Binding of Organic Electrolytes by
`Surface-Active Agent
`
`Nonionic
`
`By ARTHUR
`
`HURWITZt PATRICK
`
`DELUCAt and
`
`KOSTENBAUDER
`
`Although possible consequences of combining ionic pharmaceuticals with surface-
`active agents of opposite charge are generally recognized there has been little or no
`consideration of possible interactions between drug ions and nonionic surface-active
`agents
`In the present study cations such as chlorpromazine promethazine tetra
`caine methylrosaniline and dodecylpyridinium and anions such as naphthalene
`suffonate and methyl orange were bound to the nonionic surface-active agent poly
`sorbate 80 The degree of interaction in some cases is sufficient
`that
`to suggest
`on the stability and availability
`polysorbate 80 might have considerable
`influence
`of ionic drugs in pharmaceutical formulations
`
`agents in
`
`with
`
`agent
`
`typical nonionic surface-active
`The existence
`
`of
`
`polysorbate 80.1
`interaction between
`
`degree of
`
`JHE USE OF nonionic surface-active
`
`the formulation
`
`of various pharmaceutical
`
`some antimicrobial
`
`dosage forms is often
`routine procedure
`Their
`presence in pharmaceuticals sometimes results in
`incompatibilitiesnotably
`the inactivation
`of
`agents which are commonly
`used in pharmaceuticals for
`their preservative
`activity 116 In the majority of examples of
`incompatibility which have previously been de
`scribed the agent which is bound to the surfac
`tant is primarily in molecular or nonionized form
`interaction with the surfactant has generally been
`
`explained on the basis of either the formation of
`specific molecular complexes or the preferential
`in the
`surfactant micelle
`Persons
`
`solubility
`
`engaged in pharmaceutical
`
`formulation are gen
`erally cognizant of possible incompatibilities
`of
`this nature
`considerable dis
`Although textbooks devote
`which might
`cussion
`to incompatibilities
`from the combination of an ionic surfactant with
`drug or germicide of opposite charge there is
`usually no suggestion of possible incompatibility
`or inactivation which might arise from the com
`bination of an ionic drug or germicide with
`non-
`Recent
`ionic surfactant
`observations
`in these
`
`arise
`
`can
`
`laboratories indicated that organic ions such as
`ammonium germicides
`be
`quaternary
`by nonionic surf actants 16
`bound
`strongly
`These observations suggest
`that such generally
`incompatibilities might exist
`unrecognized
`many pharmaceutical systems and might mark
`drug from dosage
`the release of
`edly influence
`drug The
`form or might alter the stability of
`study was designed
`to investigate the
`present
`possible association of various cations and anions
`
`in
`
`Received December 18 1962 from the School of Pharmacy
`Temple University Philadelphia Pa
`for publication February 18 1963
`Accepted
`Presented to the American Association for the Advance
`ment of Science New York meeting December 1960
`Fellow of the American Foundation
`for Pharmaceutical
`of Phar
`19591960
`Present address
`Education
`School
`macy The University of Wisconsin Madison
`Karr Fellow Smith Kline and French Labo
`Walter
`ratories Philadelphia Pa
`ciba Pharma
`Present address
`ceutical co Summit
`
`893
`
`significant
`ionic drugs and
`re-evalua
`
`nonionic surfactants might
`require
`tion of the routine use of nonionic surface-active
`
`agents in pharmaceutical formulation
`
`EXPERIMENTAL
`
`promethazine
`
`Reagents.Ephedrine hydrochloride N.F sulfa
`sodium N. methylrosaniline chloride
`thiazole
`U.S.P
`U.S.P
`hydrochloride
`N.F
`diphen
`hydrochloride
`niethapyriline
`hydramine hydrochloride U.S.P chlorproiuazine
`tetracaine
`hydrochloride
`U.S.P.5
`hydrochloride
`cetylpyridinium chloride U.S. poly
`U.S.P.6
`sorbate 80
`commercial sample sodium salt of
`sulfonic acid recrystallized from
`2-naphthalene
`dodecylpyridinium bromide
`water
`laboratory
`were used
`prepared
`All other
`chemicals were
`reagent grade
`Method for Detecting
`InteractiostEquilibrium
`possible drug
`dialysis was employed
`to detect
`The
`interaction
`nonionic
`agent
`surface-active
