Characterization,stability,and pharmacokinetics of sibutramine/b -cyclodextrin inclusion complex
Yong Youn Hwang a ,b ,Dong Chul Shin b ,Yoon Sung Nam a ,c ,*,Byung-Kwan Cho a ,**
a
Department of Biological Sciences,Korea Advanced Institute of Science and Technology,Daejeon 305-701,Republic of Korea b
Life Science R&D Center,SK Chemicals,Gyeonggi-do 463-400,Republic of Korea c
Department of Materials Science and Engineering,Korea Advanced Institute of Science and Technology,Daejeon 305-701,Republic of Korea
1.Introduction
Cyclodextrins (CDs)are cyclic oligosaccharides compod of 6,7,8,or 9glucopyrano units (a -,b -g -or d -CDs,respectively)with a relatively hydrophobic central cavity and a hydrophilic surface [1].Becau CDs can form complexes with a great variety of organic molecules,they have been widely ud to increa the stability,solubility,and bioavailability of poorly soluble drugs [2].Accordingly,more than 35different drugs are currently marketed as solid or solution-bad CD complex formulations,including alprostadil,meloxicam,nicotine,omeprazole,itraconazole,aripi-prazole,and insulin [3,4].CDs having less than six units cannot be formed due to steric hindrance while the higher homologs with more than nine gluco units are very difficult to purify.The cavity size of a -CD is insufficient for many drugs,g -CD is expensive,and d -CD has weaker complex forming ability than conventional CDs [5].Among them,b -CD is widely ud for pharmaceutical applications becau of the appropriate size of its cavity [1].As for the regulatory status,b -CD is listed in a number of
pharmacopoeia sources including the US Pharmacopoeia/National Formulary (USP/NF),European Pharmacopoeia (Ph.Eur.)and Japane Pharmaceutical Codex (JPC)[6].
The International Obesity Task Force reported that more than 300million individuals worldwide are obe,and an additional 800million are overweighted [7].Obe patients have higher risks for coronary artery dia,hypertension,hyperlipidemia,diabetes mellitus,certain cancers,cerebrovascular accident,osteoarthritis,obstructive pulmonary dia,and sleep apnea [8].Sibutramine,introduced as an anti-obesity drug in 1997,is a potent inhibitor of the reuptake of noradrenaline and rotonin [9,10]and may stimulate thermogenesis through the activation of b 3-adrenocep-tors in brown adipo tissue [11,12].Sibutramine hydrochloride monohydrate,a salt form,has been ud in a commercial product (Reductil 1)becau of its improved solubility and stability [12].The sibutramine salt has a solubility of about 3mg mL À1in water at pH 5.2while the solubility of sibutramine ba is only 0.01mg mL À1[13].In addition to the investigation on other salt forms of sibutramine [14–16],the solid dispersion technique has been suggested with aiming to increa the solubility of sibutramine ba in aqueous formulations [17,18].
This report describes the first investigation on the inclusion complex of sibutramine with b -CD and the stability and pharmacokinetic properties of the complex.The stability studies under the stress con
ditions were performed in comparison with sibutramine hydrochloride monohydrate as a stable salt form.The
Journal of Industrial and Engineering Chemistry 18(2012)1412–1417
A R T I C L E I N F O
Article history:
Received 14October 2011Accepted 26January 2012
Available online 3February 2012
Keywords:Cyclodextrin
Inclusion complex Sibutramine Stability Dissolution
Pharmacokinetics
A B S T R A C T
b -Cyclodextrin (b -CD)is widely ud to increa the stability,solubility,and bioavailability of poorly
soluble drugs becau of the appropriate size of its cavity.Sibutramine is a neurotransmitter reuptake inhibitor that has been investigated as an oral anorexiant.Here we report the complexation of sibutramine ba with b -CD and the stability,dissolution,and pharmacokinetic properties of the sibutramine/CD complex.The formation of sibutramine/b -CD inclusion complexes is confirmed using differential scanning calorimetry,X-ray diffractometry,and 1H nuclear magnetic resonance.The thermal and photochemical stability of sibutramine is significantly improved by the complexation with b -CD,and the pharmacokinetic parameters (e.g.,the plasma concentration,area under the curve,and maximum concentration of two active metabolites)for humans are comparable with tho of the commercialized standard product (Reductil 1).Our study suggests that sibutramine/b -CD complexation can be of great u to increa the stability and biological efficacy of sibutramine ba.
