Pharmaceutical Research, Vol. 15, No. 7, 1998
`
`Research Paper
`
`Low Swelling, Crosslinked Guar and
`Its Potential Use as Colon-Specific
`Drug Carrier
`
`Irit Gliko-Kabir,' Boris Yagen,1 Adel Penhasi,2 and
`Abraham Rubinstein 1'3
`
`Received January 27, 1998; accepted March 26, 1998
`
`Purpose. (a) To reduce the swelling properties of guar gum (GG)
`by crosslinking it with glutaraldehyde (GA), while maintaining its
`degradation properties in the presence of typical colonic enzymes,
`(b) to characterize the modified GG and to examine its degradation
`properties in vitro and in vivo, and (c) to assess, by drug probes with
`different water solubilities, the potential of the crosslinked GG to serve
`as a colon-specific drug carrier.
`Methods. GG was crosslinked with increasing amounts of GA under
`acidic conditions to obtain different products with increasing crosslink-
`ing densities. These products were characterized by measuring (a) their
`swelling properties in simulated gastric and intestinal fluids, (b) their
`crosslinking densities, (c) the release kinetics of three different drugs:
`sodium salicylate (SS), indomethacin (Indo) and budesonide (Bud)
`from the crosslinked products into buffer solutions, with or without a
`mixture of galactomannanase and ci-galactosidase, and (d) their in
`vivo degradation in the cecum of conscious rats with and without
`antibiotic treatment.
`Results. Significant reduction in GG swelling properties, in both simu-
`lated gastric and intestinal fluids, was accomplished by its crosslinking
`with GA. The crosslinking density of the modified GG products was
`GA concentration-dependent. The release of SS from crosslinked GG
`discs was completed within 120 minutes. During the same period of
`time and for more than 10 hours the release of Indo and Bud was
`negligible. The release rate of the latter two drugs was enhanced when
`galactomannanase and a-galactosidase were added to the dissolution
`media. Discs made of the crosslinked GG were implanted in the cecum
`of rats and their degradation was assessed after 4 days. The extent of
`degradation was dependent on the amount of GA used for the crosslink-
`ing. After 4 days the same discs were recovered intact from rats exposed
`to antibiotic treatment and from simulated gastric and intestinal fluids.
`Conclusions. Reducing the enormous swelling of GG by crosslinking
`it with GA resulted in a biodegradable hydrogel which was able to
`retain poorly water soluble drugs, such as Indo and BUD, but not
`highly water soluble drugs, such as SS, in artificial gastrointestinal
`fluids. A variety of hydrogels with increasing crosslinking densities
`were produced and tested for their potential use as colon-specific drug
`platforms in vitro and in vivo. Their performance did not depend on
`creating physical barriers by means of compression.
`KEY WORDS: budesonide; colon; colonic delivery; crosslinking;
`glutaraldehyde; guar gum; hydrogel; indomethacin; sodium salicylate.
`
`INTRODUCTION
`
`Typical polysaccharidase activity in the human colon (1)
`could potentially be exploited for the specific delivery of drugs
`via the oral route into this organ. For this purpose, polysaccha-
`ride hydrogels, which can be degraded by colonic enzymes,
`are promising candidates. Enzymatic degradation has been sug-
`gested as a superior targeting mechanism to pH dependent
`carriers (2) because of the shallow pH gradient in the human
`intestine. Although the epithelium of the small intestine shows
`some glycosidase activity (3), the major hydrolysis of glycosidic
`bonds occurs in the colon. Typical colonic enzymes include
`amylase, pectinase, xylanase, p-D-xylosidase, p-D-galactosi-
`dase and P-D-glucosidase. The last three are the most active
`glycosidases (4).
`Various approaches for preparing saccharidic hydrogels as
`colonic drug carriers have recently been described (5). In a
`previous study (6), we demonstrated that a chemical modifica-
`tion of guar gum (GG) with borax does not interfere with the
`ability of GG to be degraded by galactomannanase and et-
`galactosidase, an observation which can be applied to the design
`of enzymatically-controlled colon-specific drug carriers. GG is
`a natural polysaccharide, made of a long 1,4-13-D mannopyrano-
`syl linear backbone (approximately 1,000-1,500 units) to which
`galactopyranosyl residues are attached as single unit
`side chains (7). GG is widely used as a thickening agent in
`the food and pharmaceutical industries, both in its native and
`modified forms (8). As a pharmaceutical adjuvant, it may be
`used for sustained release (9), or for colon-specific purposes,
`as has been previously suggested for acetylated galactomannans
`(10) and GG mixtures with Eudragit (11). The assumption that
`GG is useful in the area of colon-specific drug delivery stems
`from its efficient enzymatic degradation in the human large
`intestine (12,13).