`technique was similar to that described previously
`by Patel and Kosteabauder 17 in their study of
`the interaction of methyl and propyl p-hydroxy
`benzoate with polysorbate 80
`This method in
`semi
`volves equilibration of two solutions across
`permeable membrane one solution containing
`the
`agent and the other
`con
`surface-active
`solution
`Nylon membranes were
`taining only the drug
`used in this study these membranes being selected
`to permit the drug to come to equilibrium in both
`solutions but
`the nonionic
`to prevent
`passage of
`surface-active agent
`The nylon membranes were cut
`to give bags
`which held 20 ml of solution
`The bags when
`filled with the solution
`the ionic agent being
`of
`in wide mouth
`and placed
`
`studied were
`
`tied
`
`20 sorbitan monooleate is marketed as
`Polyoxyethylene
`80 by Atlas Powder Co Wilmington Del
`Tweeu
`Marketed
`as Pheuergau
`by Wyeth
`Hydrochloride
`Laboratories Philadelphia Pa
`Marketed as Histadyl Hydrochloride by Eli Lilly and Co
`Indianapolis lad
`Marketed
`by Parke Davis
`as nenadryl Hydrochloride
`and Co Detroit Mich
`Marketed
`as Thorazine Hydrochloride
`and French Laboratories Philadelphia Pa
`Marketed
`as Pontocaine Hydrochloride
`Laboratories New York
`Marketed
`as Ceepryn by The William
`Cincinnati Ohio
`
`by Smith Kline
`
`by Winthrop
`Merrell Co
`____________
`1EXHIBIT 02e31
`Iwii bL2t%I
`DATE ___________
`DAWN HILLIER RMR CRR
`
`ALKERMES Exh. 2032
`Luye v. Alkermes
`IPR2016-1096
`
`

`

`894
`
`1.5
`
`1.4
`
`1.3
`
`1.1
`
`1.0
`
`POLYSOR BATE 80
`
`10
`W/V
`
`Fig 1.Binding
`tetracaine
`hydrochloride
`polysorbate 80 at 30
`
`of
`
`by
`
`screw-cap glass bottles containing 60 ml of
`poly
`sorbate 80 solution with
`concentration
`ionic
`of
`By
`in the internal phase
`to that
`agent equal
`starting with equal concentrations
`of drug in both
`phases the equilibration or agitation time could be
`Also shorter agitation time de
`greatly reduced
`the possibility of membrane
`breakage
`creased
`The bottles were then tightly closed using
`piece
`the cap and the bottle
`film between
`of polyethylene
`The bottles were agitated
`leakage
`top to prevent
`constant
`temperature water bath at 390 for
`in
`days Aliquot portions were then taken from
`to
`both the internal and external phases and assayed
`for the ionic agent using
`spectrophotometrically
`Beckman DU spectrophotometer
`an appro
`and
`priate blank solution in each case
`few agents
`were studied in the presence
`of potassium chloride
`to determine the effect of salt on the interaction
`sodium bisulfite was
`and
`added
`to solutions
`of
`and chlorpromazine
`to retard oxida
`promethazine
`tion The spectrophotometric
`of methyl
`assays
`orange and methylrosaniline chloride were per
`formed on the Beckman model
`spectrophotometer
`The addition of polysorbate 80 2% w/v caused
`in the absorption maximum of methyl orange
`shift
`to 425 mgi and the maximum for
`from 465 mgi
`methylrosaniline chloride was shifted from 595 mgi
`The polysorbate-containing
`solutions
`to 600 mgi
`of these agents were always assayed by ndjusting
`to 2% then
`concentration
`the final polysorbate
`determining the absorbance at the shifted maximum
`The extent of binding
`is represented as Cs/Co
`of or
`where
`represents the total concentration
`on the polymer-containing side of
`ganic electrolyte
`the membrane and
`on the
`is the concentration
`calculation
`side For
`nonpolymer-containing
`of
`of binding the ratio total/
`the apparent
`degree
`free is taken as equal to the ratio C/C0
`Actually
`result of Donnan effect
`the free concentration
`as
`on the polymer-containing side of the membrane is
`less than Co and the actual
`C/
`ratio total/free
`C0
`The complexity of the systems prevents calcula
`the Donnan
`for most of
`correction
`the
`tion of
`examples of binding considered in this report but
`correction would indicate even
`if applied such
`greater binding than the apparent
`ratios reported
`here
`
`Journal of Pharmaceutical
`
`Sciences
`
`RESULTS
`
`illus
`
`is
`
`is
`
`Tetracaine Hydrochloride and Procaine Hydro
`chloride.The relative affinity of tetracaine hydro
