`
`Exhibit 1006
`
`L. M. L. Stolk et al., Dissolution Profiles of
`Mesalazine Formulation in Vitro, 12
`PHARMACEUTISCH WEEKBLAD SCIENTIFIC
`EDITION 200 (1990) (“Stolk”)
`
`

`
`Dissolution profiles of mesalazine formulations in vitro
`L.M.L. Stolk, R. Rietbroek, E.H. Wiltink and J.J. Tukker
`
`Introduction
`Sulfasalazine, a conjugate of mesalazine (5-
`aminosalicylic acid; 5-ASA) and sulfapyridine,
`has been used successfully for four decades in the
`treatment of inflammatory bowel disease. Re-
`cently, it has been demonstrated that mesalazine
`is the therapeutically active moiety of sulfasal-
`azine. In the colon the azo bond is dissociated by
`intestinal bacteria and mesalazine is set free.
`Plain mesalazine is totally absorbed in the upper
`part of the intestine. Therefore, pharmaceutical
`formulations have been designed, which can
`transport mesalazine
`undisturbed
`through
`stomach, duodenum and proximal jejunum and
`deliver high concentrations of mesalazine selec-
`tively at the inflammatory sites of the distal
`small intestine and colon [1]. These formulations
`are Pentasa | which is a 'slow-release' formu-
`lation and Asacol | and Salofalk | which are
`tablets with an acid-resistant coating.
`Pentasa | tablets contain 250 mg of mesalazine
`in microgranules (0.7-1 ram), which are coated
`with a semi-permeable membrane of ethylcellu-
`lose. Mesalazine should in principle be gradually
`released from the ethylcellulose-coated granules
`at either acid or alkaline pH and release should
`take place over the whole length of the intestine.
`Asacol | tablets contain 400 mg mesalazine and
`the tablets are coated with an acrylic resin
`(Eudragit | S), which dissolves above pH 7.0.
`Thus, in vivo it should be transported intact until
`it reaches the ascending part of the colon where
`the intraluminal pH rises above 7 and mesal-
`azine is liberated.
`Salofalk | contains 250 mg mesalazine and so-
`dium carbonate as buffering and solubilizing
`agent. The tablet has an outer coating with a
`semi-permeable membrane of ethylcellulose and
`an inner coating with an acrylic resin (Eudragit |
`L). This acrylic resin dissolves above pH 5.6. In
`this formulation mesalazine is meant to be re-
`leased in highly dispersed form in the distal
`small bowel and the colon.
`
`In our study we have investigated in vitro re-
`lease of mesalazine from Pentasa | Asacol | and
`Salofalk | tablets at pH 1.0, pH 6.0 and pH 7.5,
`intended to represent the acidity of the stomach,
`small and large bowel, respectively.
`The only official testing methods utilize the
`paddle
`and
`the
`rotating-basket dissolution
`apparatus as described in the United States
`Pharmacopeia XXI
`[2]. The
`rotating-basket
`method has inherent weaknesses, including poor
`mechanical
`stability,
`simultaneous
`disinte-
`gration and dissolution and poor homogeneity
`because of insufficient stirring. As an alternative
`for the pharmacopoeial test the flow-through or
`column-type method has been proposed [3 4]. We
`used a closed-column type method, which is kin-
`etically equivalent to a stirred-vessel system,
`but will not affect dissolution kinetics as long
`as
`'sink conditions' prevail. However,
`this
`method has the advantage of being largely auto-
`matic. No wetting agents were added to the dis-
`solution media, because there were no indi-
`cations that wettability presented a problem.
`Methods
`Materials
`Bufa
`from
`obtained
`was
`Mesalazine
`(Castricum, the Netherlands, batch no. 22K88).
`All other
`chemicals were
`analytical-grade
`quality from Merck (Darmstadt, FRG).
`Pentasa |
`tablets
`(Gist-brocades, Delft,
`the
`Netherlands, batch no. 86BG40), Asacol | tablets
`(Cedona, Haarlem, the Netherlands, batch no.
`BN87125) and Salofalk |
`tablets
`(Tramedico,
`Weesp, the Netherlands, batch no. 88G18) were
`obtained through OPG (Utrecht, the Nether-
`lands).
`Dissolution apparatus
`A Dissotest flow-through dissolution apparatus
`was used (Sotax, Basle, Switzerland; imported by
`Proton-Wilten, Etten-Leur,
`the Netherlands).
