F () 100 213120593 274120593



4 Discussion
















5 Conclusion


Data Availability




Acknowledgments


References


















































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



(a) (b)



0

20

40

60

80

100

Am

ount

of d

rug

rele

ased

()

30 60 90 120 1500Time (min)




2 4 6 8 100Time (h)

0

5

10

15

20

25

Con

cent

ratio

n of

dru

g (n

gm

l)











Rel F () 100 213120593 274120593



4 Discussion
















5 Conclusion


Data Availability




Acknowledgments


References


















































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of








Rel F () 100 213120593 274120593



4 Discussion
















5 Conclusion


Data Availability




Acknowledgments


References


















































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of









5 Conclusion


Data Availability




Acknowledgments


References


















































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of













































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of


potential of cocoa pod husk pectin-based modified release...

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