`Although it was found that the modification of GG with
`borax resulted in a product which could be degraded by the
`enzyme mixture of galactomannanase and et-galactosidase, the
`obtained product possessed a higher buffer-uptake capacity, as
`compared to native GG (6). In the present study glutaraldehyde
`(GA) was used as a crosslinker in order to decrease the swelling
`properties of GG. The use of glutaraldehyde to crosslink
`hydroxyl-containing polymers has been reported in the litera-
`ture, primarily for the crosslinking of polyvinyl alcohol (14-16).
`It should be recognized that although GA is toxic, its toxicity
`could be reduced significantly after its crosslinking (17). The
`objectives of the present study were: (a) to crosslink GG with
`GA, (b) to assess the effect of increasing amounts of GA on
`the crosslinked products by physical characterization, (c) to
`examine the degradation properties of the crosslinked GG prod-
`ucts in vitro and in vivo, (d) to assess, by different drug probes,
`whether crosslinked GG can serve as a colon-specific drug
`carrier.
`
`' The Hebrew University of Jerusalem, School of Pharmacy, P.O. Box
`12065, Jerusalem 91120, Israel.
`2 Perio Products Ltd., P.O. Box 23950, Jerusalem 91237, Israel.
`To whom correspondence should be addressed. (e-mail: avri@
`cc.huj i.ac.i1)
`ABBREVIATIONS: Bud, budesonide; GA, glutaraldehyde; GG, guar
`gum; GI, gastrointestinal; Indo, indomethacin; SS, sodium salicylate;
`TS, test solution.
`
`MATERIALS AND METHODS
`
`Materials
`
`GG was purchased from Aldrich, Milwaukee, WI; GA
`was purchased from Merck, Darmstadt, Germany; Galactoman-
`nanase (from Aspargillus niger) was purchased from Fluka
`
`1019
`
`0724-8741/98/0700-1019$15.00/0 © 1998 Plenum Publishing Corporation
`
`Exhibit 1043
`ARGENTUM
`IPR2018-00080
`
`000001
`
`(cid:9)
`(cid:9)
`

`

`1020 (cid:9)
`
`Gliko-Kabir, Yagen, Penhasi, and Rubinstein
`
`BioChemika, Germany). All other materials and reagents were
`purchased from Sigma, St. Louis, MO. Solvents were analytical
`or HPLC grade.
`
`Synthesis of Crosslinked GG Hydrogels
`
`In separate experiments, GG was crosslinked with increas-
`ing amounts of GA as follows (Scheme 1): Four g of GG were
`dispersed for 2 h at 45°C in 800 ml of double-distilled water.
`Concentrated H2SO4 (0.5 ml) was then added, followed by the
`addition of 1.2, 6, 12, 36, 60 and 84 ml (0.1, 0.5, 1, 3, 5 and
`7 equivalents) of glutaraldehyde (25% w/v) solution per half
`mole of the repeating units in the guar gum respectively. It was
`assumed that four hydroxyl groups react with a single molecule
`of GA. The reaction mixture was stirred for 30 minutes and
`kept in a sealed vessel without stirring for an additional 48
`hours at room temperature. The resulting hydrogels (denoted
`as products GG-0.1, GG-0.5, GG-1, GG-3, GG-5 and GG-7,
`respective to the equivalents of GA used) were stirred with 5%
`w/v aqueous solution of NaHSO3 for two hours and then rinsed
`with distilled water (5 L portions) until no traces of GA could
`be detected at 235 nm (polymeric GA) and 280 nm (monomeric
`GA) (Uvikon 930, Kontron Instruments, Switzerland) in the
`rinsing water (18,19). The crosslinked GG products were either
`lyophilized (to give approx. 3.5 g of dry powder of each product)
`
`A
`
`H HO
`
`0
`
`nennose
`
`n
`
`? ?
`HC(CH2)3CH+ 211*-mg=ac
`
`B
`
`RH ?H (cid:9)
`Hc(c.2)3c..z...
`
`r ?Hi
`
`C
`
`OH
`
`OH
`
`[Hci.(CH2)3H
`
`H H
`—0—?CH2)3F— 0—
`OH OH
`
`—OH (cid:9)
`
`HO—
`
`°NH(CH2I-j/C)
`C)3
`
`0—
`—O (cid:9)
`Scheme 1. A schematic representation of GG (A), isomerization of
`GA in acidic pH (B) and the typical reaction between GA and two
`adjacent hydroxyl groups (C).