`
`Keywords
`Dissolution
`Mesalazine
`Tablets, controlled release
`Dr. L.M.L. Stolk (correspondence)
`and E.H. Wiltink: Pharmacy
`Department, Academic Medical
`Centre, Meibergdreef 9, 1105 AZ
`Amsterdam, the Netherlands.
`R. Rietbroek and Dr. J.J. Tukker:
`Department of Biopharmaceutics,
`Faculty of Pharmacy, Utrecht
`University, Catharijnesingel 60,
`3511 GH Utrecht, the
`Netherlands.
`
`Stolk LML, Rietbroek R, Wiltink EH, Tukker JJ. Dissolution profiles of mesalazine
`formulations in vitro. Pharm Weekbl [Sci] 1990;12(5):200-4
`Abstract
`In vitro dissolution profiles of three controlled-release mesalazine formulations were
`determined at pH 1.0, 6.0 and 7.5. A closed-column type dissolution apparatus was used. A
`reproducible gradual dissolution profile was seen for Pentasa| at all pH values. Dissolution
`starts immediately and is complete after 20 h. Dissolution profiles at pH 1 and pH 7.5 are
`much alike and dissolution is faster than at pH 6. The behaviour of Asacol | at different pH
`values corresponds with the expectations: no release at pH 6 and pH 1, fast release at pH
`7.5. Dissolution starts after 1 h and is complete after 3 h. Mesalazine release from Salofalk |
`tablets at pH 7.5 and pH 6.0 starts after 2 and 3 h, respectively, and is complete after 5 and
`10 h. However, after a long lag-time (10 h) mesalazine is also released from Salofalk | tablets
`at pH 1 and dissolution is complete after 23 h.
`Accepted 26 July 1990.
`
`200
`
`Pharmaceutisch Weekblad Scientific edition
`
`12(5) 1990
`
`

`
`Figure 1
`Dissolution pro-
`files at pH 7.5
`(mean + SD;
`n = 5)
`
`Mesalazine dissolved
`
`%
`
`Mesalazine dissolved
`
`%
`
`~ PENTASA in SIF pH 7.5
`(=250 mg 5-ASA)
`
`ASACOL in SIF pH 7.5
`(=400 mg 5-ASA)
`SALOFALK in SIF pH 7.~
`
`~ "~--T_ ~ (=250 mg 5-ATS~..~I-.--'--~
`I f
`
`SALOFALK in 0.1 N HCI
`
`~ . ~ i T J ~ !
`
`
`
`PENTASA in O.1 N HCI
`
`1
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12 14 16
`
`18 20 22 24 26 Hours
`
`8
`
`10 12 14 16 18 20 22 24 26 Hours
`
`0
`
`4
`
`6
`
`0
`2
`Figure 3
`Dissolution profiles at pH 1.0 (mean +_ SD; n = 3)
`
`The dissolution apparatus consisted of a dissol-
`ution cell (standard cell, i.d. 22,6 ram), a pump, a
`3-1 or 5-1 vessel with dissolution medium and a
`spectrophotometer (Unicam SP 1800, Philips,
`Eindhoven, the Netherlands) with a 1 cm quartz
`flow-through cell (174-QS, Hellma, The Hague,
`the Netherlands). The cumulative dissolution
`curve was drawn by a chart recorder (Unicam AR
`25, Philips). The dissolution cell and the vessel
`were placed
`in a
`thermostatically controlled
`
`Mesalazine dissolved
`
`0/o
`
`Figure 2
`Dissolution pro-
`files at pH 6.0
`(mean + SD;
`n =3)
`
`SALOFALK in SIF pH 6.0
`
`PENTASA in SIF pH 6.0
`
`0
`
`2
`
`4
`
`6
`
`8 '10' 12' 14' 16' 18 20 22 24 26 Hours
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`The tablets were hung
`waterbath at 37.0+0.5~
`in a wire frame in the small testing chamber of
`20 ml capacity (22.6 mm diameter). The conical
`part of the testing chamber was filled with glass
`beads (diameter 1 mm) to ensure a uniform dis-
`tribution of the flow over the entire cross section
`of the cell. The dissolution medium was pumped
`around at a flow rate of 31.0_40.5 ml/min from
`the vessel with the dissolution medium through
`the spectrophotometer and then through the dis-
`solution cell back into the vessel. The solvent left
`the cell through a glass-fibre filter (Whatman
`GF/D, Proton-Wilten),
`that retained particles
`larger than 2.7 ~m to prevent removal of un-
`dissolved particles.
