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Crosslinking Studies in Gelatin Capsules Treated with Formaldehyde and in Capsules Exposed to Elevated Temperature and Humidity

CLYDE M. OFNER III, YU-E ZHANG, VALERIE C. JOBECK, BILL J. BOWMAN

Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences in Philadelphia, 600 South 43 rd Street, Philadelphia, Pennsylvania 19104

Received 11 August 1999; revised 30 June 2000; accepted 11 July 2000

ABSTRACT: Incomplete in vitro capsule shell dissolution and subsequent drug release problems have recently received attention. A modified USP dissolution method was used to follow capsule shell dissolution, and a 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay was used to follow loss of -amino groups to study this shell dissolution problem postulated to be due to gelatin crosslinking. The dissolution problems were simulated using hard gelatin capsule (HGC) shells previously treated with formaldehyde to crosslink the gelatin. These methods were also used to study the effect of uncrosslinked HGC stored under stressed conditions (37 °C and 81% RH) with or without the presence of soft gelatin capsule shells (SGC). A 120 ppm formaldehyde treatment reduced gelatin shell dissolution to 8% within 45 min in water at 37 °C. A 200 ppm treatment reduced gelatin -amino groups to 83% of the original uncrosslinked value. The results also support earlier reports of non-amino group crosslinking by formaldehyde in gelatin. Under stressed conditions, HGC stored alone showed little change over 21 weeks. However, by 12 to 14 weeks, the HGC exposed to SGC showed a 23% decrease in shell dissolution and an 8% decrease in the number of -amino groups. These effects on the stressed HGC are ascribed to a volatile agent from SGC shells, most likely formalde- hyde, that crosslinked nearby HGC shells. This report also includes a summary of the literature on agents that reduce gelatin and capsule shell dissolution and the possible

mechanisms of this not-so-simple problem. © 2001 Wiley-Liss, Inc. and the American Phar- maceutical Association J Pharm Sci 90: 79–88, 2001

Keywords: gelatin crosslinking, gelatin dissolution, amino group assay, formalde- hyde crosslinking in proteins

INTRODUCTION

After ingestion, gelatin capsule shells must dis- solve sufficiently and release their drug contents to produce a therapeutic effect. Incomplete in vitro capsule shell dissolution and subsequent drug release problems have recently received at-

Current address: Sanofi Research, 9 Great Valley Park- way, P.O. Box 3026 Malvern, Pennsylvania 19355 Correspondence to: C.M. Ofner III (Telephone: 215-596- 8881; Fax: 215-895-1100; E-mail: cofner@usip.edu)

Journal of Pharmaceutical Sciences, Vol. 90, 79–88 (2001) © 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association

tention. This problem was first reported in 1974 for a hard gelatin capsule (HGC) product contain- ing chloramphenicol 1 and in 1977 for a soft gela- tin capsule (SGC) product containing digoxin. 2 These and later cases were associated with ad- verse storage conditions of elevated temperature, humidity, or prolonged storage. But in these two cases, as in almost all others, there was no re- duced bioavailability of the drug compared with the readily dissolving product. 35 Two cases of reduced bioavailability are of his- torical interest. The first case occurred in 1985, with an HGC product containing phenytoin that

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was stored at elevated temperature and humidity and was identified in a patient hospitalized for loss of seizure control. 6 An FDA drug recall of a SGC product in 1993, because of dissolution prob- lems and reduced bioavailability, 7 is the second case. The latter led to the formation of an FDA/ Industry Gelatin Capsule Working Group to ex- amine the effects of the in vitro dissolution prob- lem on drug bioavailability. The Working Group simulated this dissolution problem with formal- dehyde crosslinked HGC and SGC containing acetaminophen and evaluated their bioequiva- lence in humans. 7,8 It is now generally believed that this in vitro dissolution problem has little risk of reduced drug bioavailability because gas- tric and intestinal enzymes that contribute to the physiological dissolution process have not been routinely used in dissolution tests. These

