b! A L 0
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`acoutical
`SClOnC6S
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`A publication of the
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`Lupin Ex. 1038 (Page 1 of 11)
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`© Copyright 1998 American Chemical Society and American Pharmaceutical Association
`
`Lupin Ex. 1038 (Page 2 of 11)
`
`

`

`(~) A publication of the
`
`American
`Pharmaceutical
`Association
`and th?
`Amer,can
`Chemical
`Society
`
`JOURNAL 0 F
`
`Pharmaceutical
`sciences
`
`May 1998
`
`Volume 87, Number 5
`JPMSAE 87(5) 531-666
`$£N 0022454g
`
`RESEARCH ARTICLES
`
`Scintigraphic Evaluation of a New Capsule-Type Colon Specific Drug Delivery System in Healthy
`Volunteers
`T. Ishibashi, G. R. Pitcairn, H. Yoshino, M. Mizobe, and I. R, Wilding* ........................................................................... 531
`
`Formation of Isomorphic Desolvates: Creating a Molecular Vacuum
`Gregory A. Stephenson,~" Edward G. Groleau, Rita L. Kleemann, Wei Xu, and Daniel R. Rigsbee ................................. 536
`
`An Investigation of FB Interactions with Poly(ethylene glycol) 6000, Poly(ethylene glycol) 4000, and
`Poly-~-caprolactone by Thermoanalytical and Spectroscopic Methods and Modeling
`F~ Lacoulonche,* A. Chauvet, J. Masse, M. A. Egea, and M. L. Garcia ............................................................................. 543
`
`Detection of Suppression of Bitterness by Sweet Substance Using a Multichannel Taste Sensor
`Sou Takagi, Kiyoshi Toko,* Koich~ Wada, Hiromi Yamada, and Kenzo Toyoshima ......................................................... 552
`
`A Study on Gelatin Capsule Brittleness: Moisture Tranfer between the Capsule Shell and Its Content
`Rong-Kun Chang, Krishnaswamy S. Raghavan, and Munir A. Hussain* ......................................................................... 556
`
`An Investigation into the Use of Low-Frequency Dielectric Spectroscopy as a Means of Characterizing
`the Structure of Creams Based on Aqueous Cream BP
`Paul L. Goggin, Renren He, Duncan Q. M. Craig,* and Donald P. Gregory ..................................................................... 559
`
`Is the Pig a Good Animal Model for Studying the Human Ileal Brake?
`Clair L. Dobson, Mike Hinchcliffe, Stanley S. Davis, Sushil Chauhan, and Ian R. Wilding* ......................................... 565
`
`Absorption and Intestinal Metabolism of Purine Dideoxynucleosides and an Adenosine
`Deaminase-Activated Prodrug of 2’,3’-Dideoxyinosine in the Mesenteric Vein Cannulated Rat Ileum
`Dharmendra Singhal, Norman F. H. Ho, and Bradley D. Anderson* ................................................................................ 569
`
`Optimization of the Local Inhibition of Intestinal Adenosine Deaminase (ADA) by
`erythro-9-(2-Hydroxy.3-nonyl)adenine: Enhanced Oral Delivery of an ADA-Activated Prodrug for
`Anti-HIV Therapy
`Dharmendra Singhal and Bradley D. Anderson* ................................................................................................................ 578
`
`Effect of Interparticulate Interaction on Release Kinetics of Microsphere Ensembles
`X. Y. Wu,* G. Eshun, and Y. Zhou ........................................................................................................................................ 586
`
`Pharmacokinetics of Aniracetam and Its Metabolites in Rats
`Taro Ogiso,* Masahiro Iwaki, Tadatoshi Tanino, Kazuyuki Ikeda, Tsuyoshi Paku, Yoshihide Horibe, and
`Hiroko Suzuki .......................................................................................................................................................................... 594
`
`The Effect of Ionic Strength on Liposome-Buffer and 1-Octanol-Buffer Distribution Coefficients
`Rupert P. Austin,* Patrick Barton, Andrew M. Davis, Carol N. Manners, and Michael C. Stansfield ........................... 599
`
`Model-Independent Estimation of Lag Times with First-Order Absorption and Disposition
`Ferenc C~izmadia and Laszlo Endrenyi* .............................................................................................................................. 608
`
`Perfusion Cells for Studying Regional Variation in Oral Mueosal Permeability in Humans. 2. A
`Specialized Transport Mechanism in D-Glucose Absorption Exists in Dorsum of Tongue
`Yufi Kurosaki, Koji Yano, and Toshikiro Kimura* .............................................................................................................. 613
`
`Contents continued on VII
`
`Journal of Pharmaceutical $~/ences /V
`Vo! 87, No. ~ May 1998
`
`Lupin Ex. 1038 (Page 3 of 11)
`
`

`

`Liposomal Delivery of a 30-met Antisense Oligodeoxynucleotide To Inhibit Proopiomelanocortin
`Expression
`Massimo Fresta~ Rosa Chillemi, Santi Spampinato, Sebastiano Sciuto, and Giovanni Puglisi* .....................................