`
`Dissolution media
`The following dissolution media were used:
`- simulated intestinal fluid USP XXI (SIF) with-
`out pancreatine, pH 7.5;
`-the
`same solution as mentioned above but
`brought
`to pH 6.0 with phosphoric acid,
`pH 6.0;
`- HCI 0.1 M in water, pH 1. In order to obtain
`'sink conditions' 5 ] dissolution medium was
`used for Asacol | and 3 1 for Pentasa | and
`Salofalk |
`
`Assay of mesalazine
`The mesalazine content of the dissolution me-
`dium was determined by measuring UV absorp-
`tion at 331 nm (pH 7.5 and pH 6.0) and at 304 nm
`(pH 1.0), respectively. The linearity and the pre-
`cision of the assay were determined by measur-
`ing the absorbance of mesalazine of known con-
`centrations four times. The detector response
`was linear in the concentration range employed:
`0-85 rag/1. The calibration lines can be described
`
`12(5) 1990
`
`Pharmaceutisch Weekblad Scientific edition
`
`201
`
`

`
`asacol
`
`"0
`0
`
`'-
`
`^ 1 |
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`2
`
`4
`
`!
`6
`
`I
`0
`8
`(hrs
`time
`--->
`Figure 4
`Dissolution profiles (computer fit) for individual Asacol | tablets. []: pH 7.5;
`0: pH 7.5 after 2 h pretreatment at pH 1.0
`
`0
`
`120
`
`salofalk
`
`~
`,A
`
`|
`
`100
`80
`60
`40
`20
`0
`
`0
`
`10
`
`20
`30
`--->
`time
`(hrs)
`Figure 5
`Dissolution profiles (computer fit) for individual Salofalk | tablets.
`O: pH 1.0; 0: pH 7.5 after 2 h pretreatment at pH 1.0; m: pH 6," D: pH 7.5
`
`pentasa
`
`o
`
`,A
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`10
`
`0
`30
`20
`(hrs)
`time
`--->
`Figure 6
`Dissolution profiles (computer fit) for individual Pentasa | tablets.
`O: pH 1.0; m: pH 6.0; [Z: pH 7.5
`
`by: y = 0.021 x + 0.007 (pH 7.5); y = 0.0145 x -
`0.0004 (pH = 6.0); y = 0.024 x + 0.004 (pH 1.0)
`[y UV absorbance; x mesalazine concentration
`(mg/l); r 0.999].
`
`Dissolution profiles
`Dissolution profiles were determined at pH 7.5,
`pH 6.0 and pH 1.0 for Asacol | Pentasa | and
`Salofalk | tablets with the Dissotest apparatus.
`Dissolution profiles of Asacol | and Salofalk |
`tablets were also determined at pH 7.5 after 2 h
`previous immersion in 0.1 M HC1 in order to
`study the influence of artificial gastric fluid. The
`tablets were placed in the 'rotating basket ap-
`paratus', described in the USP XXI filled with i 1
`0.1 M HC1 [2]. The stirring speed was 100 rev/
`min. After 2 h the tablets were transferred to the
`Dissotest apparatus with dissolution medium
`pH 7.5. Because mesalazine is released from
`Pentasa | at either alkaline or acid pH, Pentasa |
`tablets were not included in this experiment.
`Computer fit
`An emperical function was used to describe dis-
`solution profiles: the RRSBW-distribution func-
`tion [5]. The RRSBW-distribution functions were
`calculated with the program SYSTAT (SYSTAT,
`Avanston, Illinois, USA) on a Macintosh SE/30
`computer.
`Stability of mesalazine in dissolution media
`Stability of mesalazine in the dissolution me-
`dia was determined by dissolving 250 mg mesal-
`azine in 3 1 of the dissolution media. After 24 h
`in the dissolution apparatus under the same
`experimental conditions as described above, the
`mesalazine concentrations were measured. Con-
`centrations were measured with UV absorption
`and with a reversed-phase high pressure liquid
`chromatographic (HPLC) method described in
`the literature [6].
`Solubility of mesalazine at various pH values
`To 50 ml of each of the three freshly prepared
`dissolution media I g mesalazine was added. The
`resulting suspension was shaken during 10 min.
`Then the suspension was placed in a thermostati-
`cally controlled water bath of 37.0 + 0.5~
`l-ml
`Samples were
`taken after 4 and 24 h. The
`samples were filtered through a 0.8 ~m mem-
`brane
`filter
`(Sartorius,
`Etten-Leur,
`the
`Netherlands). Mesalazine concentrations were
`measured with HPLC.
