A publication of the
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`\|il(iiil|tli)ar1ml.Higuchi
`UniversityofUtah, DepartmentofPharmaceuticsand
`
`Pharmaceutical Chemistry
`Salt Lake City, UT 84112-1102
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`

`A publication of the
`@ American
`I
`Pharmaceutical
`Association
`and the
`American
`Chemical
`
`Society
`
`July 1994
`Volume 83. Number 7
`JPMSAE 83(7) 915-1068
`ISSN 0022-3549
`
`REVIEW ARTICLE
`
`Cross-Linking of Gelatin Capsules and Its Relevance to Their in Vitro—in Vivo Performance
`George A. Digenis, Thomas B. Gold, and Vmod P. Shah ...................................................................................... ..
`
`915
`
`RESEARCH ARTICLES
`
`Interaction of Anionic Compounds with Gelatin. 1: Binding Studies
`Jaya Gautam and Hans Schott
`.................................................................................................................................. ..
`
`Product Inhibition and Dose-Dependent Bioavailability of Propranolol in the Isolated Perfused
`Rat Liver Preparation
`.
`_
`Hany Ghabrial, Romina Nand, Cheryl K. Stead, Richard A. Smallwood, and Denis J. Morgan .................. ..
`
`Characterization of Wet Granulation Process Parameters Using Response Surface Methodology.
`1. Top-Spray Fluidized Bed
`Douglas M. Lipps and Adel M. Sakr ........................................................................................................................ ..
`
`Estimation and Correlation of Drug Water Solubility with Pharmacological Parameters Required
`for Biological Activity
`Maurice M. Morelock, Louisa L. Choi, Gerald L. Bell, and James L. Wright
`
`................................................... ..
`
`Synthesis of N-Heteryl-B-_[(2-alkoxyethyl)0xy]/13-[[2-(MN-difilkylamino)ethy1]oxy]acetamides as
`Possible H1-Antihistamimcs
`A. Raghu Ram Rao and V. Malla Reddy .................................................................................................................. ..
`
`Effect of Cogrinding Time on the Release of Pentoxifylline from Waxy Matrix Tablets
`Makoto Otsuka and Yoshihisa Matsuda ....................................................................................................................
`
`Interactions of Cimetidine and Ranitidinewith ‘Alu1_ni_n_um-Containing Antacids and a
`Clay-Containing Gastric-Protective Drug In an Artificial Stomach—Duodenum” Model
`J. Vatier, A Harman, N. Castela, M. T. Droy-Lefaix, and R. Farinotti ............................................................. ..
`
`Stabilization of Topotecan in Low pH LiD0S0I!1eS Composed of Distearoylphosphatidylcholine
`Thomas G. Burke and Xiang Gan ............................................................................................................................. ..
`
`Pharmacodynamic Modeling Of Bafiterial Kinetics: (3-Lactam Antibiotics against Escherichia coli
`Ronald C. Li, David E. Nix, and Jerome J. Schentag ........................................................................................... ..
`
`Use of Everted Intestinal Rings for in Vitro Examination of Oral Absorption Potential
`Paula S. Leppert and Joseph A. Fix ......................................................................................................................... ,,
`
`2_6._ A Comparison of Anticancer Activities of Several
`In the Search for New Anticancer Drugs.
`TEPA, Thio-TEPA, Seleno-TEPA, and Azetldine Analogs, Including Congeners Containing an
`Aminoxyl Moiety
`George Sosnovsky, Jan Lukszo, Maria Konieczny, Klaus Purgstaller, and Frank Laib .................................. ..
`
`922
`
`931
`
`937
`
`948
`
`953
`
`956
`
`962
`
`967
`
`970
`
`976
`
`982
`
`Contents continued an IV
`
`Journal of Pharmaceutical Sciences / 11]
`Vol. 83, No. 7, July 1994
`
`Mylan v. Qualicaps, |PR2017—00203
`QUALICAPS EX. 2013 — 4/13
`
`Mylan v. Qualicaps, IPR2017-00203
`QUALICAPS EX. 2013 - 4/13
`
`

