Journal of Suppository Formulations as a Potential Treatment for Nephropathic Cystinosis

Suppository Formulations as a Potential Treatmentfor Nephropathic Cystinosis

BARBARA BUCHAN, GRAEME KAY, KERR H. MATTHEWS, DONALD CAIRNS

Institute for Health & Welfare Research, Robert Gordon University, Aberdeen, UK

Received 22 February 2012; revised 2 May 2012; accepted 8 June 2012

Published online 6 July 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23246

ABSTRACT: Nephropathic cystinosis is a rare autosomal recessive disease characterisedby raised lysosomal levels of cystine in the cells of all the organs. It is treated by the 6-horal administration of the aminothiol, cysteamine, which has an offensive taste and smell.In an attempt to reduce this frequency and improve the treatment, cysteamine-containingpolyethylene glycol suppositories were prepared and evaluated for dissolution and stability. Theresults demonstrated that cysteamine release was complete after 30 min, and that there was auniform drug distribution within the formulations. Twelve-month stability tests highlighted apotential incompatibility among some excipients, although stability was demonstrated for thecysteamine suppositories up to 6 months. These suppositories may provide a useful alternativeto the current oral therapy for cystinosis. © 2012 Wiley Periodicals, Inc. and the AmericanPharmacists Association J Pharm Sci 101:3729–3738, 2012Keywords: Cystinosis; Formulation; PEG; Physical Stability; Dissolution; Thermal Analysis;Pediatric

INTRODUCTION

Nephropathic cystinosis is a rare genetic disease char-acterised primarily by extremely high intracellularlevels of the amino-acid cystine, electrolyte imbal-ance, proximal renal tubular dysfunction (Fanconisyndrome) and general failure to thrive.1,2 The accu-mulation of cystine as crystals in most tissues leadsto the progressive dysfunction of multiple organs.3

The incidence of cystinosis is one in 100,000–200,000live births, and affects approximately 2000 patients inthe world, although there are believed to be manymore cases undiagnosed.2,4 Some infants will die be-cause of dehydration and electrolyte imbalance fromFanconi syndrome without diagnosis.5 In the USalone, there are 500–600 reported cases, with between20 and 40 born each year.5

Cystinosis is categorised as a lysosomal storage dis-order (LSD), which is a group of progressive disordersthat share multi-organ failure as an endpoint.3,5 Aswith all LSDs, no signs of abnormality are displayedat birth; the first symptoms of glomerular dysfunc-tion begin to appear at around 6–12 months of age.2

Without treatment most children will reach end-stage

Correspondence to: Graeme Kay (Telephone: +44-1224-262548;Fax: +44-1224-262555; E-mail: [email protected])Journal of Pharmaceutical Sciences, Vol. 101, 3729–3738 (2012)© 2012 Wiley Periodicals, Inc. and the American Pharmacists Association

renal failure by age nine,5 and grow at 50%–60% ofthe expected rate. By the age of eight, an untreatedchild with cystinosis will be of the height of a 4-yearold,5 and, if inadequately treated, may not reach 4 ft.in height.

The condition is caused by a defect in the CTNSgene. This is a gene which codes for a 367-amino-acid-lysosomal-transport protein called Cystinosin.In healthy cells, the amino-acid cystine is transportedinto the cytoplasm of the cell for processing. In cysti-nosis, however, it accumulates within the lysosomesto levels of 10–1000 times those seen in healthycells, whereupon it crystallises from solution, caus-ing cell death and organ failure.6 This crystallisa-tion is widespread throughout the majority of thetissues and organs in the body.7 If left untreated,symptomatic deterioration leads to death by the sec-ond decade of life.8

The treatment for cystinosis involves the adminis-tration of the aminothiol cysteamine, which is usedtherapeutically in the bitartrate form as CystagonTM

(Mylan Laboratories, USA). With early and diligenttherapy, cystinosis patients can prevent or delaymost of the non-renal complications and extend theirlives into a fourth or fifth decade, live independentlyand some women have borne children.5,9 The re-nal damage is continuous; however, the decline isinevitable.5

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3730 BUCHAN ET AL.