`Results
`Dissolution
`The concentration of mesalazine in the dissol-
`ution media after 4 h was (mean •
`n = 3):
`11.0+0.2 g/1 (pH 1); 2.3+0.1 g/1 (pH 6); 6.5+
`0.1 g/1 (pH 7.5). After 24 h were 9.9_+0.1 g/1
`(pH 1); 2.3_+0.1 g/1 (pH 6); 6.1+0.1 g/1 (pH 7.5).
`This means that all experiments were carried
`out under 'sink conditions'.
`The dissolution profiles at pH 7.5, pH 6.0 and
`pH 1.0, respectively, are demonstrated in Fig-
`ures 1, 2 and 3. Dissolution profiles for Asacol |
`Salofalk | and Pentasa | at different pH values
`were constructed with the help of a computer fit.
`They are demonstrated in Figures 4, 5 and 6 (in-
`dividual tablets) and in figures 7, 8 and 9 (mean
`curves).
`Pentasa shows a reproducible gradual dissol-
`
`202
`
`Pharmaceutisch Weekblad Scientific edition
`
`12(5) 1990
`
`

`
`Asacol
`
`v
`
`100
`
`o
`m
`o
`
`A
`
`I
`
`80
`
`6O
`
`4O
`
`20 _J.L
`
`0
`
`2
`
`4
`
`Figure 7
`Dissolution profiles (computer fit) for Asacol | (mean •
`Q: pH 7.5 after 2 h pretreatment at pH 1.0; []: pH 7.5
`
`6
`--->
`
`i
`
`10
`(hrs)
`
`8
`time
`
`n = 5).
`
`Salofalk
`
`140
`
`120
`100
`8o
`
`60
`
`40
`
`20
`
`0
`
`,
`
`'
`
`i
`
`0
`
`10
`
`Figure 8
`n = 3).
`Dissolution profiles (computer fit) for Salofalk | (mean •
`O: pH 1.0; Q: pH 7.5 after 2 h pretreatment at pH 1.0; m: pH 6.0;
`~: pH 7.5
`
`20
`time
`
`(hrs)
`
`--->
`
`ution profile at all pH values. Dissolution starts
`immediately and is almost complete after 20 h.
`Dissolution profiles at pH 1.0 and pH 7.5 are
`much alike and dissolution is faster than at
`pH 6.0. Dissolution of Salofalk | at pH 1.0, pH 6.0
`and pH 7.5 starts after about 10, 3 and 2 h,
`respectively, and is complete after 23, 10 and 5 h.
`Salofalk |
`tablets release mesalazine more rap-
`idly when pH is rising.
`At pH 1.0 and pH 6.0 no dissolution occurred
`from Asacol | tablets. At pH 7.5 dissolution starts
`after 1 h and is complete after 3 h.
`A large variation of the dissolution profiles
`was seen for the individual Salofalk | tablets at
`
`pH 7.5 and at pH 6.0 and for the Asacol | tablets
`at pH 7.5. The dissolution curves determined at
`pH 7.5 with or without pretreatment with 0.1 M
`HC1 were almost identical. However, after im-
`mersion in 0.1 M HC] dissolution started some-
`what later.
`Stability studies
`After 24 h under experimental conditions
`mesalazine concentrations measured by HPLC
`analysis were (n = 2): 95.8% (pH 1), 94.7% (pH
`6.0), 95.6% (pH 7.5) of the original mesalazine
`level. Measured by UV absorption the figures
`are: 99.9% (pH 1), 111.9% (pH 6.0) and 100.1%
`(pH 7.5). A brown colouring of the dissolution
`medium occurred at pH 6 and pH 7.5 within 24 h.
`Discussion
`from
`release
`The
`reproducible mesalazine
`Pentasa |
`tablets is due to the principle of dif-
`fusion-regulated release from the ethylcellulose-
`coated microgranules. The slower dissolution
`rate at pH 6.0 in comparison with pH 7.5 and
`pH 1.0 can be explained by the lower solubility of
`mesalazine at pH 6.0, being nearer the isoelec-
`tric point of mesalazine (pH 4.3), where the com-
`pound's solubility is lower than at the other pH
`values used. The shape of the curve indicates fast
`desintegration into granules and gradually de-
`creasing dissolution rate [5].