ß2012The Korean Society of Industrial and Engineering Chemistry.Published by Elvier B.V.All rights
rerved.
*Corresponding author at:Department of Biological Sciences,Korea Advanced Institute of Science and Technology,Daejeon 305-701,Republic of Korea.Tel.:+82423503311;fax:+82423503310.
**Corresponding author.Tel.:+82423502620;fax:+82423505620.
E-mail address:yoonsung@kaist.ac.kr (Y.S.Nam),bcho@kaist.ac.kr (B.-K.Cho).
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1226-086X/$–e front matter ß2012The Korean Society of Industrial and Engineering Chemistry.Published by Elvier B.V.All rights rerved.doi:10.1016/j.jiec.2012.01.046
dissolution and pharmacokinetics in humans of sibutramine/b-CD formulation were evaluated compared to a commercialized standard–Reductil1,which is bad on a formulation using sibutramine hydrochloride monohydrate.
2.Experimental
2.1.Materials
Sibutramine ba and sibutramine hydrochloride monohydrate were supplied from Cipla(India)and DaeHe Chemical(Republic of Korea),respectively.b-CD(Cavamax1W7Pharma,ade) was purchad from ISP(Cologne,Germany).Lacto monohy-drate,citric acid anhydrous,sodium starch glycolate,sodium stearyl fumarate and gelatin capsule were of ade and obtained from DMV International,Merck,DMV International,JRS Pharma and Suheung capsule,respectively.Reductil1was purchad from Abbott Korea Co.(Republic of Korea).All other reagents were of analytical grade and ud without further purification.
2.2.Preparation of inclusion complexes
The inclusion complexes of sibutramine/b-CD were prepared as follows.Briefly,28.0g sibutramine ba was clearly dissolved at a concentration of100mM in aqueous hydrochloride solution(6L, 200mM),and an excessive amount of b-CD(256g)was added to the solution.The sibutramine/b-CD solution was magnetically stirred at358C for3h and cooled down to room temperature. Sodium hydroxide was then added in a molar ratio of1:2versus hydrochloride,and the solution was shaken for3h to precipitate the sibutramine/b-CD inclusion complex.Afterfiltering and vacuum drying,the content of sibutramine ba in the resulting white powder was assayed to calculate the actual stoichiometry ratio of the complex.
2.3.Characterization of sibutramine/b-CD complex
The formed complex was examined using differential scanning calorimetry(DSC),X-ray powder diffraction(XRD),and1H nuclear magnetic resonance(NMR).DSC analysis was carried out using a Perkin Elmer DSC-7System(Perkin Elmer,MA,USA)equipped with a computerized data station(Pyris ries).The thermal behavior was studied by heating the samples(1–2mg)in a aled aluminum pan from20to2008C at a rate of108C minÀ1and under nitrogen purge,using an empty pan aled as reference.X-ray powder diffraction patterns were obtained at room temperature with MAC Science,model M18XHF22diffractometer system equipped with copper(Cu)as anode material and a graphite monochromator using a voltage of45kV and a current of300mA. The diffractograms were recorded in the2u angle range between 58and608and the process parameters were t at:scan step size of 0.028;scan step time of5s.1H NMR spectra were obtained from Varian Gemini NMR(300MHz)at room temperature.The samples were dissolved in dimethylsulfoxide(DMSO)-d6.
2.4.Stability studies
The effect of inclusion complexation on the stability of sibutramine ba was examined under the following stress conditions.The thermal stability of sibutramine powder samples was examined using
a thermostatically controlled stability chamber(PSC022,SANYO Gallenkamp,UK)maintained at608C/ 75%relative humidity(RH)for7days.Thermal stability of sibutramine samples in aqueous solution was also examined using the same chamber.The solutions equivalent to1mg mLÀ1sibutramine in0.038M sodium acetate buffer(pH 5.2)were prepared in a aled glass vial and stored at608C for2weeks[19]. The photochemical stability was investigated by spreading the powder samples in a transparent Petri dish with a thickness of less than3mm.The samples were then expod to UV or visible light at 258C in accordance to ICH guideline[20].The light source with an overall illumination of about1.2million lux h and an integrated near ultraviolet energy of about200Wh mÀ2was ud in a photostability chamber(PSC062,SANYO Gallenkamp,UK).