`
`616
`
`Species Differences in Size Discrimination in the Paracellular Pathway Reflected by Oral
`Bioavailability of Poly(ethylene glycol) and D-Peptides
`Yah-Ling He, Susan Murby, Geoffrey Warhurst, Larry Gifford, David Walker, John Ayrton, Richard Eastmond, and
`Malcolm Rowland~ ................................................................................ ~. .................................................................................
`
`What Surface of the Intestinal Epithelium Is Effectively Available to Permeating Drugs?
`Ruth E Oliver, Alar~ F. Jones, and Malcolm Rowland* ~ "
`
`Spontaneous Chemiluminescence Production, Lipid Peroxidation~ and Covalent Binding in Rat
`Hepatocytes Exposed to Acetaminophen
`Yoshiyuki Minamide, Toshiharu Horie,* Atsuko Tomaru, and Shoji Awazu ....................................................................
`
`Solid State Characterization of Spray-Dried Powders of Recombinant Human Deoxyribonuclease
`(RhDNase)
`Hak-Kim Chan* and Igor Gonda ........................................................................................................... ~ ................................
`
`Solid-State Characterization of Chlordiazepoxide Polymorphs
`Dilraj Singh, Peter V. Marshall, Len ShieIds~ and Peter York* "
`
`:626
`
`634
`
`647
`
`655
`
`.,Meropenem Exists in Equilibrium with a Carbon Dioxide Adduct in Bicarbonate Solution
`Orn Almarsson,* Michael J. Kaufman, John D. Stong, Yunhui Wu, Suzanne M, Mayr, Mark A, Pe~rich, and
`J. Michael Will~ams ...................................................................... : ..................................................................................... : ....
`
`663
`
`NOTES
`
`There is no Supporting Information for this issue.
`
`In papers with more than one author, an asterisk (*) in the byline indicates the author to whom
`inquiries should be directed.
`
`Journal of Pharmaceutical Sciences/Vii
`Vo/ 87, No, 5, May 1998
`
`Lupin Ex. 1038 (Page 4 of 11)
`
`

`

`Formation of Isomorphic Desolvates: Creating a Molecular Vacuum
`
`GREGORY A. STEPHENSON,* EDWARD G. GROLEAU, RITA L. KLEEMANN, WEI Xu,t’:~ AND DANIEL R, RIGSBEE
`
`Contribution from L#ly Research Laboratories, Eli Lilly and Oompany, Lilly Oorporate Oenter, Indianapo#s, Indiana 46285, and
`Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana, 47907-1323.
`
`Received November 25, 1997, Accepted for publication February 17, 1998,
`
`Abstract [] The objective of this work was to investigate a common
`but poorly understood category of crystalline organic substances:
`isomorphic desolvates. When solvent is lost from a crystal lattice
`but the lattice retains its three-dimensional order, a lattice is created
`which is in a high-energy state relative to the original solvate structure,
`The desolvated lattice can reduce its internal energy by either resorbing
`solvent or by relaxation processes which increase the packing
`efficiency of the solid by reducing the unit cell volume. In the following
`paper, solid-state properties of isomorphic desolvates of cephalexin,
`cefaclor, erythromycin A, and spirapril hydrochloride hydrates are
`investigated. The hygroscopiCity of the compounds are evaluated
`using a vacuum moisture balance, and structural relaxation is
`measured using a combination of X-ray powder diffraction and
`isothermal microcalorimetry. The study results are explained in terms
`of Kitaigorodski’s close packing principle.