`

`In the Search for New Anticancer Drugs. 27. Synthesis and Comparison of Anticancer Activity
`in Vivo of Amino Acids, Carbohydrates, and Carbohydrate—Amino Acid Conjugates Containing the
`[N’-(2-Chloroethyl)-N’-nitrosoamino]carbonyl Group
`George Sosnovsky and C. Thomas Gnewuch ............................................................................................................ ..
`
`In the Search for New Anticancer Drugs. 28. Synthesis and Evaluation of Highly Active
`Aminoxyl Labeled Amino Acid Derivatives Containing the
`[N’-(2-Chloroethyl)-N’-nitrosoamino]carbonyl Group
`George Sosnovsky, Mustafa Baysal, and Ercin Erciyas ......................................................................................... ..
`
`Decreased Toxicity of Liposomal Amphotericin B Due to Association of Amphotericin B with
`High-Density Lipoproteins: Role of Lipid Transfer Protein
`Kishor M. Wasan, Richard E. Morton, Michael G. Rosenblum, and Gabriel Lopez-Berestein ........................
`
`Ondansetron Absorption in Adults: Effect of Dosage Form, Food, and Antacids
`Haig P. Bozigian, J. Frederick Pritchard, Ann E. Goodmg, and Gary E. Pakes .............................................. ..
`
`Evaluation of Ultrafiltration for the Free-Fraction Determination of Single Photon Emission
`Computed Tomography (SPECT) Radiotracers: ,6-CIT, IBF, and Iomazenil
`Mitchell S. Gandelman, Ronald M. Baldwin, Sam; S. Zoghbi, Yolanda Zea-Ponce, and Robert B. Innis
`
`Diabetes-Induced Reduction in the Hepatic Accumulation of 70-kDa Dextran: Role of
`Hyperglycemia and Hypoinsulinemia
`Reza Mehuar and James Reynolds ............................................................................................................................ ..
`
`1020
`
`Quantitative Analyses of the Structure—Hydrophobicity Relationship for N-Acetyl Di- and
`Tripeptide Amides
`_
`_
`Miki Akamatsu, Tadashi Katayama, Daisuke Kishimoto, Youichi Kurokawa, Hiroyuki Shibata,
`Tamio Ueno, and Toshio Fujita ...................................................................................................................................
`
`New Approaches for the Preparation of Hydrophobic Heparin Derivatives
`Jian Liu, Azra Pervin, Cindy M. Gallo, Umesh R. Desai, Cornelius L. Van Gorp, and Robert J. Linhardt
`
`Metabolites of Chloroquine: Some Observations on Desethylchloroquine and
`N-Acetyldesethylchloroquine
`O
`Aslam M. Ansari and J. Cymerman Craig ............................................................................................................... ..
`
`Liposomal Entrapment of Suramin
`Hung-Chih Chang and Douglas R. Flanagan .......................................................................................................... ..
`
`Prediction and Experimental Determination of Solute Output from a Collison Nebulizer
`Anna Y. Ip and Ralph W. Niven ............................................................................................................................... ..
`
`1026
`
`1034
`
`1040
`
`1043
`
`1047
`
`Theoretical Perspectives on Anthelmintic Drug Discovery: Interplay of Transport Kinetics,
`Physicochemical Properties, and in Vitro Activity of Anthelmintic Drugs
`Norman F. H. Ho, Sandra M. Sims, Thomas J. Vidmar, Jeff S. Day, Craig L. Barsuhn,
`Eileen M. Thomas, Timothy G. Geary, and David P. Thompson ..........................................................................
`
`1052
`
`COMMUNICATIONS
`
`Nonlinear Disposition of Hydroxyurea
`William G. Tracewell, William P. Vaughan, and Peter R. Gwilt
`
`...........................................................................
`
`1060
`
`A “Full-Space” Method for Predicting in Vivo Transdermal Plasma Drug Profiles Reflecting Both
`Cutaneous and Systemic Variability
`Patrick L. Williams and Jim E. Riviere ................................................................................................................... ..
`
`1062
`
`OPEN FORUM
`
`Comments on “Solvatochromic Linear Solvation Energy Relationship in Describing Drug
`Solubilities”
`Daniel C. Leggett
`
`.......................................................................................................................................................... ..
`
`Comments Concerning “Pharmacokinetic Characterization and Tissue Distribution of the New
`Glucocorticoid Soft Drug Loteprednol Etabonate in Rats and Dogs”
`Kenneth B. Sloan and John H. Perrin ..................................................................................................................... ..
`
`Response to the Commentary on “Pharmacokinetic Characterization and Tissue Distribution of
`the New Glucocorticoid Soft Drug Loteprednol Etabonate in Rats and Dogs”
`Guenther Hochhaus, Pascal Druzgala, John F. Howes, Nicholas Bodor, and Hartmut Derendorf ............... ..
`
`1065
`
`1066
`
`1067
`
`Journal of Pharmaceutical Sciences / V
`Vol. 83, No. 7, July 1994
`
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`