Cysteamine causes a range of side effects and thisis largely because of the high dose which is requiredas much of the drug is lost to first-pass metabolism.9

High blood levels must be achieved, as a large pro-portion of the administered drug will bind to cir-culating proteins, and cannot be up taken by thecells.10,11 Patients should aim to take their dose atregular 6-h intervals for the treatment to work effec-tively, as the plasma half-life of cysteamine is 1.88 h,7

with blood levels peaking at 1 h, and rapidly declin-ing thereafter.5 This is a lifelong commitment andrequires the patient to wake in the middle of thenight.9 The drug has a foul taste and smell akin torotten eggs, which regularly induces vomiting afteringestion.9 In approximately 10%–15% of patients,this can be severe enough to halt therapy.5 Cys-teamine and its metabolites are excreted in breathand sweat, and this is also an issue, especially whenthe child enters education. Cysteamine has the poten-tial to cause potentially serious stomach irritationssuch as gastric acid hypersecretion, reverse peristal-sis and bile reflux, and 97% of the patients report gas-trointestinal symptoms,12,13 which in many patientsis severe enough to significantly limit the therapy.14–16

Compliance can therefore be a major barrier to effec-tive treatment, and lead to significant morbidity.

The rectal route of administration can largely avoidthe phenomenon of first-pass effect. This results fromone of the three rectal veins (upper vein) draining intothe hepatic system, whereas the middle and lowerveins bypass this and drain directly into the systemiccirculation. If the suppository is positioned correctly,the drug should not be subjected to the first-pass ef-fect. This potentially allows a smaller dose to be ad-ministered, thereby reducing or eliminating some ofthe unpleasant side effects. They may also be benefi-cial for treating conditions in infancy, when capsulesare difficult to administer, or when the oral route iscompromised. Rectal formulations are useful tools,particularly in a case such as this where the tasteand side effects render the task of swallowing a tabletvery unpleasant and foreboding.

Aims

Formulation science may provide a way to improve thecurrent medication, significantly improving the livesof sufferers and those who care for them. By eliminat-ing the taste and frequency of administration throughalternative dosage forms, ease of administration ofcysteamine could be improved. The aim of this workwas to develop alternative formulations of cysteaminewhich could reduce or eliminate some of the sideeffects experienced using the current oral capsule,thereby improving the quality of life for those affected.An improvement in the ease of administration of cys-teamine to infants and young children was a centralobjective, therefore suppositories were investigated.

By avoiding the first-pass metabolism of cysteamine,a lower dose should be achievable, whereas the tasteand upper-gastric side effects should be eliminated.A study conducted by van’t Hoff et al. was previouslyundertaken where a cysteamine-loaded suppositorygel, for use in cystinosis, was evaluated. However, thisrectal formulation was eliminated before cysteamineabsorption was completed.17

MATERIALS AND METHODS

Materials

Cysteamine hydrochloride and polyethylene glycol(PEG) grades 400, 600, 1000, 1500, 3000, 4000,6000, 8000 and 14,000 were obtained from Sigma(St. Louis, Missouri). Witepsol W35 was obtainedfrom Gattefosse (St-Priest, France). Gelucire 39/01was purchased from Sasol GmbH (Witten, Germany).Poloxamer F68 was bought from BASF SE (Lud-wigshafen, Germany). Ellman’s Reagent, 5,5′-dithi-obis(2-nitrobenzoate) (DTNB) was purchased fromMolekula (Gillingham, UK). Tris buffer and Tween80 were bought from Fisher (Loughborough, UK).

Synthesis of N,N-(Bis-L-Phenylalanyl)CystamineBistrifluoroacetate (Phenylalanine Conjugate)

Cysteamine does not possess a chromophore andtherefore is ultraviolet (UV) transparent; thus, mon-itoring its release from formulations is very difficult.Initially, a phenylalanine conjugate was developed totag the molecule, allowing quantitative determina-tion of release of the active from the dosage formvia UV spectroscopy (Fig. 1). Cystamine, the oxidiseddisulphide of cysteamine, was used in the synthe-sis. The phenylalanine conjugate was subsequentlyreplaced with cysteamine hydrochloride with DTNBdetection (see the section Dissolution Studies).

To a stirring solution of cystamine dihydrochlo-ride (1 g, 0.00444 mol) in anhydrous dichloromethane(DCM) (20 cm3) at room temperature, 1,8-diazabi-cycloundec-7-ene (1.33 mL, 0.0089 mol) was added.The reaction mixture was then stirred at room

Figure 1. N,N-(bis-L-phenylalanyl)cystamine bistrifluo-roacetate, (Phenylalanine conjugate).