`chloride and procaine hydrochloride for polysorbate
`of 5%
`At
`80 is shown in Fig
`concentration
`SO approximately 20% of
`the total
`polysorbate
`con
`tetracaine is bound to the surfactant
`at
`of 10% polysorbate
`80 approximately
`centration
`30% of the tetracaine is bound
`The procaine ion
`to interact significantly with the
`does not appear
`polysorbate under the conditions of this study
`Chiorpromazine Hydrochloride..-Figure
`trates interaction of chlorpromazine
`hydrochloride
`The
`80
`with polysorbate
`in aqueous
`solution
`addition of electrolyte appears to enhance the inter
`solution of 2%
`In an aqueous
`action considerably
`polysorbate 80 approximately 50% of
`the total
`is bound to the surfactant
`chlorpromazine
`present
`when 0.1% NaHSO3
`in this system to
`is present
`retard oxidation approximately 75% of the chlor
`promazine is bound when the system consists of
`2% polysorbate
`80 0.1% NaHSO3
`and
`0.5 Al
`KCI approximately 90% of
`the chlorpromazine
`bound to the surfactant
`Promethazine Hydrochloride.The
`interaction
`with polysorbate
`of proniethazine
`hydrochloride
`At
`80 is illustrated in Fig
`concentration
`of
`2% polysorbate 80 and in the presence
`of 0.1%
`XaHSO3 to retard oxidation of
`the promethazine
`approximately 38% of
`the total promethazine
`when KC1 is added to
`bound to the surfactant
`ionic strength
`this system to establish
`total
`of
`approximately 0.6 approximately 70% of the pro
`methazine is bound to the polysorbate 80
`Dodecylpyridiniuzn Chloride and Cetylpyridiniuni
`ChlorideDeLuca
`and Kostenbauder
`previously
`presented data to illustrate the high degree
`
`of
`
`11
`
`10
`
`0.5
`1.0
`POLYSORBATE
`
`1.5
`80 V0 W/V
`
`2.0
`
`Fig 2.influence of electrolyte on binding of
`hydrochloride by polysorhate
`chloropromazine
`80
`at 30 Sodium bisulfite added as antoxidant Key
`in polysorbate 80
`in polysorbate 80 plus 0.1%
`NaHSO3
`in polysorbate 80 plus 0.1% NaHSO3
`and 0.5M ICCI
`
`

`

`Vol 52 No1
`
`September .1963
`
`the
`
`and
`interaction between cetylpyridinium chloride
`80 16 These
`polysorbate
`data are reproduced
`in Fig
`to permit coffiparison with the interaction
`dodecylpyridinium ion and polysorbate
`between
`80 At
`of 2% polysorbate
`concentration
`80
`approximately 40% of the dodecylpyridinium ion is
`bound to the polysorbate while at
`this concentra
`tion of polysorbate 80 approximately 97% of
`cetylpyridinium ion is bound
`Methyirosaniline Chloride.Figure
`illustrates
`the interaction of the cationic dye methyirosaniline
`80
`chloride with polysorbate
`These
`adsorption
`isotherms
`indicate that
`the nature of
`the binding
`may change markedly with concentration
`of
`free
`methylrosaniline ion and it
`is not possible to apply
`the relatively simple description of the binding used
`is not sufficient
`to describe the
`previously
`i.e it
`the
`in terms of
`percentage of
`binding
`definite
`methylrosaniline chloride bound to the polysorbate
`For
`concentration
`at any
`polysorbate
`specific
`example at
`of 0.25% polysorbate
`concentration
`80 the percentage of dye bound to the surfactant
`varied from approximately 70% at
`the lower dye
`to 40% at the higher dye concentra
`concentrations
`of 5% polysorbate 80
`tions At
`concentration
`approximately 90% of the dye was bound through
`out
`the concentration
`range studied
`Sodium Naphthalene Sulfonate.Sodium naph
`was
`thalene sulfonate
`included
`in this study
`to
`demonstrate that anionic as well as cationic agents
`might be bound to nonionic
`surface-active agents
`The interaction
`the naphthalene sulfonate anion
`of
`80 is illustrated in Fig
`with polysorbate
`In
`2% aqueous
`of poly
`the presence
`of
`solution
`sorbate 80 approximately 7% of
`the total naph
`thalene sulfonate ion is bound to the polysorbate
`KC1 was added to this system
`however when 0.1
`approximately 17% of
`sulfonate
`the naphthalene
`was bound to the polysorbate
`The naphthalene
`sulfonate ion was selected
`for study because of the
`absence of functional
`groups other
`than sulfonate
`which might permit hydrogen bonding to the poly