`
`or kept hydrated in a gel form. To obtain discs, the reaction
`mixture was poured into Petri dishes and left without stirring
`for 48 hours as above. The GG-0.1, GG-0.5, GG-1, GG-3, GG-
`5 and G-7 wet hydrogels obtained were then cut into discs (12
`mm OD, 3-6 mm thick), rinsed with 5% w/v aqueous solution
`of NaHSO3 as described above and oven-dried (45°C). The
`crosslinked products were characterized by measuring their
`equilibrium-weight swelling ratios and their crosslinking
`densities.
`
`Swelling Measurements
`
`Swelling properties of the crosslinked products were mea-
`sured in buffer solutions at different physical states as follows:
`(a) Products GG-1, GG-3 and GG-5 at their maximum swollen
`state (right after synthesis and rinsing, prior to drying). These
`products were placed in simulated USP gastric test solution
`fluid without pepsin, pH = 1.5 (denoted as Gastric TS) or
`simulated USP intestinal test solution fluid without pancreatin,
`pH = 7.4 (denoted as Intestinal TS) (20) until no weight gain
`could be observed (approx. 3 days). The hydrogels were then
`blotted dry, weighed and dried at 45°C until no weight loss
`could be observed (approx. 2 days). Swelling was measured
`gravimetrically and expressed in percent of wet weight over
`the dry weight of the polymers. (b) Products GG-1, GG-3 and
`GG-5 in lyophilized powder state. The powders were sieved
`through a 40 mesh STM sieve and the fraction of 40\80 mesh
`was collected. In separate studies 100 mg of each type of powder
`were dispersed in 20 ml of Gastric TS for 2 hours or Intestinal
`TS for 4 hours, after which time the swollen powders were
`blotted dry, weighed, and dried at 45°C until no weight loss
`could be observed, swelling was measured gravimetrically. (c)
`Products GG-0.1, GG-3, GG-5 and GG-7 in a film form, cut
`into 12 mm 0.D., 3-6 mm thick discs. In this case the swelling
`kinetics of the discs was measured by immersing them in Gastric
`TS or Intestinal TS fluids. At predetermined time intervals the
`discs were weighed and returned to the buffer media until no
`additional weight gain was observed. Each study was repeated
`three times.
`
`Estimation of Crosslinking Density from Swelling
`Measurements
`
`The volume fraction of the crosslinked GG before swelling,
`v2,,, and the volume fraction of the crosslinked GG at equilib-
`rium swelling state, v2 , were calculated from the following
`equations (21):
`
`v2,r Vp/Vr
`
`v2,, = Vp/Vs
`
`(1)
`
`(2)
`
`where Vp, Vr, and Vs are the volumes of the polymer in a dry
`state, relaxed (immediately after the reaction) state, and swollen
`(equilibrium) state, respectively. Vr was measured gravimetri-
`cally by weighing polymer discs (in a relaxed state) outside
`and inside a water containing picknometer. Vs was measured
`similarly for swollen discs. Vp was measured for dehydrated
`(2 days at 45°C, followed by 1 day at 80°C) discs, using a
`heptane containing pycnometer.
`Knowing v2,, and v2,„ the mean molecular weight of the
`polymer fragments between the crosslinking points (Mc) could
`be calculated from the following equation (21):
`
`000002
`
`(cid:9)
`(cid:9)
`

`

`Crosslinked Guar as Oral Colon-Specific Drug Carrier (cid:9)
`
`1021
`
`-15
`— V [1n(1 — v2,) + v2,+ X
`1 (cid:9)
`I
`2
`= —
`Mc Mn
`
`1 (.1,2
`
`I /3 (cid:9)
`
`(3)
`
`[ (VV22.: ) (cid:9)
`
`V21,)]
`
`where Mn is the number average molecular weight of GG
`[300,000 as determined by GPC (6)], V1 is the molar volume
`of the solvent used (H2O: 18 cm3/mole), -1) is the specific volume
`of the bulk polymer at swollen state [0.63 cm3/g for GG (22)]
`and X i is the Flory-Huggins polymer-solvent interaction param-
`eter which decreases with an increase in the polymer-solvent
`interaction. GG is an hydrophilic polymer which forms disper-
`persions in water in concentrations of 0.5% and above. There-
`fore a x value of 0.8, similar to polyvinylalcohol (23), was
`chosen.