2.5.High performance liquid chromatography(HPLC)
At predetermined time intervals,samples were taken and dissolved with methanol,filtered through0.45m m membrane filters and analyzed by HPLC.The HPLC system(Waters,Milford, MA,USA)compod of2695paration module,2695auto sampler,2996PDA detector,and Empower2chromatography manager for processing data.A C18column(Hypersil BDS, 250mmÂ4.5mm i.d.,5m m particle size,Thermo Hypersil-Keystone,Germany)was ud at358C with aflow rate of 1.0mL minÀ1.The mobile pha was a35:65(v/v)mixture of phosphate buffer(pH6.0)and
联系的普遍性
acetonitrile,and the elution of sibutramine was detected at225nm.
2.6.Formulation of sibutramine/b-CD inclusion complex
The composition of the capsule dosage form containing sibutramine/b-CD inclusion complex was determined bad on compatibility screening between sibutramine/b-CD and candidate excipients.Citric acid was ud to adjust the pH becau sibutramine ba is very soluble in acidic solution.In addition, citric acid is known to increa the aqueous solubility of sibutramine ba in solid dispersion systems[17,18].The composition of the sibutramine/b-CD complex formulation includes sibutramine/b-CD inclusion complex,lacto monohy-drate,citric acid,sodium starch glycolate and sodium stearyl fumarate in size2hard gelatin capsules.The capsule dosage form was manufactured through dry granulation process using a roller compactor under KGMP condition.The amount of sibutramine ba per capsule was12.55mg.
2.7.In vitro dissolution testing
In vitro dissolution tests were performed using Ph.Eur7.0, dissolution apparatus II with900mL buffer(pH1.2,4.0,and6.8)as a dissolution medium at37.0Æ0.58C.The speed of the paddle was fixed to50rpm.The sibutramine/b-CD complex formulation and Reductil1containing the equivalent do of1
2.55mg sibutramine ba were inrted into a sinker and placed in a dissolution tester(VK 7025,Vankel,USA).At predetermined time intervals,5mL of the medium was centrifuged at13,000rpm,and the drug concentration in the supernatant was then analyzed using the same HPLC system as described above.A C18column(Capcellpak UG120,150mmÂ4.5mm i.d.,5m m particle size,Shiido,Japan)was ud at358C at a flow rate of1.5mL minÀ1.The mobile pha was a64:35:10(v/v/v) mixture of phosphate buffer(pH3.0),acetonitrile,and tetrahydrofu-ran.
2.8.Pharmacokinetics
A single-center,randomized,open-label,2-period,comparative crossover study was conducted with2week washout period in accordance with ethical principles and standards described in the Declaration of Helsinki and the International Conference on Harmonization(ICH)/Good Clinical Practice(GCP)and with the approval of the institutional review board of Asan Medical Center,
Y.Y.Hwang et al./Journal of Industrial and Engineering Chemistry18(2012)1412–14171413
Seoul,Republic of Korea.All subjects received a single12.55mg oral do of sibutramine ba(sibutramine/b-CD formulation)and 15mg oral do of sibutramine hydrochloride monohydrate(the commercial product)equivalent to sibutramine ba12.55mg.All subjects were adm
itted to the study center on the evening before the day of drug administration.The next morning,they received a single oral do of the sibutramine/b-CD complex formulation or Reductil1according to the randomized schedule.An indwelling angiocatheter with a normal saline lock was inrted into a brachial vein.Blood samples were collected immediately before drug administration(baline)and at0.5,1,2,3,4,5,6,8,10,12, 24,48and72h after drug administration.After1mL blood from the angiocatheter was discarded,6mL blood was collected in a heparinized tube.The plasma was parated by centrifugation at 1800Âg at a temperature of48C within10min of collection and was stored atÀ708C until analysis[21,22].