enzymes break down the swollen and poorly soluble film of the capsule shell, sometimes called

a pellicle, that usually retards or prevents drug

release under in vitro dissolution conditions. Carstensen and Rhodes, 9 as well as others, have suggested introducing such enzymes into in vitro dissolution testing. One result of these sugges- tions and of the Working Group investigation was the initiation of the regulatory process to allow

so-called two-tier testing; that is, the use of a sec- ond USP dissolution test, which contains an en- zyme if the product failed the original monograph dissolution test because of this problem. 7,10 Although this in vitro dissolution problem is unlikely to produce biological consequences, it re- mains a theoretical safety issue and its causes are

a concern. There is a growing body of evidence

that this problem is primarily caused by gelatin crosslinking in the capsule from aldehyde impu- rities that form the pellicle barrier to drug re- lease. 9,11 Crosslinking gelatin leads to an intri- cate network of high molecular weight that pro- duces a swellable hydrogel but substantially reduces, or even prevents, dissolution of the gela- tin. 1214 An earlier review of gelatin crosslinking and capsule dissolution, 11 as well as related stud- ies with a different focus have been reported. 1517 Recently, approaches to minimize this crosslink- ing and dissolution problem using additives 18 and by accelerated detection testing 19 have also been reported. This report has three goals. The first is to de- scribe and disseminate an in vitro evaluation of gelatin crosslinking in the formaldehyde-treated capsules used in the Working Group acetamino- phen biostudy. The second goal is to report the

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application of the methods just described to evaluate crosslinking and shell dissolution in plain, uncrosslinked capsules that have been “stressed” by exposure to elevated temperature and humidity. The last goal is to collect the dis- parate literature on this issue for a discussion and comparison of these results for this not-so- simple dissolution problem. The ideal evaluation of crosslinking in gelatin would include determi- nation of the crosslinking extent, identification of the agent or cause and the specific reaction mechanism, as well as identification of the site, the molecular weight, and the number of such crosslinks. A macroscopic and molecular ap- proach was used in the current investigation to elucidate some of these aspects. The macroscopic approach was to evaluate dissolution of the cap- sule shell alone, without the presence of drug or excipients, using an assay for the dissolved gela- tin. This gelatin dissolution is used as an indica- tion of crosslinking 13 without regard to specifics such as mechanism or site. The molecular ap- proach was to assay for the potential loss of amino groups due to crosslinking because formaldehyde (and other aldehydes) reacts with amino groups on lysine and hydroxylysine amino acid residues of gelatin during crosslinking. 2022 Such specific- ity suggests that this formaldehyde crosslinking can be quantitatively determined. The formalde- hyde–gelatin crosslinking mechanism, however, is not entirely clear; non-amino group crosslink- ing also has been reported. 21 A comparison be- tween results of the two methods in this investi- gation could corroborate such non-amino group crosslinking. Another approach to enhance our understanding of this dissolution problem is to apply these methods in an exploratory manner to stressed, plain, uncrosslinked capsules. Specifi- cally, the theoretical possibility of self-cross- linking occurring without an agent 11,12 can be ex- amined. Under the stressed conditions of this study, a crosslinking agent was generated from a SGC shell that induced crosslinking in HGC shells. This stressed-induced crosslinking can be directly measured, which to the authors’ knowl- edge has not yet been reported.