`u
`
`NATIONAL LIBRARY OF M DICINE
`
`\H \\\l\
`
`W E;
`
`I‘
`
`I
`
`V
`
`s«~
`
`NLH nnaaavaa 3
`
`acknowledge
`
`Parke-Davis Pharmaceutical Research
`
`A Division of the Warner-Lambert Company
`
`SmithKline Beecham Pharmaceuticals
`Research and Development
`
`for support of the
`Journal ofPharmaceutical Sciences
`
`.« ;.._.::...e_-_e.e,_e.-
`c
`T - ua '
`QUALICAPS EX. 2013 - 6/13
`
`Mylan v. Qualicaps, IPR2017-00203
`QUALICAPS EX. 2013 - 6/13
`
`

`

`A publication of the
`American
`Pharmaceutical
`Association
`and the
`American
`Chemical
`Society
`
`J O U R N A L O F
`
`Sciences
`
`July 1994
`Volume 83, Number 7
`
`REVIEW ARTICLE
`
`Cross-Linking of Gelatin Capsules and Its Relevance to Their in Vitro-in Vivo
`Performance
`
`GEORGE A. DIG EN IS.^, THOMAS B. GOLD', AND VINOD P. SHAHS
`Received October 14, 1993, from the "Division of Medicinal Chemistty and pharmaceutics, College of pharmacy, University of
`Kentucky, Lexington, KY 40536, and *Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug
`Accepted for publication February 23, 1994".
`Administration, Rockvilk, MD 20855.
`
`Abstract 0 The present revlew deals with the chemistry of gelatin
`cross-linking under conditions that are relevant to pharmaceutical
`situations. Mechanistic rationalizations are offered to explain gelatin
`cross-linking under "stress" conditions. These include elevated
`temperature and high humidity conditions. In addition, the chemical
`interactions between gelatin and aldehydes, such as formaldehyde
`and other formulation excipients, are discussed. The literature on the
`in vitro and in vivo dissolution and bioavailabili of a drug from stressed
`gelatin capsules and gelatin-coated tablets is reviewed. Cross-linking
`phenomena, occurring in stressed hard gelatin capsules and gelatin-
`coated tablets, could cause conslderable changes in the in vitro
`dissolution profiles of drugs. However, in a few cases, the bioavailability
`of the drug from the stressed capsules is not significantly altered when
`compared to that obtained from freshly packed capsules. It is
`concluded that, as with other drugdeiivery systems, careful attention
`should be paid to the purity and chemical reactivity of all excipients
`that are to be encapsulated in a gelatin shell. It is suggested that in
`vitro dissolution tests of hard gelatin-containing dosage forms be
`conducted In two stages, one in a dissolution medium without enzymes
`and secondly in dissolution media containing enzymes (pepsin at pH
`1.2 or pancreatin at pH 7.2, representing gastric and Intestinal media,
`respectively) prior to in vivoevaluation. Such in vitro tests may constitute
`a better indication of the in vivo behavior of gelatln-encapsulated
`formulations. Furthermore, testing for contamination with formaldehyde
`as well as low molecular weight aldehydes should be a standard part