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SUPPOSITORY FORMULATIONS AS A POTENTIAL TREATMENT FOR NEPHROPATHIC CYSTINOSIS 3731

temperature for 1 h. To this, butoxycarbonyl-L-phenylalanine-N-hydroxysuccinimide ester (3.22 g,0.0089 mol) was added. After thin layer chromato-graphic analysis, the reaction mixture was then parti-tioned between DCM (20 mL) and water ( 50mL). TheDCM extracts were washed with water (3 × 50 mL)and dried over magnesium sulphate. The resultantsolution was applied to a silica gel chromatographycolumn (4 × 30mL) prepared with DCM. The col-umn was initially eluted with 100% DCM followedby 2% graduations of methanol, with the protectedcompound eluting at 9:1 DCM–methanol.

The protected compound was dissolved in triflu-oroacetic acid (5 mL) at room temperature. After3.5 h, the trifluoroacetic acid was removed by co-evaporation with ethanol (3 × 20 mL). Triturationwith diethyl ether followed by filtration and drying(vacuum oven at 50◦C) gave the target compound asan off-white solid. Yield: 42%.

Mass spectroscopy data: m/z 447.2 (100%); 224.4(50%). M: C22H32N4O2S2

+; Exact Mass: 448.2.Nuclear magnetic resonance data: All analyses

were undertaken using a Bruker Topspin Ultrashield400MHz (Billerica, Massachusetts). 1H NMR spec-trum (CD3OD) (400 MHz) *: 2.55 (m, CH2 S2, 2H);2.95 (m, CH2 Ar, 1H); 3.05 (m, CH2 Ar, 1H); 3.25(NH CHH, 1H); 3.45 (m, NH CHH, 1H); 3.95 (t,"-CH, 1H); 7.25 (m, Ar H’s, 5H). 13C NMR spec-trum (CD3OD) (400 MHz) *: 37.7(CH2S2); 38.8 ($-CH2); 39.5 (NH CH2); 55.8 ("-CH); 128.9 (Ar CH);130.1 (Ar CH); 130.5 (Ar CH); 135.6 (Ar CH); 169.7(CO). Melting range: 100–104◦C.

Suppository Preformulation and Manufacture

To allow for wastage, the weight of eight supposito-ries was calculated for a six-form mould. The cali-bration value was calculated for each mould, and thedisplacement value was calculated for each drug. Thebases were weighed accurately on a Mettler AE50analytical balance, along with the active. The baseswere heated and blended together, before addition ofeither cysteamine or phenylalanine conjugate. Thismix was then thoroughly stirred and the resultantliquid poured into a mould and allowed to cool. Thetops were then trimmed with a spatula, and the fi-nal forms were removed from the mould, bottled andlabelled [British Pharmacopeia (BP), 2005]. The sur-factant Tween 80 was used in some formulations toensure that the fatty bases were sampled accuratelyfrom the dissolution medium.

Lipophilic bases, hydrophilic bases and blends ofdifferent PEG grades were examined for optimumcharacteristics. As 10 mg kg−1 was used for previousrectal delivery of cysteamine, 60 mg was used as anominal value.17 Until further tests are carried outin vivo, the correct dose cannot be determined.

Suppository hardness was tested on an ErwekaTBH28 hardness tester (Heusenstamm, Germany).A hardness value of at least 1.8–2 kg is consideredacceptable for unmedicated suppositories.18 If the in-ert suppository blend produced unsatisfactory resultsbeyond this range, no further testing was performed.The suppositories with the best characteristics, basedon hardness tests and appearance were selected forfurther testing.

Dissolution Studies

Dissolution tests were performed in a six-chamberedSotax CH-4123 dissolution bath (Basel, Switzerland).The dissolution test method followed the standard BPtest for suppositories: one suppository in a stainless-steel basket, rotating at 100 rpm in a 1 L beaker con-taining medium at 37◦C, sampling at 5 min inter-vals for 1 h (BP, 2005 ed.). The dissolution mediumwas altered depending on the active used. The re-lease was measured by UV spectroscopy. The sampleswere returned to the bath after analysis. Supposito-ries containing the phenylalanine conjugate were ini-tially tested in distilled water, using UV absorbanceat 256 nm corresponding to the λmax.