EXPERIMENTAL SECTION

Materials

Type B gelatin granules, with a bloom strength of 254 and an approximate moisture content of 11% (w/w) determined by loss on drying at 105 °C for

72 h, were supplied by Kind and Knox (Sioux City, IW, sample #T7468, lot #1). Capsugel AG (Basel, Switzerland) supplied formaldehyde- treated HGC and controls that were prepared as described later. Plain uncrosslinked HGC used in the stressed conditions study were purchased from Lilly. Untreated SGC were supplied by Ban- ner Pharmacaps Inc. (High Point, NC) and con-

tained a poly(ethylene glycol) (PEG) fill liquid and

a shell composed of gelatin, glycerin, sorbitol, and water. The BCA (bicinchoninic acid) protein assay kit and 2,4,6-trinitrobenzenesulfonic acid (TNBS) was purchased from Pierce Chemical Company (Rockford, IL). Water was purified by reverse os- mosis. All other chemicals were at least ACS re- agent grade. Formaldehyde-treated HGC were prepared by Capsugel, 23 by mixing formaldehyde (HCHO) with lactose at different levels. The HCHO/lactose mixture was filled into empty HGC capsules that were then exposed briefly to 40 °C and 75% RH, then to 25 °C and 50% RH for 6 weeks, at which time the HCHO/lactose mixture was removed from the capsules. The treatment levels of HCHO in the mixtures were 0, 20, 30, 120, 200, 400, and 1000 ppm. The 0, 20, and 120 ppm levels were evaluated in the Working Group Biostudy. The control passed the in vitro dissolu- tion tests in the absence and presence of enzyme (P/P). The 20 ppm capsule failed the conventional dissolution tests but passed with the addition of enzyme (F/P). The 120 ppm capsule failed disso- lution tests in the absence and presence of en- zyme (F/F). The 20 ppm capsules were found bio- equivalent to the uncrosslinked controls, whereas the 120 ppm capsules were not bioequivalent.

Stressed Hard Gelatin Capsules

A saturated ammonium sulfate solution was used

in glass desiccates to produce 81%RH at 37 °C. The desiccates were stored in an incubator at 37 °C. Gelatin granules and plain HGC, in separate petri dishes, were placed in a desiccator desig- nated as “HGC stored alone”. Gelatin granules, plain HGC, and untreated SGC, in petri dishes, were placed in the desiccator designated as “HGC stored with SGC”. Prior to this exposure, the SGC were cut in half and rinsed with small amounts of methanol to remove residue of the PEG fill liquid. HGC and granule samples were removed at de- sired times and evaluated by shell dissolution and amino group assay.

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Capsule Shell Dissolution

A modified USP paddle method was used to

evaluate dissolution of the gelatin from the cap- sule shell. The Van-Kel apparatus (Chatham, NJ) used consists of a 200-mL kettle (20-cm height, 4-cm inner diameter), an adapter ring to allow the “mini” kettle to fit into the water bath system for conventional kettles, 100 mL of purified water at 37 °C, a 100 rpm speed for the minipaddle, and Teflon-coated coilers to weight the empty cap- sules to the bottom of the kettle. After removal of 1-mL samples, this volume was replaced with warmed purified water. Dissolved gelatin was de- termined from predetermined calibration plots by BCA protein assay or ultraviolet (UV) absorbance measurements at 214 nm. In the resulting cap- sule dissolution profiles, each point is the mean of six samples and error bars represent standard de- viation (SD).

Gelatin Amino Group Assay

The uncrosslinked, or free, -amino groups of gela- tin were assayed by a method reported previ- ously 24 This method uses TNBS as a UV chromo- phore that reacts with primary amino groups and, after acid hydrolysis and organic extraction, re- mains in the aqueous phase reacted with -amino groups on lysine residues in peptide fragments.

Statistics

The results were analyzed by a one-way analysis

of variance (ANOVA). Multiple comparisons be-

tween means were evaluated by a Neuman–Keuls test. 25 For the few cases of non-normal variance, the multiple comparisons between means were evaluated using a Kruskal–Wallis nonparametric ANOVA test with log-transformed data. 25

RESULTS

Evaluation of Formaldehyde-Treated Capsules

Capsules crosslinked with various amounts of HCHO were evaluated using gelatin shell disso- lution as a general indicator of crosslinking and the -amino group assay as an indicator of specific functional group participation. The dissolved cap- sule profiles in Figure 1 show no difference be- tween the control, untreated capsules and those treated with the two lower amounts of 20 and 30 ppm HCHO. Additional, but unsuccessful, experi-