`
`Introduction
`Hydrates which desolvate yet retain their original crystal
`lattice are common. The dehydrated structure is extremely
`hygroscopic when reexposed to elevated humidities. The
`Latin proverb "natura vacuum abhorrer" provides insight
`into this solid-state behavior. A paper by Pfeiffer et al.
`characterizes such systems as a "frequently undetected or
`poorly described property of powder technology’.1 A crys-
`talline form is described therein which is stoichiometrically
`solvated while in equilibrium with a saturated solution,
`loses most of its solvent upon drying, but does not convert
`to a different crystalline form. The term isomorphic
`desolvate accurately defines a desolvate which retains the
`structure of its parent solvate form, since it indicates that
`the desolvated structure retains the three-dimensional
`order of the original crystal, as defined by space group
`symmetry and the lattice parameters. Pfeiffer further
`emphasizes the importance of referring to the parent
`solvated structure when naming the desolvated structure.
`The "desolvated acetonitrilate of cephalexin" would be used
`to designate a form which maintains the structure of the
`acetonitrile solvate yet no longer possesses the solvent of
`crystallization.
`The relationship of an isomorphic desolvate to its sol-
`vated form is readily apparent through similarities in X-ray
`diffraction patterns. Despite crystallogrdphic similarities,
`the physical properties of the solvate and desolvate may
`differ substantially. One/difference is the indiscriminate
`voracity for small molecules to fill void space in the
`desolvated crystal lattice. In the case of the acetonitrile
`disolvate of cephalexin, the acetonitrile molecules can be
`
`*Corresponding author. Telephone: (317)27%3841. E-maih
`Stephenson_Gregory_A@lilly.com.
`* Purdue University.
`* Current Address: Merck Research Laboratories, Mersk and
`Company, WP78-302, West Point, PA, 19486.
`
`removed and replaced with a different solvent without
`altering its crystal lattice substantially from that of its
`original solvate form.1 The reduced packing efficiency of
`the desolvated lattice results in a net decrease in lattice
`energy; i.e., it becomes less stable, relative to the solvated
`structure. The thermodynamic driving force, or molecular
`vacuum, created by desolvation can be relieved through
`density-increasing processes, either by incorporation of
`small molecules into the lattice (resolvation) or by struc-
`tural relaxation (annealing). The formation of isomorphic
`desolvates can result in an extremely hygroscopic solid or
`reduced chemical stability.~-4 The following paper will use
`the concepts developed by Kitaigorodski et al.s to under-
`stand ~he physical properties of isomorphic desolvate
`crystalline forms of cephalexin monohydrate, cefaclor
`monohydrate, erythromycin A dihydrate, and spirapril
`hydrochloride menohydrate.. Erythromycin A dihydrate
`and spirapril hydroch!oride monohydrate provide an in-
`teresting perspective on isomorphic desolvate systems since
`their crystallographic structures have been determined.
`Packing Efficiency--Kitaigorodski developed a theory
`of crystal packing for organic molecules that states that
`the lowest energy stucture is that which minimizes the void
`space in the lattice and maximizes the number of close
`contacts with neighboring molecules.5 Calculations of
`packing coefficients, as in eq 1, make., the assumption that
`molecular volume does not change from one crystal form
`to another. Gavezzotti has demonstrated that this is a
`reasonable assumption through his analysis of organic
`molecular structures.6,7 By statistical analysis of 204
`polymorphic pairs, he showed a linear correlation of unit
`slope between the difference in densities of polymorphic
`pairs and difference in packing coefficients for polymorphic
`pairs. The density rule is used as an indicator of the
`relative stability of crystalline polymorphic forms.6,s The
`rule states that the modification which has the lowest
`density will be the least stable one at absolute zero. The
`theory is often applied to polymorphic forms at room
`temperature; the polymorphic form having the closest
`packing, highest density at a given temperature is consid-
`ered to have the lowest overall free energy at that tem-
`perature. Exceptions to the rule can occur due to neglect
`of consideration of the entropic contribution to the free
`energy of polymorphs~ or when strong hydrogen bonds are
`present in the less densely packed structure.9 The density
`rule still remains one of the common criteria for assessing
`relative stabilities of polymorphic systems.