`The crosslinking density, P, was calculated from the fol-
`lowing equation (21):
`
`P = (cid:9)
`
`Mc (cid:9)
`
`(4)
`
`Drug Release Studies
`
`The release kinetics of SS (from GG-3, GG-5 and GG-7),
`Indo (from GG-0.1 and GG-1) and Bud (from GG-0.5) were
`studied in 10 ml of PBS pH = 6.4 (0.2 M), or 10 ml of a
`mixture of 0.175 U/m1 of galactomannanase and 0.033 Wm'
`of cx-galactosidase (from E. coli) in the same buffer solution
`(except for the SS, which was studied in buffer solution pH =
`6.4 only). The studies were conducted separately in sealed glass
`beakers mounted in a shaking (100 rpm) bath at 37°C. Samples
`(200 µ,1) of SS were withdrawn at 0, 15, 30, 45, 60, 90, 120, 180,
`240, 300, and 360 minutes and centrifuged with the supernatant
`collected for drug analysis. The same procedure was repeated
`for the Indo and Bud sampling with the time intervals for
`withdrawal at: 0, 30, 60, 90, 120, 180, 240, and 360 minutes.
`Withdrawal volumes were replenished with an equal volume
`of fresh dissolution medium.
`At the end of the release studies, each disc residue was
`digested with an excess of galactomannanase and a-galactosi-
`dase mixture (3.3 and 17.5 U/ml, respectively) and the residual
`drug left in the discs was determined to verify the observed
`value of the total amount of drug released.
`
`Estimation of the Crosslinking Density from Elasticity
`Measurements
`
`Drug Assays
`
`Specimens (13 mm long X 4 mm wide) of the various
`crosslinked products were soaked in distilled water at 37°C
`until equilibrium and their modulus of elasticity [G*],c could
`be measured (Instron, Mini, model 44, Buckinghamshire, U.K.).
`The effective network density was calculated using the follow-
`ing equation (24):
`
`vN = (cid:9)
`
`[G*].
`A4KT(v2„)2/3
`
`(5)
`
`where vN is the effective network density or the molar concen-
`tration of the elastic (effective) chains in 1 cm3 of polymer,
`is the structure factor (in the case of highly swollen network,
`selected for our case, A:4, = 1 — 2/4)), .1) is the crosslinking
`functionality (in our case, for glutaraldehyde, (cid:9)
`= 4), K is
`the Boltzman constant, and v2,, is the volume fraction of the
`crosslinked GG at equilibrium swelling state, calculated from
`Equation 2.
`
`Drug Loading of the Hydrogels
`
`Discs taken from the GG-3 and GG-7 products (for sodium
`salicylate, denoted as SS), GG-0.1 and GG-1 products (for
`indomethacin, denoted as Indo) and GG-0.5 (for budesonide,
`denoted as Bud) immediately after completion of their synthesis,
`as described above, were oven-dried (45°C, overnight). The
`discs were then immersed in the drug solutions as follows: SS:
`the discs were immersed in 10 ml of 10 mg/ml SS in water
`overnight; Indo: the discs were immersed in 10 ml of 0.8 mg/
`ml Indo in PBS pH = 7.8 overnight; Bud: the discs were
`immersed in 10 ml of 0.1 mg/ml Bud in water:ethanol, 5:1 v:v
`overnight. The recovered drug-loaded discs were oven-dried at
`45°C for 24 h. Drug excess (assumed to be on the surface of
`the discs) was removed by rinsing the discs three times with
`10 ml portions of PBS pH = 6.4. The amount drug loaded
`was 357, 146 and 65 mg/g dry polymer for sodium salicylate,
`indomethacin and budesonide respectively.
`
`SS in the withdrawn samples was determined spectropho-
`tometrically (X = 296 nm) after suitable dilution with PBS pH
`= 6.4. Indo was determined spectrophotometrically (X = 318
`nm) after suitable dilution with PBS pH = 7.4. Bud was deter-
`mined spectrophotometrically (X = 247 nm) after suitable dilu-
`tion with a water:ethanol (52:48 v:v) mixture. When samples
`taken from enzyme-containing dissolution media were mea-
`sured, enzyme solutions (containing equal concentrations and
`handled similarly) served as blank solutions.