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8 2 OFNER ET AL. Figure 1. Gelatin shell dissolution profiles of formal- dehyde treated hard

Figure 1. Gelatin shell dissolution profiles of formal- dehyde treated hard gelatin capsules in 37 °C water (bars are SD, n 6).

ments were conducted to enhance differences be- tween these profiles using different paddle speeds, an acid medium, capsule strips, fillers, and different weights of fillers (data not shown). The next levels of 120 and 200 ppm, however, produced substantial reductions leading to 8 and 2%, respectively, of dissolved gelatin by 45 min, and only 15 and 4%, respectively, of dissolved gelatin by 2 h. In addition, the variation among replicates in the higher two levels of crosslinking was less than that of the controls and two lower crosslinking levels. The SD for the controls ranged from 4 to 11, that of the 20 ppm capsules ranged from 1 to 7, and that of the 120 and 200 ppm capsules ranged from 0 to 2. The loss of -amino groups in the HCHO- treated capsules is shown in Figure 2. The un- treated control capsules have 32.9 × 10 5 mol/g of free, uncrosslinked -amino groups. This value corresponds to 32.9 -amino groups on one gelatin molecule of 1000 amino acid residues with an ideal molecular weight of 100,000. The value is virtually identical to the 33.0 × 10 5 mol/g previ- ously reported for Type B gelatin granules. 24 The variation is also the same with a relative SD (RSD) of 2.6%. There is no measurable difference between the control capsules and those treated with 120 ppm HCOH, and by inference, the capsules treated with lower amounts. There is, however, a small but measurable loss of -amino groups for the cap- sules treated with 200 ppm. The loss continues with increasing HCHO treatments to a final value of 27.4 × 10 5 mol/g for capsules treated

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OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 1, JANUARY 2001 Figure 2. Loss of -amino groups in

Figure 2. Loss of -amino groups in formaldehyde treated hard gelatin capsules (bars are SD; n 5; *, p < 0.05).

with 1000 ppm. This is a 17% loss of -amino groups compared with untreated capsules. The SD for these maximally treated capsules is 0.29.

Evaluation of Stressed Hard Gelatin Capsules

Figure 3 illustrates the conditions of HGC expo- sure to 37 °C and 81% RH in the presence of SGC. A separate desiccator experiment was conducted without SGC to evaluate changes induced in gela- tin by only the elevated temperature and humid- ity. Figure 4 shows the percent dissolved of cap- sule gelatin at 45 min as a function of weeks of exposure to the stressed conditions. The HGCs stored alone showed little change in their com- plete dissolution up to 21 weeks of exposure. The HGC stored with SGC showed dissolution re-

of exposure. The HGC stored with SGC showed dissolution re- Figure 3. Stressing of hard gelatin

Figure 3. Stressing of hard gelatin capsules and gelatin granules at 37 °C and 81% RH in the presence of soft gelatin capsule shells.

Figure 4. Dissolution of stressed hard gelatin cap- sule shells during storage at 37 °C

Figure 4. Dissolution of stressed hard gelatin cap- sule shells during storage at 37 °C and 81% RH up to 21 weeks in the presence and absence of soft gelatin cap- sule shells.

duced to 77% at 12 weeks followed by a return to complete dissolution by 19 weeks. A very similar trend is found for the loss of -amino groups in the gelatin capsule shells ex- posed to these conditions. Figure 5 shows little if any amino group loss from exposure to the el- evated temperature and humidity in the absence of SGC. For exposure with SGC at these condi- tions, there is a small but real (8%) loss of -amino groups by 14 weeks. As was observed in the dis- solution experiments, these measurements re- turn to their normal values at 19 weeks. This return to normal values for both measures is at- tributed to hydrolytic degradation of the cross-

is at- tributed to hydrolytic degradation of the cross- Figure 5. Loss of -amino groups in

Figure 5. Loss of -amino groups in stressed hard gelatin capsule shells during storage at 37 °C and 81% RH up to 21 weeks in the presence of soft gelatin cap- sule shells.