`When chemical composition changes, as in the case of
`comparing a solvate to its isomorphic desolvate form,
`density comparisons are inappropriate. Packing coef-
`ficients provide a convenient means for comparing struc-
`tures of different composition. In an effort to understand
`the thermodynamic instability imparted on ~he crystal
`lattice due to reduced packing efficiency of isomorphic
`desolvates, one may turn to Kitaigorodski’s packing coef-
`ficient, as in eq 1.
`
`536 / dournal of Pharmaceutical Sciences
`Vol, 87, No. 5,, May 1998
`
`$0022-3549(97)00449-B CGC: $~5.00
`Pub#shed on Web 04/07/1998
`
`© 1998, American Chemical Society and
`American Pharmaceutical Association
`
`Lupin Ex. 1038 (Page 5 of 11)
`
`

`

`Ck = ZVm/go
`
`where Z is the number Of molecules present in the unit
`cell, Vm is the volume which is occupied by the molecule,
`and Vo is the volume of the unit cell of the crystal lattice.
`Thermodynamic calculations of lattice energetics include
`a van der Waals term, a Coulombic term, and a hydrogen-
`bdnding term in the energy expression; see eq 2.
`
`Elattiee = Even der Weals -[- ECoulombic -1- Ehydrogen bonding (2)
`
`The van der Waals contribution, being the term of greatest
`magnitude in the expression, is the primary term consid-
`ered in ~he derivation of the close-packed principle.
`The van der Waals term is often expressed using the
`Lennard-Jones functional form, as shown in eq 3,
`
`Evan der WealsF ~ At-12 - Br-6
`
`(3)
`
`where r is the interatomic separation of atoms and A and
`B are Lennard-Jones coefficients, The van der Waals
`interactions fall off rapidly as interatomic separation
`increases. Given a crystal lattice of the same molecular
`content, the lattice energy is reduced as cell volume; i.e.,
`atomic separation, increases. A corollary may be applied
`to solvates and their isomorphic desolvates. If the unit cell
`volume remains essentially constant but the molecular
`volume present in the lattice is decreased, the lattice
`energy is reduced. The lattice energy difference created
`by desolvating a solvate to its isomorpbAc desolvate, therein
`reducing the molecular volume present in the unit cell,
`creates a high-energy state that can be relaxed through
`processes which increase packing efficiency. Specific objec-
`tives of this paper are to provide an understanding of why
`isomorphic desolvates have the general charac.teristic of
`extreme hygroscopicity and to provide a basic understand-
`ing of what happens to the crystal lattice in the event that
`it is not allowed to rehydrate,
`
`Materials and Methods
`
`Materials--Cephalexin monohydrate and cefaclor monohydrate
`were provided by Eli Lilly and Co, Spirapril hydrochloride was
`provided by Sandoz Pharmaceuticals Corp. as the monohydrate.
`Erythromycin A was purchased from Sigma Chemical Co. The
`dihydrate sample was prepared by placing the material in water
`at room temperature and allowing the sample to reach equilibrium
`over a period of approximately 1 week; the sample was then
`filtered and allowed to dry in a desiccator at 55% relative humidity
`(RH).
`X-ray Powder Diffraction--Hydrated samples were mixed
`with fluorophlogopite, the NIST #675 Iow,angle diffraction stan-
`dard, and packed into a sample holder, and data were collected
`under ambient conditions. The sample, while still in its holder,
`was placed in a desiccator over P205, The sample was taken from
`the desiccator and analyzed periodically to monitor changes in its
`diffraction pattern.
`For each analysis, the dehydrated sample was placed in the
`diffractometer~s Anton-Paar environmental chamber containing
`2-4 cups of P20~ as a desiccant. The Anton-Paar chamber
`allowed the diffraction experiment to be conducted under con-
`trolled humidity conditions, that is ~0% RH. Each sample was
`allowed 4 h for equilibration within the chamber prior to data
`acquisition. The NIST #675 standard was used to correct for any
`20 displacement error, This standard ensured a high degree of
`accuracy in the d spacings measured from the diffraction experi-
`ment.