`of excipient evaluation procedures.
`
`Introduction
`Gelatin is a mixture of water-soluble proteins derived primarily
`from collagen. It is obtained by boiling skin, tendons, ligaments,
`bones, and other similar products in water. Gelatin is extensively
`used in solid dosage forms: hard and soft gelatin capsules and
`
`Abstract published in Aduance ACS Abstracts, April 15, 1994.
`
`gelatin-coated tablets. It is a favorable material for use as the
`outer layer of drug formulations because of its glossy appearance,
`ability to hold dye color, neutral taste, and processing conven-
`ience. Gelatin has also been used as a stabilizer, thickener, and
`texturizer in foodstuffs.
`Due to the apparent dissolution problems in in vitro testing
`of hard and soft capsules, questions have been raised about the
`potential of cross-linking effects in gelatin.' The current
`literature tends to indicate that these effects primarily impact
`the in vitro testing methodology rather than the in vivo
`bioavailability of drugs formulated in hard gelatin capsules.
`Nevertheless, this chemical reactivity of gelatin must be taken
`into account when designing the final drug formulation.
`In general, the amino acid content of gelatin is glycine, 25.5 % ;
`proline, 18%; hydroxyproline, 14.1%; glutamic acid, 11.4%;
`alanine, 8.5 % ; arginine, 8.5 % ; aspartic acid, 6.6% ; lysine, 4.1 % ;
`leucine, 3.2% ; valine, 2.5 % ; phenylalanine, 2.2 % ; threonine,
`1.9%; isoleucine, 1.4%; methionine, 1.0%; histidine, 0.8%;
`tyrosine, 0.5%; serine, 0.4%; and cystine and cysteine, 0.1%.2
`Gelatin may be produced by lime hydrolysis of bones and animal
`skins (type B, isoelectric point = 4.7-5.3) or by acid hydrolysis
`of animal skins (type A, isoelectric point = 6.0-8.0).1 Hard gelatin
`capsules are usually prepared from a mixture of type A and B
`gelatins.
`The reactivity of the gelatin molecule appears to arise from
`the trifunctional amino acids it contains, specifically, lysine. In
`type B gelatin, the lysine-derived eamino function content was
`found to be 33.0 mol/gelatin molecule of 1000 amino acid
`residues.3 Carboxyl groups are far more plentiful than amino
`groups in gelatin but appear to be less reactive in reactions that
`involve cross-linking.' The amino groups arising from histidine
`residues are generally thought not to play as important a role
`in gelatin cross-linking as the c-amino groups of lysine.1 The
`highly basic guanidino group of arginine is protonated at neutral
`pH and consequently is thought to be unreactive unless a very
`high pH is employed.1 More recently, however, the participation
`
`0 1994, American Chemical Society and
`Amerlcan Pharmaceutical Association
`
`0022-3549/94/ 12069 15$04.50/0
`
`Journal of pharmaceutical Sciences / 915
`Vol. 83. No. 7, July 1994
`
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`