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links and regeneration of the amino groups. Dis- solution and amino assay results for gelatin gran- ules were very similar to that of the capsules and are not shown.

DISCUSSION

Evaluation of Formaldehyde-Treated Capsules

Formaldehyde crosslinking in the gelatin shell forms a three-dimensional molecular network of a higher average molecular weight than the origi- nal molecules that dissolve at this temperature. In addition, this crosslinking reduces hydrophilic- ity of the molecules by loss of ionizable amino groups. Both these effects lead to formation of the nondissolving layer, or pellicle, that hinders or prevents drug release. The HCHO from the treated lactose induces crosslinking on the inner capsule shell surface to form the nondissolving layer that extends further into the shell as the HCHO treatment levels are increased. Treatment of 120 ppm extended the crosslinking throughout the gelatin shell, which substantially reduced gelatin shell dissolution to only 15%. The thin

layer from 20 and 30 ppm HCHO exposure prob- ably represents only a small fraction of the gela- tin that is not distinguishable from 100% dissolu- tion of untreated shells using the shell dissolution method. The higher levels of crosslinking from 200 to 1000 ppm decrease free -amino groups, which substantiates the participation of these groups in

a covalent crosslinking reaction. The downward

trend also suggests that not all potential crosslinking sites reacted with HCHO, even at the 1000 ppm level. It is interesting to estimate the HCHO content of these treated capsules and compare these amounts to the loss of amino groups. Assuming that all HCHO in the lactose reacts with the gelatin shell, a lactose fill weight

of 400 mg, a capsule weight of 75 mg, and a mois-

ture content of 10%, the calculated HCHO in the shells is 0.4, 2.4, and 20 × 10 5 mol/g gelatin for 20-, 120-, and 1000-ppm treatments, respectively. These values are not far from the amino group losses of 0.3, 0.9, and 5.5 × 10 5 mol/g gelatin for

the 120-, 200-, and 1000-ppm treatments, respec- tively. Gold et al. 15 measured HCHO crosslinking in HGC using near-infrared spectrophotometry after exposing the capsules to 150 ppb HCHO at- mosphere. These exposures substantially reduced in vitro release of incorporated amoxicillin. Al- though this method is more sensitive for detecting

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HCHO crosslinking, it does not identify and quantitate participation of a specific functional group. Crosslinking clearly occurs from 20- and 120- ppm HCHO treatments as shown by results from the Working Group Biostudy, and in Figure 1 for the 120 ppm treatment. However, the -amino group assay only measures a loss of these groups for treatments >200 ppm. The assay can measure

a loss of one -amino group from the initial 33

groups on lysine and hydroxyl lysine residues per

average gelatin molecule of 1000 residues (see Figure 2). This number of gelatin residues per

molecule is based on the ideal gelatin molecule of 100,000 g/mol (three molecules from a parent triple helical collagen molecule). 26 Two average gelatin molecules crosslinked to each other by as little as one -amino group on each gelatin mol- ecule should then be detectable. This would be analogous to the -amino group loss at 200 ppm shown in Figure 2. These two crosslinked gelatin molecules would represent a small crosslinked unit that should be detectable in this situation. Consequently, and in contrast to these results, the extensive crosslinked network producing the substantial dissolution reduction observed from the 120-ppm treatment would be expected to have