`Data were collected on a Siemens D5000 X-ray diffractometer
`from 3° to 45° 20. The samples were illuminated with Cu K~
`radiation (2 = 1.54056/~) at 50 kV and 40 mA. A nickel filter
`was used to reduce the Kfl contribution to the X-ray signal and a
`scintillation counter was used for detection. Variable divergence
`
`and antiscatter slits maintained 6 mm illumination of the sample
`throughout analysis. The detector slit was fixed at 0.1 mm.
`Moisture Sorption--Data were generated ufiing a VTI vacuum
`moisture balance (MB300G). Between 15 and 26 mgwas analyzed
`with a sample temperature of 25 °C and a drying temperature of
`30 °C. Sorption/desorption was observed over a range of 0-95%
`RH with a 5% RH step interval. The weight was sampled every
`10 min. The equilibrium criterion was satisfied if not more than
`a 5 /~g difference was observed for three successive sampling
`inter~als;
`Additional analyses were conducted on erythromycin A and
`spirapril hydrochloride. For erythrqmycin A the drying step was
`eliminated and the instrument wa~equilibrated under vacuum.
`Following equilibration, the instrument ramped up to 20% relative
`humidity at the fastest rate possible (approximately 1%/min).
`Spirapril hydrochloride was examined by drying at 30 °C and
`ramping to 20, 40, 60, or 80%
`Isothermal Microcalorimetry-All samples were initially,,
`equilibrated to 33% RH by storing overnight over a saturated ~a~
`solution of magnesium chloride in a desiccator. A portion of this
`material was placed under vacuum for, 5 h over P205; the rest was
`kept in the desiccator. The "dried" and "as-is" samples were
`transferred into glass ampules in a glovebag purged with N2 at
`the appropriate relative humidity. The ampules were then loaded
`into the calorimeter for equilibration to 40 °C.
`The thermal activity was monitored using a Thermometric
`isothermal microealorimeter equipped .with a computer for data
`acquisition. The instrument was electronically calibrated accord-
`ing to the instrument manual. All four channels were used to
`obtain replicate results from each sample. Typically, data were
`recorded over a period of several days.
`Calculation of Packing CoefficientS--The number of mol-
`ecules in the unit cell was determined by single-crystal X-ray
`diffraction. Molecular volumes were calculated from the single-
`crystal data using the Cerius~,~ Connolly surface module.~ Unit
`cell volumes were determined experimentally from the X-ray
`powder diffraction patterns and calculated using the standard
`equations for orthorhombic unit cells.
`
`Results and Discussion
`
`Crystalline forms which are presented herein are often
`called pseudopolymorphs,~ nonstoichiometric solvates?~;~
`or solid solutions.~4 Such terminology indicates that
`crystalline: forms of ~his category are not discrete phases
`but rather fall into a gray area of solid-state chemistry.
`The number of solvent molecules in the crystal lattice is
`variable, being dependent on the activity of the solven~
`molecules in the surrounding environment. Contrary to
`intuition, the solvent molecules in the structures of eryth-
`romycin A dihydrate and spirapril hydrochloride monohy-
`drate are not randomly located in the crystal lattice. The
`water molecules are localized in the lattice and. form
`hydrogen bonds. By and large, we find that solvates exist
`as stoichiometric solvates when first isolated from the
`environment from which they were recrystallized; however,
`when removed from a state of equilibrium into a nonequi-
`librinm state, deficient of the solvent of recrystallization,
`they take on the nonstoichiometric characteristic. In the
`case of isomorphic desolvates, the: degree to which they are
`dehydrated is a function of the relative humidity.14b Their
`nonstoichiometric nature may be attributed to a tendency
`to establish equilibrium with the environment and a low
`activation energy for solvation!desolvation, since structural
`transformation is not prerequisite. The. structures of
`erythromycin A dihydrate~ and spirapril hydrochloride
`monohydrate~ have been determined and serve as model
`structures which form isomorphic desolvates; see Figures
`1 and 2.