2.9.Blood sample analysis
Plasma concentrations of two active metabolites(denoted M1 and M2)were analyzed using a modified high-performance liquid chromatographic mass spectrometric(LC–MS-MS)method[22–24]validated according to ICH guideline[25]and in accordance with Good Laboratory Practice(GLP)environment.For the analysis, 4mL tert-butylmethylether and30m L desipramine methanol solution(50ng mLÀ1)as the internal standard were quentially added to0.5mL plasma.After vigorous vortexing for20min in a mixer(C-SGM,JISCO,Republic of Korea),the mixture was centrifuged,and the organic pha was transferred to a clean glass tube and dried under aflow o
f nitrogen gas(MG-2100,Eyela, Japan).The dried residue was reconstituted with300m L of50% acetonitrile,and10m L aliquot of this solution was injected into the Thermo Finningan LC–MS-MS system(Thermo Electron,Japan). The compounds were parated on a Capcell pak C18column (150mmÂ2.0mm,5m m,Shiido,Japan)with a45:55(v/v) mixture of acetonitrile and water(adjusted to pH 4.0with trifluoroacetic acid)as a mobile pha.The column was heated to 308C,and the mobile pha was eluted at0.2mL minÀ1.The mass spectrometer with an electrospray source was run in the positive ion mode,and m/z266,252,and267were monitored M1,M2,and internal standard,respectively.轮胎排名
2.10.Pharmacokinetic data analysis and statistical analysis
The concentrations of M1and M2in plasma and their pharmacokinetic characteristics were compared in two sibutra-mine formulations becau the metabolites of sibutramine were pharmacologically active compared with the parent drug,sibu-tramine[26].The area under the drug concentration–time curve from zero to the last measurement(AUC last)or infinity(AUC inf)and the half-life(t1/2)were calculated using a noncompartmental analysis(WinNonlin Pharsight Corporation,US).Values for C max and T max were estimated directly from the obrved plasma concentration–time data[27].Levels of statistical significance (p<0.05)were assd using the Student-t-test between the two means for unpaired data.All data are expresd as the mean Æstandard deviation(S.D.)or a
s the median(ranges)for T max.
3.Results and discussion
3.1.Characterization of sibutramine/CD complex
The1H NMR spectrum of the sibutramine/b-CD complex is shown in Fig.1.The characteristic peaks of the aromatic ring of sibutramine ba were found at7–8ppm,and b-CD has the characteristic proton peaks at4–6ppm.The theoretical peak ratio was1:7for a1:1complex.From the1H NMR spectra for the
optimized sibutramine/b-CD inclusion complex,the molar ratio of sibutramine ba and b-CD in inclusion complex was expected to be1:2.The DSC thermograms of sibutramine ba,b-CD,a1:2 mixture of sibutramine ba and b-CD,and the sibutramine/b-CD inclusion complex were shown in Fig.2.Crystalline powder of男女对唱歌曲大全
sibutramine ba appeared a sharp endothermic melting peak
around538C.The broad endothermic peak of b-CD was obrved around1208C.This peak was likely to be associated with the loss of
bound water.Their physical mixture clearly exhibited two
endothermic peaks corresponding to sibutramine ba and b-CD,though the bound water peak slightly decread.This result
eduyunindicates that there was no strong interaction between sibutra-
mine ba and b-CD in the mixture.Interestingly,the sibutramine/ b-CD complex has no apparent endothermic peaks,indicating the effective inhibition of crystalline phas of individual molecules
through the formation of inclusion complex between sibutramine
and b-CD.The XRD patterns of sibutramine ba,b-CD,a1:2 mixture of sibutramine ba and b-CD,and the sibutramine/b-CD inclusion complex are shown in Fig.3.The prence of inten and sharp peaks in the XRD of sibutramine ba(2u=14.2,20.4,and 24.38),and b-CD(2u=9.1,12.8,23.1,27.1,and34.98)indicates that the compounds existed in a crystalline form.Their physical mixture exhibited the superposition of the individual components. The sibutramine/b-CD complex showed distinctive peak
patterns Fig.1.1H NMR spectrum of sibutramine/b-CD inclusion complex in
DMSO-d6.