`

`Scheme 1
`of arginine in the hardening of gelatin in the presence of
`formaldehyde has been shown to be eminent.P6
`
`Chemistry of Gelatin Cross-Linking
`The cross-linking of the gelatin molecule is a well-known
`phenomenon that may occur by one or several chemical
`reactions? One possible reaction may involve the formation of
`desmosine-type cross-linking, known to occur in elastin fiber.8
`Lysine residues which are proximal to each other are oxidatively
`deaminated to yield terminal aldehyde groups. One of the
`aldehyde groups could then be attacked by a free €-amino group
`of a neighboring lysine to yield an imine which subsequently
`undergoes a series of aldol-type condensation reactions to produce
`a cross-linked product containing pyridinium ring(s): (Scheme
`1). Another possible gelatin cross-linking event involves the
`reaction of a lysyl t-amino group with an aldehyde (Scheme 2).
`(Trace quantities of formaldehyde may be present in corn
`starch,%12 which is an excipient in many drug formulations.)
`The initial product, a (hydroxymethy1)amino group (Scheme 2),
`eliminates water to give a cationic imine. The imine can react
`with another (hydroxymethy1)amino lysine residue to yield a
`dimethylene ether.' This ether may then rearrange to form a
`methylene link between two t-amino groups of lysine.
`
`016 /Journal of pharmceutlcel Sciences
`Vol. 83, No. 7, July 1994
`
`A third example of gelatin cross-linking is the formation of an
`imine arising from the reaction of the eamino group of lysine
`and an aldehyde, with subsequent formation of an aminal, the
`amine form of an acetal (Scheme 3). Step 1 of Scheme 3 involves
`the formation of a carbinolamine, which is rate limiting in acid
`pH, while step 3 involves the formation of the imine with
`concurrent water 10ss.13.14 Step 2 is rate limiting in base.l3J4
`The final step (step 4) requires the attack of a free amine on
`the cationic imine to give the aminal (Scheme 3). The optimal
`pH of formation of the aminal would inevitably be close to 7
`because each step (step 1 or 2) becomes rate limiting on either
`side of neutral pH. Aminal formation, like that of an acetal, is
`reversible at low pH. The acidic milieu combined with the
`presence of enzymes in the stomach explains why cross-linking
`in gelatin capsules may exert little influence on the in vivo
`dissolution rate of a drug.
`Glucose or other aldose sugars are often included in drug
`formulations and may provide yet another possibility for gelatin
`cross-linking. The aldehyde functional group of these saccharides
`may react with a free €-amino group to give an imine intermediate,
`which, through an Amadori rearrangement,15 produces a ketose
`sugar (Scheme 4). The formed aminoglycoside (ketose sugar)
`is then free to react with another amine through its carbonyl
`functionality to form the cross-linked gelatin.g.15
`
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`

`

`- CH, - .CH,-
`
`9
`
`R = alkvl
`
`Q-
`
`OH
`I
`-H
`R-N-C
`
`I
`I
`H R
`
`0
`R - N =C -H
`I
`I
`
`H R
`imine
`
`Scheme 2
`
`imine
`
`dimethylene ether
`
`cross-linked gelatin
`
`B
`n 8-
`R = CH2-CH2-CH2-CHz-@ B
`
`OH
`
`R-N-6-H + -k H+
`'
`I
`
`H H
`carbinolamine
`"hemiaminal"
`
`step 2
`
`I
`H
`
`I
`H
`
`
`
`
`
`-H,O Itstep 3
`
`I1
`0
`R-NNC-H
`
`'
`I
`H H
`cationic imine
`
`H
`cationic irnine
`
`H
`
`aminal
`cross-linked gelatin
`
`Scheme 3
`
`Types of Gelatin Cross-Linking
`
`In general, cross-linking within the gelatin polypeptide may
`occur in one of two ways. Bridging may take place within the
`same polypeptide strand (intrastrand, intramolecular cross-
`linking), or amino acid residues from two neighboring peptide
`strands may form a bridge (interstrand, intermolecular cross-
`linking) (Figure 1).
`Model reactions of the hardening of gelatin with W-labeled
`formaldehyde have recently been monitored by using 13C NMR
`spectroscopy in solution and solid state.5 It was found that when
`a 12% gelatin solution is treated with 0.5% W-labeled form-
`aldehyde, the latter first formed methylols (carbinolamines) of
`lysine residues and subsequently of arginine residues. After
`longer incubation periods with formaldehyde (72 h) lysine-
`arginine aminals (cross-links) are formed (Figure 1). During
`the drying process of hardened gelatin, in addition to the lysine-
`
`arginine cross-link, an arginine-arginine cross-link appears to
`form (Figure 1). Humidity strongly influences the rate of this
`cross-linking process.5
`
`Agents Which Catalyze Gelatin Cross-Linking
`
`Gelatin capsules undergo conformational change and cross-
`linking when stored under high-humidity conditions, as dem-
`onstrated by gemfibrozil, hydrochlorothiazide, and diphenhy-
`dramine hydrochloride capsules.le The cross-linking process
`causes formation of a swollen, rubbery, water-insoluble mem-
`brane (pellicle) during dissolution testing.17-19 This water-
`insoluble gelatin film acts as a barrier, restricting drug release.
`The mechanism through which humidity probably acts is by
`indirect catalysis of imine formation, which is the first inter-
`mediate in all the cross-linking reactions listed in the previous
`
`Jownai of phsrmaceutical Sciences / 017
`Voi. 83, No. 7, Ju& 1994
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`