a measurable loss of amino groups involved in

such crosslinking. Formaldehyde was chosen as the crosslinking agent in these capsules because it has been iden- tified as the causative agent of this dissolution problem in two cases, 3,16 and to model the more complex problem of crosslinking under stressed conditions. The mechanistic details of HCHO-to- gelatin crosslinking, however, are not entirely clear. A likely explanation for the apparent insen- sitivity of the amino group assay is that HCHO crosslinks with other functional groups in combi- nation with, or independent of, -amino groups. In a 13 C nuclear magnetic resonance (NMR) study of 13 C-labeled HCHO reacting with gelatin in so- lution and the solid state, no evidence was found of HCHO crosslinking between amino groups on lysine residues; 21 however, strong evidence of ar- ginine (guanidino functional groups)-to-lysine crosslinks and arginine-to-arginine crosslinks was reported. In fact, these investigators reported that optimum crosslinking of both reactions was achieved at 30 °C and 60–70% RH. In addition, only arginine-to-arginine crosslinks were mea- sured after 24 h at 30 °C and 100% RH. These conditions are important to the current investiga- tion because the HCHO-treated capsules were

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also exposed to elevated humidity as part of their treatment. Whereas the HCHO concentrations in the cited study are much higher than those used in the current study, the earlier study demon- strates the possibility of the arginine-to-arginine reaction in the solid state. Gold et al. 22 recently obtained corroborating evidence in solution ex- periments for the lysine-to-arginine crosslinks for HCHO concentrations as low as 100ppm, but did not report any evidence for arginine-to-arginine crosslinks. The indication of crosslinking in the current study at the 120-ppm treatment from the capsule dissolution, but the absence of -amino groups participating at this HCHO level supports the presence of arginine-to-arginine crosslinks. The two methods used in the current investi- gation are probably less sensitive than the USP drug dissolution test as an indicator of gelatin shell HCHO crosslinking. In a report of acetamin- ophen dissolution using these HCHO treated cap- sules, the 20-ppm treated capsules demonstrated impaired dissolution in water and only 50% re- lease of drug by 45 min. 22 Acetaminophen release from the capsules treated at the higher level of 120 ppm was <1% under these conditions. Such reductions in the rate and extent of drug release reflect effects of the crosslinked gelatin layer im- peding drug diffusion already described. These cited acetaminophen dissolution tests were con- ducted in water, as were the current capsule shell dissolutions. The amoxicillin release reported by Gold et al. 15 also illustrates the sensitivity of the USP dissolution test to HCHO crosslinking in gelatin shells.

Evaluation of Stressed Hard Gelatin Capsules

Evaluation of the HCHO-treated capsules illus- trates the uses and limitations of the current study methods to evaluate gelatin shell crosslink- ing. The amino, carboxylic acid, and perhaps other groups on the gelatin molecule raise the possibility that gelatin may crosslink with itself under the so-called stressed conditions of elevated temperature and humidity. It has been suggested that amino groups might undergo oxidative deamination to an aldehyde, which could then in- duce a crosslink. 11 High-temperature exposure of gelatin (105 °C and reduced pressure) has pro- duced insoluble, but swellable gelatin that is as- cribed to a self-crosslinking mechanism. 12,13 The HGC stored alone at 37 °C and 81% RH show no evidence of self-crosslinking. Crosslinking is clearly present in the HGC shells exposed to SGC under these conditions.