Fig.2.DSC thermograms of sibutramine ba(A),b-CD(B),the physical mixture(C), and the inclusion complex of sibutramine and b-CD(D).
Y.Y.Hwang et al./Journal of Industrial and Engineering Chemistry18(2012)1412–1417 1414
with relatively broad bands,indicating the structural changes of sibutramine and b -CD as they form an inclusion complex.
3.2.Thermal and photochemical stability
The inclusion complexation with b -CD can affect the physical and chemical properties of guest molecules (e.g.,aqueous solubility and stability).Although it can negatively affect the chemical stability of guest molecules,the formation of an inclusion complex usually leads to the retardation of degradation process [28–31].As shown in Fig.4,the effect of the elevated temperature on the degradation of sibutramine powder was examined at 608C for 7days.All of the samples showed excellent stability in the form of powder.Particularly,there was no obvious change of the sibutramine/b -CD complex,whereas the degradation products of sibutramine ba incread by about 0.13%in a week.The thermal
stability of sibutramine was also investigated in aqueous solution (acetate buffer at pH 5.2).Although sibutramine ba had a dramatic degradation of more than 15%,the sibutramine/b -CD inclusion complex showed no significant change for 2weeks at 608C.Extended stability test showed that there was no further change in the sibutramine/b -CD inclusion complex for 2months.Sibutramine/b -CD inclusion complex was very stable against UV illumination though sibutramine itlf quickly degrades when expod to UV light.From the HLPC chromatograms from the stability studies,the main degradation product was identified to be desmethyl sibutramine (M1).It is likely that the dimethylamino group of sibutramine was positioned in the cavity of b -CD and thus protected from chemical and photochemical stress.The stability of sibutramine against elevated temperature,hydrolysis,and light was effectively incread through its complexation with b -CD.
Molecular modeling was performed using the X-ray structure of b -CD as an initial conformation [32].As mentioned above,b -CD and sibutramine form complexes at a molar ratio of 2:1,making three possible types of b -CD dimers.Molecular mechanical analysis on both of the (R)isomer and (S)isomers of sibutramine were carried out to obtain the energetically favorable conforma-tion.The Surflex-Dock in SYBYL8.1.1was ud to perform docking studies to obtain the complex structure between b -CD dimer and sibutramine.The complex formation of one sibutramine molecule with face-
to-face b -CD dimers showed the optimal docking mode (Fig.5A).In addition,the docking modes of the (R)and (S)forms of sibutramine were similar to each other.The molecular structure of the 2:1inclusion complex between b -CD and sibutramine is shown in Fig.5B.
3.3.In vitro dissolution study
The in vitro relea profiles of the sibutramine/b -CD complex formulation and Reductil 1were compared (Fig.6)using the difference factor (f 1)and similarity factor (f 2),as defined by the
following equation [33,34]:f 1=[P (R t ÀT t )/P
R t ]Â100and
f 2=50Âlog{[1+P
(R t ÀT t )2]À0.5Â100},where n is the number of time points,and T t and R t are percentage releas at time point (t )for the sibutramine/b -CD complex and Reductil 1,respectively.In general,to ensure a similar correlation between the profiles,f 1should be in the range of 0–15,and f 2in the range of 50–100.As shown in Table 1,f 1values between the sibutramine/b -CD complex and Reductil 1were 5.02,5.50,19.83and 18.32,at
pH
Fig.3.X-ray powder diffraction spectra of sibutramine ba (A),b -CD (B),the physical mixture (C),and the inclusion complex of sibutramine and b -CD
(D).
Fig.4.Thermal and photochemical stability of sibutramine powder at 608C and 75%RH and sibutramine solution in acetate buffer (pH 5.2)at 608C (A),and UV or visible light at 258C
(B).
Fig.5.Schematic reprentations of a b -CD dimer (A)and the 2:1inclusion complex between b -CD and sibutramine (B).
Table 1
Difference and similarity factors between the sibutramine/b -CD complex formulation and Reductil 1.