Cysteamine hydrochloride release can be mea-sured via free sulphydryl concentration throughthe use of DTNB [Ellman’s Reagent, 5-(3-carboxy-4-nitrophenyl)disulphanyl-2-nitrobenzoic acid]. Basi-cally, 1 mol of DTNB reacts with 1 mol of cysteamineto release 1 mol of 2-nitro-5-thiobenzoate (NTB). Theconcentration of NTB and hence the concentration ofcysteamine can be determined using UV–visible spec-troscopy. Calibration measurements, using an analyt-ical wavelength of 440 nm, gave a linear response,R2 = 0.9991. The dissolution medium was 90%deionised water and 10% 1 M Tris buffer solution atpH8. Because of the susceptibility of DTNB to pho-tolysis when in solution, the tank was surrounded byaluminium foil and protected from light.

The Higuchi equation was used to determine therate order of the drug release. A result below 0.45 isindicative of Fickian diffusion.19

Active Dispersion Studies

In a suppository, there is a possibility that the tipmight contain more drug than the rest of the formbecause of the gravitational settling.18 To ensure uni-form drug loading, three separate areas of the suppos-itory were sampled and compared using a DSC Q100differential scanning calorimeter (TA Instruments,New Castle, Delaware) and the experiment carriedout in triplicate. Coupled with this, differential scan-ning calorimetry (DSC) studies of suppositories werecarried out to determine the thermal characteristicsof each suppository blend.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 10, OCTOBER 2012

3732 BUCHAN ET AL.

Table 1. A Summary of the Batches Made and Their Characteristics

SuppositoryBatch Composition (% w/w)

Hardness WithoutActive

Hardness Including Active(Cysteamine HCl) Appearance

A PEG 8000 40% 19 N, 1.94 kg 9 N, 0.92 kg Uniform whitePEG 600 60%

B PEG 14,000 40% 17 N, 1.73 kg 12 N, 1.22 kg Uniform whitePEG 600 60%

C PEG 1500 100% 30 N, 3.06 kg 27 N, 2.75 kg Opaque

Stability Tests

Stability studies were conducted over a 12-month pe-riod. Upon manufacture (time zero, T0), the supposito-ries were stored under three different environmentalconditions to determine their stability; a refrigera-tor at 4◦C and 8% relative humidity (RH), a storecontaining a desiccator with saturated sodium hydro-gen sulphate solution at 20◦C and 52% RH,20 and anEnvironmental Test Chamber (Copely, Nottingham,England) set at 30◦C and 75% RH.21 These condi-tions were selected to simulate cold storage, typicalhome storage and accelerated testing. As the sup-positories had low melting points, it was decidedto reduce the commonly used accelerated tempera-ture from 37◦C to 30◦C. The temperature in each ofthe storage areas was monitored daily. DSC and in-frared (IR) spectroscopy were undertaken every weekfor 1 month, every month for 3 months and sub-sequently every 3 months for 12 months, and com-pared with the T0 results.21 IR spectroscopy, wasundertaken using a Nicolet IS10 IR from Thermo Sci-entific (Fisher) with a Smart iTR attachment anda diamond HATR (horizontal attenuated total re-flectance). Thiol groups absorb at a wavenumberof 2600–2550 cm−1, whereas disulphides absorb be-tween 620 and 600 cm−1.22 These measurements wereused as the basis for the comparisons made betweensamples.

Samples of uniformly less than 10 mg were placedin a standard aluminium pan for DSC analysis. TheDSC was set to a heat–cool–heat cycle, where the sam-ple was equilibrated at 0◦C, then heated to 100◦C at10◦C/min, equilibrated at 100◦C, then cooled at 10◦C/min to 0◦C, before being heated again to 100◦C at10◦C/min.

RESULTS AND DISCUSSION

The final three suppositories selected for further char-acterisation were blend “A” (40% PEG 8000, 60% PEG600), blend “B” (40% PEG 14000, 60% PEG 600) andblend “C” (PEG 1500) (Table 1). There is a large vari-ation in release onset in forms A, B and C, which is il-lustrated by large standard deviation values. T100 forPEG blends A, B and C were observed at 20, 45 and60 min, respectively (n = 5–9). PEG blend C releasedthe phenylalanine conjugate more slowly than PEGblends A and B. This may be because of the greaterhardness of blend C (Table 2).