`the polysorbate and because this
`ether portion of
`ion is essentially in the completely dissociated form
`
`LI
`
`solution thereby eliminating the possi
`in aqueous
`being attributed to binding
`of an interaction
`bility
`of undissociated molecules to the polysorbate
`Methyl OrangeeThe interaction of
`the anionic
`dye methyl orange with polysorbate 80 is shown in
`As with niethylrosaniline chloride the
`Fig
`of dye bound to the surfactant
`at any
`fraction
`changes markedly with
`polysorbate concentration
`the concentration of unbound dye in the presence
`of 5% polysorbate 80 the percentage of bound dye
`ranged from 96% at
`low dye concentrations
`83% at higher dye concentrations
`Drug Ions Not Bound to Polysorbate 80.No
`interaction was observed for the following
`significant
`drug ions in the presence
`solutions of
`of aqueous
`polysorbate 80 ephedrine hydrochloride sulfathia
`hydrochloride and di
`zole sodium methapyrilene
`phenhydramine hydrochloride
`
`to
`
`DISCUSSION
`with Polysorbate 80
`
`Nature
`Interaction
`of
`The interaction
`of drug ions with polysorhate 80
`appears to be similar to the interaction of cetyl
`pyridinium ion with polysorbate 80 as descrihed by
`DeLuca and Kostenbauder 16 It appears that
`large hydrophobic group can undergo
`ions having
`association with the polysorbate micelle to form
`ions such
`type of mixed-micelle Binding of
`dodecylpyridinium and
`sulfonate
`naphthalene
`which exist entirely in dissociated form in aqueous
`solution and have no other
`functional groups whicb
`to hydrogen bond to the ethylene
`might be expected
`oxide portion of the polysorbate indicates that the
`feature for an organic ion to
`structural
`necessary
`interact with
`nonionic
`surface-active
`agent
`that of
`portion
`relatively large hydrophobic
`
`as
`
`is
`
`20
`
`15
`
`LI 10
`
`0.1
`
`0.5
`1.0
`IOLYSOR BATE 80
`
`1.5
`WIV
`
`20
`
`Fig 3.Binding of promethazine
`by polysorhate 80 at 30 Solid symbols correspond
`to systems containing 0.1% NaHSO3 as antoxidant
`0.1%
`open
`symbols represent
`systems containing
`plus sufficient KC to produce an ionic
`NaHSO3
`strength of 0.6
`
`hydrochloride
`
`POLYSORBATE
`
`80
`
`WJV
`
`Fig 4.Relative binding by polysorbate 80 of
`and
`dodecylpyridinium chloride
`cetylpyridinium
`chloride at 30 Binding data for cetylpyridinium
`chloride reproduced from DeLuca and Kostenbauder
`16
`
`

`

`896
`
`Journal of Pharmaceutical
`
`Sciences
`
`L3
`
`1.4
`
`LI 1.3
`
`1.2
`
`1.1
`
`1.0
`
`POLYSOItBATE 80
`
`W/V
`
`Fig 6.-.--Binding of sodium naphthalene sulfonate
`80 at 30 Key
`with 0.1M
`by polysorbate
`without XCI
`KCI
`points obtained by
`Donnan correction to the binding data
`applying
`obtained in absence of KCI
`
`of
`
`shown in Fig
`the binding data in absence
`salt when corrected for the Donnan effect
`are in
`excellent agreement with the degree of binding ob
`swamping elec
`served in the presence
`of 0.1
`The effect of electrolyte
`on the apparent
`trolyte
`snlfonate
`of binding of naphthalene
`to
`80 can therefore be attributed almost
`polysorbate
`entirely to elimination of the Donnan effect
`amenable
`Other
`studied
`are not
`systems
`to
`correction for this Donnan effect Where there is
`high degree of interaction with the polysorbate 80
`is almost
`certainty that there is considerable
`as there would
`be in
`retention just
`is not
`surfactants While
`Donnan correction it
`can be
`feasible to apply
`Donnan effect
`that any correction for
`predicted
`could only lead to an even greater degree of
`inter
`by the ratio Ct/C0
`action
`than
`that
`indicated
`Ct/C in all cases
`therefore total/free
`Where there is considerable counter-ion
`retention
`by bound hydrophobic
`ions added electrolyte may
`further effect as indicated by application of
`have
`the law of mass action to micelle formation Where
`ion and
`the
`represents the hydrophobic
`counter-ion the interaction with polysorbate may
`be represented as
`
`.01
`
`.001
`
`.001
`
`.01
`
`.1
`
`Co GM./L
`
`Fig 5Binding of methylrosaniline chloride by
`80 at 300
`Open
`polysorbate
`symbols
`represent
`0.25% polysorbate 80 solid
`containing
`solutions