The reductions in dissolved gelatin shells and in -amino groups are striking because they occur at virtually identical times. Because such reductions are absent in HGC stored alone, it is concluded that the SGC exposure under these conditions in- duced the crosslinking. In addition, the crosslink- ing substance must be volatile because the HGC and SGC are physically separated but share the same atmosphere. The magnitude of the -amino group reduction indicates extensive crosslinking in the HGC shell. This result is somewhat sur- prising because a 400 ppm HCHO treatment was required to produce a similar reduction. Yet the reduced shell dissolution is not as extensive as would be expected by this level of HCOH treat- ment. It is conceivable that the HGC surface in contact with the petri dish was protected from the volatile crosslinking agent to allow dissolution of this portion of the shell, and perhaps dissolution of the inner surface of the shell. Such an atmo- spheric transfer of the crosslinking agent is un- likely to be influenced by the powder fill. In addi- tion, it should be noted that generation of this crosslinking agent from the SGC also appears to crosslink the SGC. These SGC were not analyzed for crosslinking due to interferences from non- gelatin components of the shell. A report of formaldehyde generated from a sur- factant in a HGC capsule suggests a plausible ex- planation for the volatile crosslinking agent origi- nating in the SGC. 3 Polysorbate 80 in a gemfibri- zole HGC formulation was shown to produce HCHO, insoluble gelatin films, and impaired cap- sule dissolution after 1 month of storage at 37 °C and 80% RH. The authors attributed the HCHO production to auto-oxidation of the end hydroxyl groups in the polyoxyethylene (POE) chains of polysorbate 80. The SGC capsules in the current study initially contained PEG as the fill liquid. The PEG molecule is essentially identical to the POE chains of polysorbates. Although the SGC were opened and washed at the start of the ex- periments, PEG could have previously migrated into the SGC shell because of the 6–12% water in typical SGC shells. In this case, the end groups of PEG could auto-oxidize under these conditions to produce HCHO. An earlier report also implicates HCHO as the causative agent because storage of the SGC at 40 and 60 °C produced reductions in SGC shell disintegration, equilibrium swelling, and shell dissolution to the same extent as that produced from exposure to HCHO. 28 In addition, it has been suggested that the PEG fill in SGC may contain formaldehyde and induce crosslinking. 16

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Agents That Reduce Gelatin Dissolution and Possible Mechanisms

Aldehydes and Covalent Crosslinking

The unintended influence of aldehyde impurities leading to dissolution problems has occurred from

a surprising range of sources. One of the earliest

reports already noted was the generation of HCHO from polysorbate 80. In addition, a poly- hydroxyl pentose, as an intermediate from cellu- lose in rayon coilers, has also been shown to form the aldehyde furfural and a crosslinked, poorly soluble gelatin product after storage at 40 °C and 75% RH for 2 months. 29 Another potential source of formaldehyde is hexamethylenetetramine, which may be added to cornstarch as a stabilizer and which may decompose to formaldehyde under humid conditions. 11 Additional compounds that have been reported to promote crosslinking in gelatin include glucose, hydrogen peroxide, ben- zene, sulfonic acid, p-toluene sulfonic acid, and guanidine HCl. 30 Aldehydes have a long history of crosslinking, but the exact crosslinking mechanisms are not entirely clear. For example, the dialdehyde glu- taraldehyde is recognized to react with primary amino groups, but its mechanism, first suggested in 1968, 31 was more recently suggested to involve four different crosslinks from 12 different reac- tions. 32 Gelatin crosslinking studies with formal- dehyde and the dialdehyde glyoxal have been re- ported as early as 1963. 33 The lysine–arginine gelatin crosslinks from HCHO already discussed were first suggested in 1978, 34 and studies have continued to elucidate the process. 2022 The cur- rent investigation lends support to the lysine– arginine and arginine–arginine crosslinks sug- gested earlier.

High Temperatures and Covalent Crosslinking

Exposure to 105 °C and reduced pressure of 10 m Hg has produced insoluble gelatin that was

ascribed to an amino group-to-carboxylic acid con- densation crosslinking mechanism. 12 In addition,

it has been suggested that oxidative deamination

between two adjacent amino groups on lysine residues would form an aldehyde and then a crosslink during exposure to elevated tempera- tures. 11 However, no measurable loss of gelatin amino groups was reported for gelatin exposed to these conditions for up to 5 days, 35 which sug- gests a crosslinking mechanism without an amino

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group occurs to produce insolubility at high tem- peratures.