`The
`release mechanism of Asacol |
`and
`Salofalk | tablets is a dissolution-regulated reser-
`voir system. After dissolution of the coating, re-
`lease is very fast. A possible explanation for the
`large variation of the dissolution profile of the in-
`dividual Asacol | and Salofalk | tablets could be
`the variation of the thickness of the coating layer
`of the metacrylic metacrylate esters. The typical
`sigmoid shape of the curves indicates that desin-
`tegration and dissolution are of comparable rate
`[5]. The behaviour at the different pH values of
`Asacol | corresponds with the expectations: fast
`release at pH 7.5 and no release at pH 6.0 and
`pH 1.0. As expected, we found fast release for
`Salofalk | at pH 7.5 and pH 6. However, after a
`long lag-time release was also measured at
`pH 1.0. Perhaps the coating is somewhat perme-
`able at lower pH Values. After fluid penetration
`the presence of sodium carbonate may be re-
`sponsible for fast dissolution. No large changes of
`the dissolution profiles at pH 7.5 for Asacol | and
`Salofalk | were seen after previous immersion in
`0.1 M HC1. We have no explanation for the
`longer lag-time of Asacol | and Salofalk | after
`pretreatment with 0.1 M HC1.
`The plateau value of about 110% at pH 6.0 can
`be explained by the forming of a degradation
`product of mesalazine with high UV absorption
`at 331 nm. The same phenomenon was also found
`by Terpstra et al. [7]. They determined dissol-
`ution profiles during 24 h with
`the paddle
`method (USP XXI) at pH 2.0, 6.0 and 7.5. The re-
`sults for Pentasa | were essentially identical to
`our results. For Asacol | and Salofalk |
`the dissol-
`ution was significantly faster than in our exper-
`iments. This might be caused by use of the paddle
`method, thus putting more stress on the tablets
`and decreasing desintegration
`time of
`the
`
`12(5) 1990
`
`Pharmaceutisch Weekblad Scientific edition
`
`203
`
`

`
`Pentasa
`
`...
`
`120
`
`100
`
`80
`
`60
`
`40
`
`2O
`
`,A
`
`0
`
`0
`
`3O
`20
`time
`(hrs)
`--->
`Figure 9
`Dissolution profiles (computer fit) for Pentasa | (mean • SD; n = 3).
`O: pH 1.0; R: pH 6.0; [~: pH 7.5
`
`10
`
`tablets. Duchateau et al., determined dissolution
`profiles with the USP rotating-basket method at
`pH 5.0; 5.5; 6.0; 6.5; 7.0 and 7.5 during 3 h [8].
`The results of this investigation are in agree-
`ment with our results. However, caution should
`be exercised in extrapolation of results in vitro
`
`to the situation in vivo. Recent investigations in-
`dicate pH values that deviate from the generally
`assumed figures: 6.6 to 7.5 in the small intestine
`and 6.4 to 7.0 in the colon. Moreover, a large
`interindividual variation was measured [9].
`References
`1 Jarnerot G. Newer 5-aminosalicylic acid based drugs in
`chronic inflammatory bowel disease. Drugs 1989;37:73-
`86.
`2 Anonymous. United States Pharmacopeia XXI.
`Rockville: United States Pharmacopeial Convention,
`1989.
`3 Langenbucher F, Rettig H. Dissolution rate testing with
`the column method: methodology and results. Drug Dev
`Ind Pharm 1977;3:241-63.
`4 FIP Working group V Dissolution Tests. Guidelines for
`dissolution testing of solid oral products. Pharm Ind
`1981;43:334-43.
`5 Langenbucher F. Parametric representation of dissol-
`utiou curves by the RRSBW distribution. Pharm Ind
`1976;38:472-7.
`6 Lee EJD, Bang SB. Simple and sensitive high-perform-
`ance liquid chromatograph assay for 5-aminosalicylic
`acid and acetylaminosalicylic acid
`in serum.
`J
`Chromatogr 1987;431:300-4.
`7 Terpstra IJ, Bavelaar JF, Klooster NTM, Groenendaal
`JW, Hespe W. In vitro dissolution of 5-aminosalicylic
`acid delivering compounds [Abstract]. 13th Inter-
`national Congress of Gastroenterology. Rome, Sept
`1988.
`8 Duchateau A, Philipse R, Van der Hoek E, Conemans J.
`pH influence on in vitro release of 5-ASA (Mesalazine)
`[Abstract]. Pharm Weekbl [Sci] 1989;11(Suppl E):E1O.
`9 Evans DF, Pye G, Bramley R, Clark AG, Dyson TJ,
`Hardcastle JD. Measurement of gastrointestinal pH
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`
`204
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`Pharmaceutisch Weekblad Scientific edition
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`12(5) 1990