The fatty bases Witepsol (Gattefosse) and Gelucire(Sasol GmbH) produced a more homogenous blendand melted instantly (data not shown), whereas thePEG bases produced a more prolonged dissolutionprofile and dissolved more slowly over time (Fig. 2).It was decided to continue studies using PEG basesonly, as they produced a more prolonged release of20–60 min compared with 2 min for Witepsol (Gatte-fosse) and Gelucire (Sasol GmbH). This slower disso-lution of the PEGs allows the drug to be in contactwith the rectum for longer where it is more likelyto be absorbed into the body. A sudden melt over2 min would increase the chances of the drug beinglost through expulsion.

Figure 3 illustrates the release of the active fromthe three suppository bases over time. Cysteaminehydrochloride was used as the active, formulated intosuppository bases PEG blends A, B and C.

All three PEG blends containing cysteamine hy-drochloride as the active displayed more reproducibleprofiles than dissolution using the phenylalanine con-jugate. The PEG bases should dissolve at the samerate in every replicate, and this may have been due

Table 2. Summary of Phenylalanine Conjugate Release Studies

Time to Release Phenylalanine Conjugate (Minutes, ±SD)

Percentage Release PEG Blend A PEG Blend B PEG Blend C Gelucire Witepsol

10% 2 (±7.1) 2 (±6.3) 3 (±5.1) 0.5 0.525% 5 5 9.5 1 150% 7 7.5 12.5 2 275% 10 (±5.6) 10 (±5.4) 17 (±8.6) 2.5 2.5100% 20 (±0.6) 45 (±0.8) 59 (±0.7) 4.5 4.5



Figure 2. Percentage release of the phenylalanine conjugate from PEG blends A, B and Cover time (n = 7, 9 and 6, respectively).

to the non-uniform drug loading of the phenylalanineconjugate. The phenylalanine conjugate was devel-oped initially to allow release from the dosage forms,to be monitored spectrophotometrically. It should benoted that cysteamine hydrochloride is a difficultdrug to formulate. At room temperature it undergoesoxidation to a disulphide form, cystamine, which itselfhas been shown to deplete cells of cystine but whichis not licensed for use.5,23,24 Cysteamine is also ex-tremely hygroscopic and deliquescent. However, theintroduction of DTNB as a quantitative reagent al-lowed measurement of cysteamine directly. A sum-mary of the release times obtained is shown in Table 3.The data produced using Ellman’s reagent demon-strated excellent reproducibility (average standarderror of the mean for PEG blend: A–0.39, B–0.58 andC–0.14).

Active Dispersion Studies

To ensure cysteamine hydrochloride was evenly dis-persed throughout the suppository, samples weretaken from three separate portions of the suppositoryand analysed using DSC (Fig. 4). There was minimaldifference between the tip, edge and middle sections

of the suppository suggesting a uniform dispersion ofactive within the PEG suppository.

Stability Tests

The temperature and RH in each of the three storagechambers were monitored continuously throughout a12-month period. The average results obtained areshown in Table 4.

DSC Results: T0, T6 Months, T12 Months Comparison

The thermal properties of the suppositories were as-sessed at T0, 6 (T6) and 12 (T12) months storage at4◦C/8% RH, 21◦C/52% RH and 30◦C/75% RH. Themain melting endotherm of PEG in the broad rangeof 30◦C–60◦C was analysed in terms of the onset ofthe melt (Tonset), the peak temperature (Tmax) and thetotal enthalpy (W/g). Selected data are presented inTable 5 as an increase or decrease in temperature andenthalpy compared with samples at T0. An exemplarythermogram of PEG blend C suppositories at T0, T6and T12 is also included (Fig. 5).

Reference to the data in Table 5 indicates signifi-cant changes to Tonset and Tmax for aged samples ofPEG blend A at 30◦C, blend B at 21◦C and blend Cat both 21◦C and 30◦C. Suppositories stored at 4◦C


3734 BUCHAN ET AL.

Figure 3. Percentage release of cysteamine from PEG blends A, B and C over time (n = 6, 5and 5, respectively).