`symbols represent 59 polysorbate 80
`
`substituent
`
`Ions such as ephedrine sulfathiazole methapyrilene
`diphenbydramine and procaine apparently are not
`to associate with poly
`hydrophobic
`sufficiently
`The presence
`sorbate 80 micelles
`the n-butyl
`of
`the p-amino group of
`tetracaine
`on
`character
`confers sufficient hydrophobic
`apparently
`this ion to associate with the polysorbate
`for
`The importance of the hydrophobic
`character of the
`ion is further illustrated in the comparative
`binding
`of cetylpyridinium ion and dodecylpyridinium ion
`Salt Effects.For
`bound electrolyte
`weakly
`such
`as sodium naphthalene
`sulfonate enhanced
`binding to polysorhate 80 in the presence of added
`electrolyte might be accounted for on the basis of
`Donnan
`swamping
`Adsorption
`of
`effect
`of
`ions would in effect
`cause the poly
`hydrophobic
`sorbate 80 to act as an ionic polymer and necessitate
`Donnan
`studies
`the binding
`correction for
`If
`ion binds few counter
`the adsorbed hydrophobic
`is assumed that
`ions and if
`the hydrophobic
`ion
`it
`under study does not form micelles at the concentra
`Donnan correction can be
`tions considered then
`applied in the following manner
`
`Let
`
`C0
`
`C1 tree
`
`ion in the
`concentration of hydrophobic
`or nonpolymer containing
`outside
`compartment
`concentration
`of total hydrophobic
`ion
`in the inside compartment and con
`sists of bound plus free
`concentration
`free hydrophobic
`of
`in inside compartment
`
`ion
`
`Donnan equilibrium
`
`C2
`
`C11 Ire
`
`Ci tree
`is therefore less than
`total/free drug are greater
`Ct/co
`This Donnan
`
`correction
`
`naphthalene
`
`was
`sulfonatepolysorbate
`
`and actual
`ratios of
`than the observed ratio
`
`to the
`applied
`80 data
`As
`
`degree
`
`it
`
`counter-ion
`
`micelles
`
`of
`
`ionic
`
`it
`
`aAbBTwrsABTw ab
`ABc Tie
`Bjb Tie
`
`The concentration
`
`is
`
`of bound hydrophobic
`At Bib Tie
`
`ion
`
`and an increase in the concentration
`of the counter-
`on addition of an electrolyte
`such
`as
`ion
`KCI would be expected
`to increase markedly the
`binding of the hydrophobic
`ion
`strongly bound electrolyte
`such as chlor
`For
`proma.zine hydrochloride the effect of added elec
`trolyte on the observed ratio Ct/C0 might therefore
`the Donnan
`to swamping of
`be attributed in part
`
`

`

`Vol 52 No
`
`September 1963
`
`897
`
`a-
`
`.1
`
`.01
`
`.01
`
`Co GM/L
`
`.1
`
`effects
`
`on drug stability which might arise from
`drugs with surf actant micelles
`of
`association
`Riegelman18 Nogami eta 19 20 and Naka
`Jima 21 have shown that
`number of drugs with
`labile ester linkages can be significantly stabilized
`against ester hydrolysis when solubilized in aqueous
`solutions of micellar nonionic surface-active agents
`On the other hand Motsavage and Kostenhauder
`22 have recently
`demonstrated
`that binding
`of
`to nonionic micelle-forming agents
`alkyl sulfates
`may increase the rate of the hydronium ion catalyzed
`the sulfate ester as much as 60-fold
`hydrolysis of
`Whether
`drug is more or less stable when asso
`ciated with the nonionic
`surfactant micelle will
`depend on the positioning of the labile group in or
`on the micelle and on the nature of the decomposi
`tion to which it
`is subject
`
`Fig 7.Binding of methyl orange by o% poly
`sorbate 80 at 3O
`
`SUMMARY
`
`Data have been presented to show that drug ions
`solution with
`nonionic
`can interact
`in aqueous
`to an
`extent which might
`agent
`surface-active
`markedly influence the stability of the drug or the
`release of the drug from formulation
`Only those drug ions having
`relatively large
`to interact with
`found
`group were
`hydrophobic
`polysorbate 80 The mechanism of the interaction
`appears to be similar to the mixed micelle formation
`which occurs
`on interaction of cetylpyridinium ion
`with polysorbate 80 Addition of electrolyte ap
`pears to enhance the interaction
`considerably
`The following ions were found to bind to poly
`sorbate 80 in aqueous
`solution
`cetylpyridinium
`methylrosaniline methyl orange
`chlorpromazine
`dodecylpyridinium promethazine tetracaine and