`Solvate crystals often have large solvent channels which
`are readily observed by examining the crystallographic
`packing.~ The lattice oferythromycin A shows that water
`molecules must traverse a solvent tunnel which zigzags
`
`Journal o~ Pharmaeeutica/ Sciences /537
`Vol. 87, No. 5,. May ~98
`
`Lupin Ex. 1038 (Page 6 of 11)
`
`

`

`Vapor Sorption Kinetics
`
`~. 2
`
`,~.~"
`
`1 ]
`
`o
`
`¯
`20% relative humidity
`o 80% relative hu~dity
`
`Time (~nutes)
`Fiflur~ ~--Watar uptako el tho isomorphic 6~solvat~ of sNrapril hg6rochlori6a
`monohNrat~ when ~x~os~6 to 6ifferent r~latiw humi6iti~s,
`
`close to equilibrium, into an ambient environment and into
`ambient humidity, the "hydrate" itself is perceived as
`hygroscopic. The nonequilibrium state of the bulk drug
`substance can persist for days or months, depending upon
`the moisture permeability of its container, its liner, and
`the relative humidity of its storage environment. The
`hygroscopicity of the compound makes accurate weighing
`difficult and creates difficulties for formulators and ana-
`lytical chemists.4,1s Truly the hydrate itself is not hygro-
`scopic, but rather the desolvated form is hygroscopic.
`Protection of an isomorphic desolvate from moisture merely
`delays the transition toward equilibrium with the ambient
`environment.
`Other problems may result from a lattice which is void
`of solvent. Since oxygen is a small molecule and partici-
`pates in many solid-state reactions,, chemical instability
`may be altered by providing accessibility.17 For example,
`water typically increases the rate of chemical decomposi-
`tion through its interaction with amorphous components;19
`however, in tunnel solvates the solvent may impart a
`stabilizing effect.2°,21 !n hydrocortisone tert.butyl acetate
`form II, the solvent acts as a stabilizer.2 When desolvation
`occurs, an isomorphic desolvate is formed and oxygen is
`able to penetrate the lattice and cause oxidation.
`Due to the extreme hygroscopicity of isomorphic desel-
`vates, even a minor fraction of the bulk existing in the
`desolvated state may result in "hygroscopic" behavior.~
`Figure 3 illustrates the hygroscopic behavior of spirapril
`hydrochloride monohydrate’s isomorphic desolvate form.
`The rate of moisture sorption of the desolvate exposed to
`20% RH shows that the compound increases weight at a
`rate of approximately l%/min until a stoichiometric mono-
`hydrate water level is achieved. The rate of moisture
`sorption was examined at constant humidities of 20, 40,
`60, and 80% and showe.d little difference in the rate of
`sorption. Apparently, the rate is limited primarily by the
`rate of diffusion of water molecules into the solvent tunnels.
`Sorption and desorption isotherms were measured on
`each of the isomorphic desolvate/solvate systems. Figure
`4 provides the sorptlon isotherms for the different isomor-
`phic desolvates. Equilibration of isomorphic desolvates at
`different reIative humidities is rapid and reversible. The
`desorption isotherms, not shown, show Iittle sign of hys-
`teresis with respect to their adsorption isotherms~ The
`adsorption characteristics show substantial increase in
`water content in going from 0 to 10% RH. Generally, the
`rapid rise of water absorption at low relative humidities
`
`Lupin Ex. 1038 (Page 7 of 11)
`
`Figure 1--Molecular modeling perspeotive of the solvent tunnel of erythromyoln
`A dihydrate, The two crystallographically independent water molecules are
`shown as ball-and-stick, whereas erythromycln molecules are depicted as
`cylinders,
`
`Figure 2~Moleoular modeling of spirapril hydrochloride monohydrate stucture
`shows that a relatively small solvent tunnel runs through the crystal lattice.
`
`along the a-axis of the crystal in order for it to form its
`isomorphic desolvate. The tunnel’s cross section is ap-
`proximately circular with a radius of 4 ~. In the lattice of
`spirapril hydrochlorlde monohyd~rate, t~he Water tunnel is
`approximately rectangular, 10 A × 5