`
`In Vivo Degradation Analysis
`
`Pre-weighed discs (8 mm OD X 2 mm thick) of GG-0.5,
`GG-1 and GG-3 were hydrated over 2 hours in PBS pH = 7.4
`and tested for biodegradation in the cecum of Sabra rats using
`a previously described method (25). Briefly, the discs were
`mounted in gauze bags which were individually implanted in
`the cecum by attaching them to the organ wall of an anesthetized
`rat (a single disc/rat) with 3/0 silk sutures. The rats were allowed
`to recover and kept on a normal diet for 4 days, after which
`they were sacrificed and the bags opened. In the bags where
`disc residues were found, residues were collected and dried
`until no further weight loss was observed. Sabra rats which
`were treated with an antibiotic cocktail (300 ml of intra-cecal
`administration of ampicillin 250 mg/ml, chloramphenicol 0.5
`mg/m1 and cefazolin 250 mg/m1) were used as controls. In the
`"antibiotics" study discs of the same size were weighed and
`implanted. The residues were then dried and weighed after 4
`days. The antibiotic cocktail was also added to the drinking
`water of the control rats for the 4 days of the study. A parallel
`in vitro control study was performed by soaking discs of the
`same crosslinked products and similar sizes in PBS, pH = 7.4
`for 4 days.
`
`RESULTS
`
`The reduction in swelling properties of GG as a result of
`its crosslinking with GA is shown in Figure 1. Swelling was
`
`000003
`
`(cid:9)
`

`

`1022 (cid:9)
`
`Gliko-Kabir, Yagen, Penhasi, and Rubinstein
`
`16000
`
`14000
`12000
`
`10000
`
`8000
`6000
`
`4000
`2000
`
`0
`
`A
`
`1:1 Gastric TS
`II Intestinal TS
`
`3 (cid:9)
`
`5
`
`Equivalents of glutaraldehyde
`
`A
`
`3000"
`
`2000
`
`1000
`
`f
`
`O Gastric TS
`
`▪ Intestinal TS
`
`1
`0 (cid:9)
`Equivalents of glutaraldehyde
`
`B
`
`2000
`1800
`1600
`1400
`1200
`1000
`800
`600
`400
`200
`0
`0
`
`C
`
`2000
`1800
`1600
`1400
`1200
`1000
`800
`600
`400
`200
`0
`0 (cid:9)
`
`16000 B
`14000
`12000
`
`10000
`5000
`
`21 0 6000
`49
`4000
`2000
`0
`
`2000
`1500'
`1000'
`500'
`0
`
`5
`
`-
`=
`
`,f)
`
`0 (cid:9)
`
`5
`
`1 (cid:9)
`3 (cid:9)
`Equivalents of glutaraldehyde
`Fig. 1. Buffer (Gastric TS, 2 hours or Intestinal TS, 4 hours) uptake
`of GG crosslinked with increasing amounts of GA in a swollen state
`[A] and in a powder form after lyophilization [B]. Expanded scale of
`the results obtained for the crosslinked products is included for the
`powder form. Numbers at the x-axis are the equivalents of GA used;
`GG-0 is native GG. Shown are the mean values ± S.D., n = at least
`three different batches.
`
`evaluated by measuring buffer (Gastric TS and Intestinal TS)
`uptake. The GA concentration-dependent reduction in buffer
`uptake was much more marked in the swollen (Figure I A) and
`lyophilized (Figure 1B) states. Also, crosslinking diminished
`the effect of pH on the swelling properties of GG (Figure 1B).
`The reduction in swelling properties of GG in a film form
`as a result of its crosslinking with GA and the swelling kinetics
`of films made of four crosslinked GG products in Gastric TS
`and Intestinal TS are shown in Figure 2. Apart from the observa-
`tion that the higher the crosslinking, the lower the buffer uptake
`(Figure 2B and C), it can be seen that in all cases the swelling
`reached equilibrium within 90 minutes. The GA concentration
`dependent changes in Mc (the mean molecular weight of the
`polymer fragment between crosslinking points), the crosslink-
`ing density (p), the modulus of elasticity [G*] and the effective
`network density (v/V) are summarized in Table I and Table II.
`Figure 3 shows that the release of the soluble drug marker
`SS out of discs made of two highly crosslinked products (GG-
`3 and GG-7) was relatively rapid, complying similar kinetic
`profiles. Indo release kinetics from discs made of two low
`crosslinked products, GG-0.1 and GG-1, in buffer solutions with
`and without guar-hydrolyzing enzymes, are shown in Figure 4.