Dyes, Noncovalent Binding, and Light

Gelatin carboxyl and amino groups have been shown to interact with functional groups on dyes via ionic, hydrogen, and van der Waals bonds to reduce gelatin solubility. As early as 1973, the dyes FD&C Red No.3 and FD&C Red No.40 were shown to interact with gelatin and reduce its solu- bility. 36 Gautam and Schott 37 reported that dye binding by ion pairing, hydrogen bonds, and other secondary valence forces rendered the gelatin less ionic and less hydrophilic. These interactions cor- roborate a report of the cationic compounds of ce- tylpyridinium Cl and dodecylammonium Cl re- ducing both the initial swelling rate and equilib- rium swelling of uncrosslinked anionic gelatin. 38 A high-dose, water-insoluble drug filled into cap- sules containing FD&C Red No.3 stored at 80% RH and 30 °C under fluorescent light produced only 20% drug dissolution by 60 min. 39 Substan- tial dissolution reductions were also reported for these capsules exposed to high humidity at ambi- ent, as well as UV light. Less severe dissolution reductions were noted for these capsules stored at 35% RH and when protected from light, indicat- ing the accelerating effect of moisture and light on this dye interaction. Capsules containing FD&C Blue No.1 also demonstrated substantial dissolu- tion reductions but only from exposure to fluores- cent light at high humidity. 40 Storage for 8 days at 40 °C and 75% RH in the presence of UV and visible illumination reduced drug dissolution to <5% for a SGC and <60% for a HGC product by 60 min. 19. Also, proteins are frequently crosslinked by exposure to UV light. This mechanism is as- cribed to free radical formation and subsequent crosslinking. However, the participating residues and the composition, number, and size of crosslinks are not fully understood.

CONCLUSIONS

The capsule dissolution test measured reduced dissolution from 120-ppm HCHO-treated cap- sules. This test is more sensitive than the -amino group assay that required a 200-ppm HCHO treatment to measure an effect. However, com- paring these results with a report of a USP dis- solution test of such HCHO-treated capsules con- taining acetaminophen indicated that the USP

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test was more sensitive to this crosslinking than the methods used in this study. The inability of the shell dissolution test to detect crosslinking in 20- and 30-ppm HCHO-treated capsules is prob- ably due to a thin crosslinked and insoluble film on the inner shell wall that prevented drug re- lease. This film may represent only a small frac- tion of the total soluble gelatin in the shell. The -amino group assay results verify participation of these groups in HCHO crosslinking and sup- port earlier reports of HCHO crosslinking in gela- tin between amino groups of lysine and guanidino groups of arginine, and between the guanidino groups in arginine-to-arginine crosslinks. Un- treated, plain gelatin capsules stored at 37 °C and 81% RH up to 21 weeks show no evidence of self- crosslinking. This crosslinking mechanism, how- ever, should not be completely ruled out because of theoretical possibilities, evidence of high tem- perature self-crosslinking, and the sensitivity and mechanism specific limitations of the methods used in this study. Volatile agent(s) generated from SGC at 37 °C and 81% RH can cause sub- stantial and reproducible -amino group cross- linking and dissolution reductions in nearby HGC. Auto-oxidation of residual PEG to form HCHO and induce the crosslinking is a likely ex- planation. Capsule shell dissolution might be used to distinguish shell dissolution problems from other dissolution problems, such as formu- lation or degradation. Proper containers and stor- age are important issues to minimize this disso- lution problem in gelatin capsule products.

ACKNOWLEDGMENTS

Presented in part at the 1997 annual meeting of the American Association of Pharmaceutical Sci- entists (AAPS) in Boston, MA. Supported in part by USPHS Contract FDA-223-95-3006 (to CMO). We gratefully acknowledge Dr. Ewart T. Cole (Capsugel AG, Basel, Switzerland) for donation of the formaldehyde-treated hard gelatin capsules, Banner Pharmacaps (Highpoint, NC) for donation of soft gelatin capsules, and Dr. J. Michael Dunn (Kind & Knox, Sioux City, IO) for donation of the gelatin granules.

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