三年级列式计算
Dissolution medium
pH 1.2
pH 4.0
pH 6.8
Water Difference factor (f 1) 5.02 5.5019.8318.32Similarity factor (f 2)
59.63
66.57
45.35
40.51
Y.Y.Hwang et al./Journal of Industrial and Engineering Chemistry 18(2012)1412–1417
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1.2, 4.0,6.8and water,respectively.Furthermore,they had f 2values of 59.63,66.57,45.35and 40.51at pH 1.2,4.0,6.8and water,respectively.Thus,the sibutramine/b -CD complex and Reductil 1showed a similar correlation of dissolution profiles at pH 1.2and 4.0but a different correlation at pH 6.8and deionized water,caud by the difference of dissolution rate during the first 10min.The different correlation at pH 6.8and deionized water ems to result from the decread micro-environmental pH,which is caud by free hydrochloride relead from Reductil 1,around sibutramine in dissolution medium.
3.4.Pharmacokinetics
Fig.7shows the mean plasma concentration–time profiles for M1,M2,and M1+M2following the oral administration of the sibutramine/b -CD complex and Reductil 1at an equivalent do of 12.55mg sib
utramine ba in humans.The pharmacokinetic
parameters for M1,M2and M1+M2are summarized in Table 2.The in vivo effects of sibutramine are predominantly the results of the action of the two metabolites [12,22,26,35].In human subjects,sibutramine is rapidly metabolized to an N -mono-desmethyl compound (desmethylsibutramine,M1)and an N ,N -didesmethyl compound (didesmethylsibutramine,M2).The AUC and C max values of M1and M2for the sibutramine/b -CD complex were not significantly different from tho for Reductil 1.The mean log-transformed ratios of the parameters and their confidence intervals were all within the predefined bioequivalence range of 80–125%[36].The pharmacokinetic parameters calculated for the active moiety by the summation of M1and M2concentrations were also found to be biologically equivalent in considering that the effects of sibutramine are predominantly the result of the actions of M1and M2[11,37].Sibutramine hydrochloride monohydrate and sibutramine ba may have different pharma-cokinetic characteristics as a result of potentially
different
Fig.6.Dissolution profiles of sibutramine from the sibutramine/b -CD complex formulation and Reduct
il 1at pH 1.2(A);pH 4.0(B);pH 6.8(C);deionized water (D).Each value reprents the mean ÆS.D.(n =6).
Table 2
Pharmacokinetic properties of desmethylsibutramine (M1),didesmethylsibutramine (M2)and the summation of desmethylsibutramine and didesmethylsibutramine (M1+M2)after oral administration of the sibutramine/b -CD complex and Reductil 1in 22healthy male humans.Each value reprents the mean ÆS.D.
Parameters
AUC last (ng h mL À1)
AUC inf (ng h mL À1)
C max (ng mL À1)
T max (h)
t 1/2(h)M1
Complex 40.6Æ17.843.62Æ19.42 2.81Æ1.02 3.5Æ1.117.4Æ3.85Reductil 1
38.12Æ17.44
40.9Æ18.87
2.69Æ1.06
去韩国3.28Æ1.06
17.39Æ3.37
Ratio (90%CI) 1.07(96.2–118.4)
1.07(96.0–118.7)
1.06(97.2–115.5)
M2
Complex 127.5Æ31.55137.11Æ35.467.36Æ2 3.63Æ118.98Æ3.42Reductil 1
113.73Æ22.58
122.68Æ24.89
6.58Æ1.6
3.37Æ1.21
19.07Æ3.75
Ratio (90%CI) 1.11(107.6–114.6)
1.11(106.8–114.5)
1.11(105.0–117.2)
M1+M2Complex 168.24Æ38.76180.52Æ45.2910.12Æ2.2 3.5Æ0.9118.52Æ3.32Reductil 1
151.97Æ29.61
163.33Æ34.41
9.1Æ1.83
3.42Æ1.21
18.49Æ3.3
Ratio (90%CI)
1.10(105.5–114.6)
1.10(104.8–114.7)
1.11(105.3–116.4)
Y.Y.Hwang et al./Journal of Industrial and Engineering Chemistry 18(2012)1412–1417
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