`While almost no drug was released in the buffer solutions due
`to the low water-solubility of Indo, the addition of enzymes
`accelerated the release kinetics of the drug, with the total amount
`
`GA equivalents:
`0.1
`3
`5
`—o-- 7
`
`60 (cid:9)
`
`180
`120 (cid:9)
`Time, minutes
`
`240
`
`300
`
`GA equivalents:
`•-- 0.1
`•-- 3
`5
`—0--- 7
`
`60 (cid:9)
`
`240
`
`300
`
`180 (cid:9)
`120 (cid:9)
`Time, minutes
`Fig. 2. The reduction in the swelling properties of GG in a film form
`as a result of its reaction with 1 equivalent of GA (A) and the swelling
`kinetics of crosslinked GG discs in Gastric TS (B) or Intestinal TS
`(C) buffers of the following products: GG-0.1, GG-3, GG-5 and GG-
`7. Shown are the mean values ± S.D. n = at least three different batches.
`
`Table I. The Volume Fraction v2 , (After Swelling), the Mean Molecu-
`lar Weight of the Polymer Fragments Between the Crosslinking Points
`(Mc), as Calculated from Equation 3, and the Crosslinking Density P
`as Calculated from Equation 4 of the Four Crosslinked GG Products:
`GG-0.5, GG-1, GG-3, and GG-5
`
`Product
`
`GG-0.5
`GG-1
`GG-3
`GG-5
`
`Mc (cid:9)
`(g/mole) (cid:9)
`
`P X 104
`(mole/cm3)
`
`0.003 ± 0.0006
`0.006 ± 0.0002
`0.010 ± 0.002
`0.012 ± 0.002
`
`25,208 ± 3040
`14,231 ± 539
`9,774 ± 1340
`5,088 ± 582
`
`0.64
`1.12
`1.68
`3.24
`
`Note: Shown are the mean values ± S.D., n = 10 measurements.
`
`000004
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`Crosslinked Guar as Oral Colon-Specific Drug Carrier (cid:9)
`
`1023
`
`Table H. The Modulus of Elasticity of the Four Crosslinked GG Prod-
`ucts: GG-0.1, GG-0.5, GG-1, and GG-3, and Their Effective Network
`Density (vN) as Calculated from Equation 5
`
`Product
`
`GG-0.1
`GG-0.5
`GG-1
`
`[G*] (MPa)
`
`0.004 -± 0.0008
`0.02 ± 0.0005
`0.08 ± 0.015
`
`(vN) X 104
`
`0.19 ± 0.001
`0.71 ± 0.03
`2.7 ± 0.03
`
`Note: Shown are the mean values ± S.D., n = 10 measurements.
`
`100
`
`N 80
`
`60
`
`E 4°
`ts° 20
`
`of Indo released increased by 9.7-fold and 6.7-fold for the GG-
`0.1 and GG-1, respectively. Similar results were obtained for
`Bud. Its release kinetics from discs made of GG-0.5, with and
`without enzyme mixture, are shown in Figure 5. In the presence
`of enzyme mixture the total Bud released was 7.3-fold greater
`than the total drug release in the buffer solution without
`enzymes.
`The in vivo degradation of discs made of the crosslinked
`products GG-0.5, GG-1 and GG-3 in the cecum of the rat, with
`and without antibiotic treatment, is summarized in Table III,
`which shows a complete degradation of GG-0.5 and GG-1
`discs, and partial degradation of the GG-3 discs. Dosing the
`rats with antibiotics caused a significant decrease in the
`implanted discs' degradation (from 100% without antibiotics,
`
`60 (cid:9)
`
`120 (cid:9)
`
`180 (cid:9)
`
`240 (cid:9)
`
`300
`
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`
`% Cumulative drug release
`
`Time, minutes
`Fig. 3. SS release kinetics from discs made of GG-3 (closed circles)
`and GG-7 (open circles) into PBS pH = 6.4.
`
`100
`
`80
`
`60
`
`40
`
`20
`
`% Cumulative release
`
`200 400 600 800 1000
`Time, minutes
`Fig. 5. Bud release kinetics from discs made of GG-0.5 in PBS pH
`= 6.4, with (closed circles) and without (open circles) enzymes mixture.
`Shown are the mean values of three different measurements ± S.D.
`
`to 13.0 ± 5.3 and 7.0 ± 2.7% with antibiotics for GG-0.5 and
`GG-1, respectively. The GG-3 disc did not degrade at all). Table
`III also shows that the same discs were resistant in a pH = 6.4
`buffer solution over 4 days.