`naplithalene sulfonate
`The following ions did not appear to interact with
`significant extent
`polysorbate 80 to
`ephedrine
`sulfathiazole diphenhydramine methapyriline and
`procaine
`
`REFERENCES
`
`and Mirimanoff
`
`Pharm Pharmaccl
`
`and Miritnanoff
`
`Phann Ada
`
`of
`
`is
`
`effect and in part to the effect of increasing concen
`on the equilibrium as pre
`tration of counter-ion
`dicted by the law of mass action
`of These Interac
`Pharmaceutical
`Significance
`the most obvious consequence
`tionscPerhaps
`such as those described in this paper
`interactions
`the influence of
`the binding on the release
`of
`drug from
`nonionic
`formulation containing
`germi
`surfactant or the availability or activity of
`in such
`formulation
`Interactions
`cidal agent
`in
`large percentage of the drug is bound to
`which
`in the formulation would be expected
`component
`to
`modify greatly the release of the drug and subsequent
`This
`to the site of action
`absorption
`or access
`might be particularly true in topical
`formulations
`agents may he em
`where nonionic
`surface-active
`of 5% or more in the
`ployed in concentrations
`phase
`Of course absorption of drugs or
`aqueous
`release of drugs from formulations is an extremely
`complex process and binding of drug to surfactant
`is only one aspect of the problem it may well be
`factors to be considered make it highly
`that other
`be employed in
`desirable that
`the surfactant
`large concentration
`in spite of possible interactions
`with the drug
`Nevertheless
`is important
`to
`recognize that
`nonionic
`surfactants
`can
`interact
`with ionic drugs and that
`this interaction might be
`to exert
`considerable
`on
`the
`expected
`influence
`release of some drug ions from such systems
`The very pronounced effect of interaction
`between
`and quaternary anamonium
`nonionic
`surfactants
`and antimicrobial
`compounds
`on the availability
`the quatetnary nmmonium compounds
`activity of
`previously 16 Such
`has
`been
`demonstrated
`interactions
`between nonionic agents and quaternary
`ammonium compounds
`or other materials having
`antimicrobial activity are not always undesirable
`the inactivation of quaternary ainmonium com
`common
`pounds by addition of
`nonionic agent is
`procedure in carrying out sterility
`tests where there
`normally would be interference from the antimicro
`in the formulation
`bial agent present
`second important consequence
`of drug-surfac
`interactions
`is the effect of such interaction on
`tact
`the drug Although no data can
`the stability of
`be cited for systems discussed in this paper Riegel
`man 18 has demonstrated
`the very significant
`
`it
`
`Bolle
`6851950
`Bouchardy itt
`HeIr 26 2841951
`ibid 26 2841951
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`Lawrence
`and Erlandson
`423521953
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`Yoshioka
`Pharm Soc Japan 76 9391956
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`deNavarre
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`Chemists
`ibid 8681957
`deflavarre
`Twa JoURNAL
`and rice
`narr
`1957
`10 Moore
`MIg Chemist
`and Hardwick
`ibid 29 1941958
`27 3051956
`11 Gershenteld
`and Stedman
`1949
`12 Davies
`Hyg 47 2711949
`App Microbiol
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`1141956
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`Sac Cosmetic Chemists
`2101958
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`and Robinson
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`JOURNAL 49 4b01960
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`2891958
`18 Riegelman
`ibid 49 3391960
`19 Nogami
`Awazu
`Watanabe
`IC Chem Pharm Bull Tokyo
`11361960
`20 Nogami
`Awazu
`ibid 10
`and Nakajima
`5031962
`21 Nakajima
`Pharm Soc Japan 81 16841961
`22 Motsavage
`and Kostenbauder
`Colloid Sci
`in press
`
`THis JOURNAL
`
`and Matsuzaki
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`Soc Cosmetic
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`46 445
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`ibid 121 249
`
`Soap Perfumery
`
`Twa
`
`ibid 47
`
`and Sato
`
`and Kostenbauder
`
`