`R-NH,
`
`I -
`
`C-H
`
`HO-C-H
`I
`R
`aldose sugar
`
`R
`I
`N O
`11
`C - H
`I
`HO-C-H
`I
`R
`imine
`
`- -
`
`imine-enamine
`
`R
`I
`N-H
`I
`C-H
`11
`tautomenzation
`HO- C
`I
`R
`enamine (enol)
`
`keto-enol
`
`tautomerization
`
`R
`I
`N-H
`I
`H-C-H
`I
`o= c
`I
`R
`ketose sugar
`(aminoglycoside)
`
`R-NH,
`
`-H2O
`
`N-H
`I
`
`I
`R
`
`R
`I
`N-H
`I
`H - C - H
`I
`C=N-R
`I
`R
`cross-linked product
`
`Scheme 4
`
`;iI!l "f""" H
`
`I
`
`I "
`
`I
`
`
`
`I
`
`"
`I
`H O L H
`
`I i v 1
`'
`H H O H H
`
`1
`
`
`
`@= site of possible peptide bond scission by pepsin
`@= site of possible peptide bond scission by pancreatin
`
`H I
`
`I
`H
`
`Flgure 1
`
`section. For example, corn starch, a drug excipient, may contain
`traces of the stabilizer hexamethylenetetramine,9-12 which
`decomposes under humid conditions to form ammonia and
`formaldehyde (Scheme 5).20 The latter may react with lysine
`residues present in gelatin to form imines, which subsequently
`
`can undergo any of the possible cross-linking reactions described
`above (Schemes 1-4).
`The presence of aldehydes, among other reactants, leads to
`an increase in the molecular weight of gelatin, likely due to
`formation of interstrand bridges across its polypeptide backbone?
`
`918 / Journal of Pharmaceutical Sciences
`Vol. 83, No. 7, July 1994
`
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`QUALICAPS EX. 2013 - 10/13
`
`

`

`Reversibiiity of Cross-Linking
`reversibility of the aminal reaction
`1. Nonenzymatic-The
`(Scheme 3, step 4), as well as that of the imine functionality,
`shows a definite pH dependence (Scheme 3, steps 2 and 3). In
`general, the aminal decomposition (Scheme 3, step 4) is reversible
`under basic c0nditions~3.14 while steps 2 and 3 (imine to
`carbinolamine) are reversible in acidic media. Carbinolamine
`decomposition (step 1) occurs under basic conditions.13J4
`2. Enzymatic-The
`impeding barrier that is exerted upon
`drug molecules by a highly cross-linked gelatin capsule wall can
`be alleviated by the presence of pepsin and/or pancreatin.2ss2s.29
`Gastric juice, at pH 1.2, consists mainly of the enzyme pepsin,
`an acid-dependent endopeptidase which cleaves peptides proxi-
`mal to hydrophobic and aromatic amino acids (Figure 1).30
`Likewise, pancreatin, which contains the protease enzyme
`trypsin, an endopeptidase which operates at neutral pH and
`preferentially cleaves the carboxyl