`
`DISCUSSION
`
`GG is degraded by colonic bacteria (4,6,13). However, its
`enormous swelling is a drawback in its use as a microbially
`controlled colon-specific delivery system because of the possi-
`ble risk that an entrapped drug will leak out prior to arriving
`at the colon. In the present study, glutaraldehyde (GA) was
`used as a crosslinker to decrease GG swelling properties. The
`reaction with GA takes place with hydroxyl groups of the
`galactose or mannose subunits of GG (Scheme 1), typical of
`the GA reaction with polyols at acidic conditions (14-16).
`The swelling properties of the resulting crosslinked GG
`products were studied in buffer solutions at three different
`physical forms: swollen state; dried discs; and lyophilized form.
`The latter was performed to account for intrinsic swelling char-
`acteristics of GG and its crosslinked derivative. In such a physi-
`cal state, the difference between native GG and the crosslinked
`product is most profound due to the reduced number of entangle-
`ments which may restrict the equilibrium degree of swelling
`that occurs upon reconstitution. As expected, the higher the
`amount of GA, the lower the buffer uptake observed, indicating
`an increase in the crosslinking density (Figure 1 and Figure 2).
`Figure 2 clearly shows that the drying procedure involved in
`
`Table III. The Degradation of Discs Made of GG-0.5, GG-1, and GG-
`3 in the Cecum of the Rat, Exposed (+) and Not Exposed (-) to
`Antibiotic Treatment, and in PBS pH = 6.4 (Not Implanted), as
`Detected 4 Days After Implantation
`
`Weight loss
`- antibiotic
`(%)
`
`100
`100
`38.5 ± (cid:9) 11.5
`
`Product
`
`GG-0.5
`GG-1
`GG-3
`
`Weight loss
`+ antibiotics
`(%)
`
`13.0 ± 5.3
`7.0 ± 2.7
`0
`
`Weight loss
`in buffer
`(%)
`
`0
`0
`0
`
`Note: Results are presented as a fraction of weight loss during the
`study, expressed in % of initial weight. Shown are mean values (for
`the degraded products) ± S.D. (n = 3 rats).
`
`100
`
`300 (cid:9)
`200 (cid:9)
`Time, minutes
`Fig. 4. Indo release kinetics from discs made of GG-0.I and GG-1 in
`PBS pH = 6.4, with (closed circles and triangles) and without (open
`circles and triangles) enzyme mixtures. Shown are the mean values of
`three different measurements ± S.D.
`
`400 (cid:9)
`
`500 (cid:9)
`
`600
`
`000005
`
`(cid:9)
`

`

`1024 (cid:9)
`
`Gliko-Kabir, Yagen, Penhasi, and Rubinstein
`
`the disc preparation prevented the hydrogels from returning to
`their original volume (swollen state, Figure IA). Thus, the
`drying introduced irreversible changes to the hydrogels. For
`example product GG-5 swelled 28-fold at equilibrium, 7.8-fold
`in lyophilized form, and only 2.7-fold in a dry disc form (Figure
`1 and Figure 2). A similar trend in equilibrium swelling was
`reported by Peppas and Korsmeyer who crosslinked poly (vinyl
`alcohol) with GA. They concluded that such a pattern is a
`characteristic of physical crosslinking rather than a change in
`the crystalline structure (26). This means that physical entangle-
`ments in the polymer network are responsible for the changes
`in the degree of swelling of the various crosslinked GG products.
`The lyophilized GG-5 swelled 3.6-fold less than the same prod-
`uct in the swollen state. That is, physical entanglements occurred
`even in the lyophilized product, although lyophilization is sup-
`posed to keep the network structure of the polymer as intact
`as possible. The order of magnitude decrease in the equilibrium
`swelling of GG-5 in the disc form compared with the swollen
`state, and also compared with the 1.9-fold reduction observed
`in GA crosslinked PVA (26), may be explained by the much
`higher molecular weight of GG and the fact that it is a
`branched polysaccharide.
`The non-ionic nature of crosslinked GG causes the buffer
`uptake rates of products GG-3, GG-5, and GG-7 to be similar
`(Figure 2). The swelling rate of GG-0.1, however, was signifi-
`cantly lower in Gastric TS, probably because in this loosely
`bound product it is easier for GA to continue to react in an
`acidic environment (Scheme 1).
`The degree of crosslinking of the GG hydrogels was
`assessed from swelling and modulus of elasticity calculations.