demonstrated less certain variation in Tonset and Tmaxas may have been expected for samples stored at thisrefrigerated temperature. Although the bulk of thetemperature variations were considered to be withinthe error of measurement for DSC analyses, it isclear that changes to the degree of crystallinity at thehigher storage temperatures had occurred. A deple-tion in the crystalline character of PEG suppositoriesmay be expected to be based on the lengthy exposureto high humidity and temperature, exacerbated by thehygroscopic (deliquescent) nature of contained cys-teamine hydrochloride. There is some evidence of in-creased crystallinity for PEG blend B stored at 21◦C,considered as a `̀lamellar thickening” of ordered ethy-lene oxide units,25 but the overall dataset suggests

a decrease in the molecular order of cysteamine con-taining PEG suppositories at elevated temperaturesover time. Increased enthalpies for the melt transitionin all samples are a consequence of a broadened en-dotherm, symptomatic of increased disorder in crys-talline domains rather than increased order whichwould produce clear increases to values of Tonset andTmax and a sharpening of the melting event. Suchchanges are not evident.

IR Spectroscopy After 1 Week, 3 Months, 6 Months and12 Months

Infrared spectroscopy utilises a set of unique peakson a spectrum which can be used for identifyinga compound. Each of the suppository samples was

Table 3. Summary of Cysteamine Hydrochloride Release Studies

Time to Release Cysteamine Hydrochloride (Minutes)

Percentage Release PEG Blend A PEG Blend B PEG Blend C

10% 1.75 2.5 1.7525% 5 6.5 4.550% 9 10 775% 13 15 9.5100% 26 26 17



Figure 4. Section comparison of the melt phase between the tip, middle and edge areas of thePEG blend A suppository containing cysteamine hydrochloride.

determined by IR spectroscopy and added to a com-pound library. A percentage match was then per-formed on each sample (Table 6).

The oxidation of cysteamine to cystamine was thebasis of comparisons made between samples. This re-action produces a peak in the region of disulphidebond stretch, that is, 620–600 cm−1, and allows a sim-ple identification of sample degradation.

Each suppository was analysed over time using theIR spectrometer, and compared with the supposito-

ries at time zero. The results are shown in Table 6(selected examples only).

The stability tests using IR analysis demonstratedthat PEG blend C was the most stable in all con-ditions, with minimal changes over 6 months atroom temperature and accelerated tests, and up to12 months stability when stored at 4◦C. PEG blendB shows evidence of stability over 12 months atroom temperature. PEG blend A shows evidence ofdegradation when subjected to a range of storage

Table 4. Average Temperatures and Relative Humidities in Each StorageChamber, With Standard Deviations (n = 3)

Refrigerator Store ETCa

Average temperature 3.7◦C (±0.8) 20.3◦C (±1.9) 29.9◦C (±1.0)Average relative humidity 9.2% (±0.3) 52% 76.4% (±2.0)

aEnvironmental Test Chamber.

Table 5. DSC Analysis Data of Selected Suppository Samples Over Time (n = 3)

T0 T6 Months T12 Months

Suppository Sample Tonset (◦C) �H (J/g) Tmax (◦C) Tonset (◦C) �H (J/g) Tmax (◦C) Tonset (◦C) �H (J/g) Tmax (◦C)

PEG blend A, 4◦C storage 50.6 71.8 56.4 +0.4 +4.2 +0.3 −0.9 +9.4 −1.3PEG blend A, 21◦C storage 50.7 83.8 57.7 +1.1 +9.0 +0.4 −0.2 +13.3 −0.5PEG blend A, 30◦C storage 50.5 75.3 56.6 −1.9 +8.8 −0.8 −5.9 +14.5 −5.1PEG blend B, 4◦C storage 51.4 73.4 57.8 +0.8 +7.3 −0.1 −0.3 +9.1 −1.3PEG blend B, 21◦C storage 50.4 67.2 56.7 +2.3 +19.2 +1.7 +0.6 +22.8 +0.2PEG blend B, 30◦C storage 50.7 80.1 57.6 +0.9 +6.1 −0.5 −0.8 +10.9 −1.7PEG blend C, 4◦C storage 41.2 159.4 50.3 +1.6 +20.4 −0.3 +0.9 +26.5 −0.2PEG blend C, 21◦C storage 41.1 165.9 50.3 −1.5 +6.5 −1.1 −3.5 +9.0 −2.9PEG blend C, 30◦C storage 50.0 164.5 40.9 −0.7 +6.1 −2.5 −15.3 −8.1 +5.3


3736 BUCHAN ET AL.

Figure 5. An Exemplary thermogram of PEG blend C supositories.

conditions, and therefore is unsuitable for the rectaldelivery of cysteamine.

Correlation Between DSC Plots and IR PercentageMatch Results

The IR percentage match data support the DSC plots.This is evidence of correlation between the two meth-ods and allows a more conclusive result to be pro-duced. For example, PEG blend C stored at 21◦C dis-played a 98.94% match at 6 months (Table 6).

These stability test results correlate with the hard-ness testing, and indicate that PEG blend C wasthe most stable formulation over a 12-month period.The suppositories stored at 4◦C were generally morestable than those stored at higher temperatures, al-though PEG blend C displays long-term stability evenat room temperature. PEG blend B suppositories werestable over time at room temperature. PEG blend Asuppositories displayed long-term instability underall storage conditions. PEG blend C displays ideal sta-bility over time in a range of storage conditions andis proposed as the optimal formulation in this study.

CONCLUSIONS

Liquid gel suppositories containing cysteamine havepreviously been investigated for the treatment ofcystinosis.17 However, because of the osmotic natureof the suppository vehicle, the doses were rapidlyexpelled and cysteamine blood plasma concentra-tions were insufficient to produce a reduction inmean leukocyte cystine concentration. In this project,the suppository bases Witepsol (Gattefosse), Gelu-cire (Sasol GmbH) and PEG were investigated fortheir suitability as vehicles for the incorporation ofcysteamine hydrochloride and a phenylalanine–cys-tamine conjugate. Melting point, hardness, stabilityand appearance were analysed, and the three sup-pository bases with the most suitable characteristicswere chosen for further study. PEG blends A, B andC were made and characterised. Blend C displayedgood qualities (i.e., complete, reproducible release af-ter 30 min, stability over 12 months at 4◦C/8% RH)required for the delivery of cysteamine hydrochlo-ride to the rectum. DSC analysis indicated that cys-teamine hydrochloride was dispersed uniformly in the

Table 6. Suppository Stability Over Time, Presented as Percentage Match toSample at T0 (Selected Data Only)

Suppository Blend/Storage Conditions Percentage Match to Sample at T0

PEG blend C, 4◦C/8% RH 99.2% at 3 months 98.84% at 12 monthsPEG blend A, 4◦C/8% RH 97.4% at 3 weeksPEG blend C, 21◦C/52% RH 98.94% at 6 monthsPEG blend B, 21◦C/52% RH 97.95% at 12 monthsPEG blend C, 30◦C/75% RH 98.94% at 6 months



suppositories. Dissolution studies using the pheny-lalanine–cystamine conjugate revealed 20–60 min re-lease, whereas cysteamine hydrochloride as the ac-tive component demonstrated 17–26 min release onaverage. The stability tests indicate that 4◦C/8% RHprovided the ideal storage conditions over a 12-monthperiod. Formulation C was the most stable over time,whereas blend A was the least stable form, and evenwhen stored in the refrigerator was subject to degra-dation. There was also evidence of an incompatibilitybetween cysteamine hydrochloride and PEG blendsA and B, and this may be a combination of physicalageing of the PEG and syneresis of cysteamine.26–29

There was evidence that crystal ripening over time isforcing the expulsion of the cysteamine hydrochlorideto the outside surfaces of the suppository. The highlydeliquescent cysteamine then quickly dissolves in en-vironmental moisture, forming droplets of liquid onthe surface of the suppositories. For these reasons,PEG blends A and B would not be suitable for therectal delivery of cysteamine. Analysis indicates thatPEG blend C would be an ideal suppository base forthe delivery of cysteamine hydrochloride.

These tests demonstrate that cysteamine hy-drochloride can be formulated as a suppository, andthat the bases can tolerate varying amounts of drugloading. This will be of particular benefit when treat-ing cystinosis during infancy, and should allow a sig-nificant reduction in side effects, improving compli-ance and morbidity. In addition, suppositories mayhelp to eliminate the overnight treatment breakwhich is difficult to overcome with the oral capsules.Future work with the suppositories will involve thedevelopment of in situ gelling forms, and should in-clude in vivo testing or an in vitro–in vivo correlationto investigate the potential benefits these forms mayhave compared with the current oral treatment.

ACKNOWLEDGMENTS

The authors gratefully acknowledge financial supportfrom the Cystinosis Foundation, UK and TENOVUS,Scotland.

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