`The number average molecular weight between two adjacent
`crosslinking points, Mc, of the crosslinked products was calcu-
`lated according to Bray and Merrill (27), as used by Peppas
`and Merrill (21). Table I shows that increasing amounts of GA
`in the products caused a decrease in the Mc values, as would
`be expected from Flory's theory (28). That is, an increase in
`the concentration of the crosslinker in the reaction mixture
`resulted in a densely crosslinked product (lower Mc, higher p
`and higher v2,, values). The degree of crosslinking was also
`derived from the modulus of elasticity and the effective network
`density (v/V) measurements. As shown in Table II increase in
`the amount of GA used in the reaction resulted in increase in
`the mechanical strength and effective network density for the
`various crosslinked products.
`The fact that high amounts of GA (more than 1 equivalent)
`were required for the crosslinking reaction suggests that the
`crosslinking efficiency was low. This could be attributed to (a)
`low reactivity of the GG hydroxyl groups as a result of the
`limited water solubility of the polymer, (b) GA polymerization
`during the crosslinking process and (c) possible masking effect
`of the hexose units of the branched polymer.
`Figure 3 demonstrates that, due to rapid drug diffusion
`through its network, crosslinked GG cannot be used for the
`specific delivery of highly water-soluble drugs, such as SS,
`into the colon. Total drug release from the most dense products
`GG-3 and GG-7, was accomplished within 180 minutes. In
`terms of GI transit time, this is barely sufficient for the polymer
`to arrive at the mid ileum. The fast release of SS is a result of
`the relatively rapid swelling kinetics of the two crosslinked GG
`products tested, as shown in Figure 2, a phenomenon which
`allows a highly water-soluble drug to easily leach out of the
`
`carrier. The release kinetics of two poorly water-soluble drug
`probes, Indo and Bud, in the presence or absence of specific
`enzymes, was therefore evaluated. The enhanced drug release
`kinetics of the two drugs in the presence of the enzyme mixture,
`as demonstrated in Figure 4 and Figure 5, indicate that (a) the
`ability of GG to be degraded by specific enzymes of the colon
`was maintained despite its modification with GA, (b) cross-
`linked GG can potentially be used as a colon-specific carrier
`only for poorly-water-soluble drugs. The ability of the cross-
`linked GG to be degraded by the enzymes could be explained
`by the assumption that after crosslinking, the polymeric network
`was composed of two parts, an unchanged GG section and a
`crosslinked section, mixed together randomly. McCleary (29),
`who investigated the enzymatic degradation mechanism of
`carob-gum galactomannans, suggested that enzymes cleave the
`polymer at a distance of three hexose units from the branching
`point. Assuming that this is the situation with crosslinked guar,
`the mean distance between the crosslinking points of product
`GG-0.5 is 25208/160 = 157 sugar units (160 is the molecular
`weight of a single hexose unit, and 25208 is the calculated
`Mc value for GG-0.5, Table I). It is speculated that this distance
`is large enough for the enzyme to penetrate into the network
`and cleave the GG section.
`Discs of products GG-0.5, GG-1 and GG-3 were also
`analyzed for their enzymatic degradation properties in the
`cecum of the rat. One group of rats was kept on a normal diet,
`while the control group was treated with antibiotics to reduce
`its cecal microbial flora. Table III shows that out of the three
`products used in this section of the study, GG-3, which was
`crosslinked with the largest amount of GA, was not completely
`degraded four days after implantation. The other two products,
`GG-0.1 and GG-0.5, which were crosslinked with lower
`amounts of GA, were degraded completely. This, together with
`the observation that in positive control studies (Table III) the
`crosslinked products were not degraded over four days, indi-
`cates that degradation of crosslinked GG in the rat cecum
`(Figures 4 and 5) results from enzymes of bacterial origin.
`
`CONCLUSIONS
`
`In this study we reduced the swelling properties of GG by
`crosslinking it with increasing amounts of GA. The crosslinked
`products retained the ability of GG to be degraded in vitro by
`a mixture of galactomannanase and a-galactosidase. This was
`verified by drug release studies, which showed that crosslinked
`GG can retain a poorly water-soluble drug load, such as Bud
`and Indo, but not highly water soluble drugs such as SS. This,
`together with the observation that crosslinked GG discs are
`degradable in vivo in the cecum of the conscious rat, suggest
`that crosslinked GG with GA can potentially be used for the
`specific delivery of poorly water-soluble drugs to the colon.
`Compared to plain GG tablets (30), this new colonic delivery
`system is unique in its ability to function specifically without
`relying on c