ACKNOWLEDGMENTS AND ADDRESSES
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`Received May 19, 1975, from the Pharmaceutics Research Unit,
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`University of Nottingham, University Park, Nottingham, NG7 2530,
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`United Kingdom.
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`Accepted for publication August 5. 1975.
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`For the gifts of compounds used in this study, grateful acl-:nowl-
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`edgment is made to the Boots Co. Ltd.
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`‘ To whom inquiries should be directed.
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`Absorption and Distribution of Radioactivity from
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`Suppositories Containing 3H-Benzocaine in Rats
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`JAMES W. AYRES ", DUANGCHIT LORSKULSINT, ALBERT LOCK,
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`LYNDA KUI-IL‘, and PAUL A. LASKAR
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`Abstract I: The effects of the suppository vehicle. drug concentra~
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`tion. and nonionic surfactants on in oitro benzocaine dialysis through
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`a cellulose membrane and on rectal absorption in rate of total radio-
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`activity following administration of 3H-benzocaine were investigated.
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`In oitro dialysis correlated quite well with in oioo absorption. and drug
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`release was greater from water-soluble" vehicles than from olcaginous
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`vehicles. Inclusion of a nonionic hydrophilic or lipophilic surfactant
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`in cocoa butter resulted in a statistically significant increase for in
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`Ultra drug release, while a lipophilic surfactant showed little effect
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`in also and a hydrophilic surfactant depressed release in oiuo. Both
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`types of surfactant had small effects on release from polyethylene
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`glycol. In oitrc release of benzoceine from some commercially avail-
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`able suppositories was compared with experimental preparations.
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`Variation in blood radioactivity following administration of the same
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`concentration of 3H—benzocaine in the same dosage form in male and
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`female rats is reported.
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`Kayphrasesl:IAhsorption—benzocaine from suppositories, effect
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`of vehicle, drug concentration, and nonionic surfactants, rats D Dis-
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`tribution—benzocaine from suppositories, effect of vehicle, drug
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`concentration, and nonionic surfactants. rats El Ben2.ocaine—ab-
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`sorption and distribution from suppositories, rats I:I Suppositories-
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`absorption and distribution of benzocaine, rats D Dosage forms-——
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`suppositories. absorption and distribution of benzucaine, rate
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`It is well recognized that formulation factors can in-
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`fluence the availability of a" drug from a dosage form.
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`Surface-active agents included in dosage forms may
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`exert their effects on the active ingredient, the dosage
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`form itself, or the membrane at the absorption site.
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`Surfactants have been reported to increase and to de—
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`crease the absorption of drugs (1). Moreover, varying
`the concentrations of a surfactant can enhance or retard
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`drug absorption, depending on the type of surfactant
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`and whether or not micelle formation occurs (1).
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`The complex mechanisms of surfactant effects on
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`drug absorption were reviewed previously (2). The in
`oitro release of benzocaine from ointment vehicles was
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`reported (3) and compared (4) to the rate of absorption
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`and resulting total blood level radioactivity following
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`rectal administration of 20% 31-Lbenzocaine (ethyl p-
`aminobenzoate} from ointment vehicles in rats. This
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`paper reports the effects of suppository vehicles, vari-
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`ations in drug concentration, and the presence of a
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`nonionic hydrophilic or lipophilic surfactant on the in
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`oitro dialysis of henzocaine and the absorption of 3H-
`benzocaine in rats.
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`832 /' Journal of Pharmaceutical Sciences
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`Page 1 of 8
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`EXPERIMENTAL
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`Dosage Form Pl-e]:Iara.I.ion—Allsuppositories were prepared by
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`the fusion method, and commercial products were used as received.
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`The reagents and equipment used were similar to those reported
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`previously (3. 4). Additional materials used in the present experiment
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`were: dialysis membrane, available as s 2.54-cm (1-in.) X 30.5-n1
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`(1{)D—ftl roll‘; cocoa butter‘; polysorbate 803: and sorbitan l‘|’l0l'I00le-
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`ate‘.
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`For in oiuo studies, 3H—benzocaine was dissolved in the polyethylene
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`glycol vehicle (75% polyethylene glycol 1000 and 25% polyethylene
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`glycol 4000) or suspended in the cocoa butter vehicle. Suppository
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`vehicles containing 3H-benzocaine were poured into plastic, dispos-
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`able, U4-ll) insulin syringes, which were refrigerated until completely
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`congealed. The tips of the syringes were cut. off, and the excess
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`semisolid was removed.
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`A suppository volume of Ill") ml was used for the experiment. The
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`amount of surfactant used was too small to weigh directly, and the
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`aliquot method was used for preparation.
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`In Vitro Dialysis—Dialysis tubing was cut into ll}-cm lengths and
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`soaked for at least 24 hr in distilled water. At the time of the test, the
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`tubing was closed and weighted at one end by tying with a thin strip
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`of the dialysis tubing to a glass stopper. The suppository was intro-
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`duced into the tubing followed by 2.5 ml of distilled water. The top
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`was tied to form a container, which was as nearly full as possible
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`without loss of water.
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`The sample was then placed in a 600-ml beaker containing 500 ml
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`ofdistilled water maintained at 37.5”. It floated upward, being held
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`near the center of the container by the glass stopper weight. At the
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`appropriate time periods, 5-ml samples were pipetterl from the beaker
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`and 5 ml of distilled water l3'I.5°) was returned to the beaker. Care
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`was taken to draw each sample from as close to the same place in the
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`beaker as possible and to avoid stirring.
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`Analytical MeI.ho£l—~The analysis of the benzocaine released
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`during the in Ultra test was carried out by the method of Matsumoto
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`et a1. {5}. Aliquot portions of a sample solution were pipetted into a
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`test tube followed by 2 N HCI (2 roll and 0.2% NaNCI2 (0.4 ml), and
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`the mixture was shaken for 5 min. Then 0.5% NH4S0,-,-NH2 (9.4 ml}
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`was added, and the mixture was shaken for 3 min. N-(2-Diethylamh
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`noethyl)—1—naphthylamine hydrochloride (1.0 ml of 0.5%} was then
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`added with shaking.
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`After 30 min of intermittent shaking, the percent transmittance
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`was measured at 550 nm and the concentration of benzocaine was
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`determined from a standard curve.
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`In Vivo Studics—F‘emale Sprague—Dawley rats were used for all
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`experiments except the male versus female study. Animal weights
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`varied between 100 and 280 g. Surgical preparation, cannulation_
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`’ Seamless regenerated cellulose dialysis tubing, Catalog No. 25225-226, VWR
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`Scientific Supplies. Portland, Ore.
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`’ Hershey Food Corp., Hershey, Pa.
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`3 Tween 80. .1, T, Baker, Phillipsburg. NJ.
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`' Span 8:1, J. T. Baker. Phillipsburg, NJ.
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`SENJU EXHIBIT 2141
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`LUPIN V. SENJU
`
`IPR2015-01100
`
`Page 1 of 8
`
`SENJU EXHIBIT 2141
`LUPIN v. SENJU
`IPR2015-01100
`
`

`
`Table I—Composition and Benzocaine Concentration of Various Types of Suppository Bases
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`Sup-
`posi—
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`tor}:
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`A
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`B
`C
`D
`E‘.
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`F
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`Vehicle
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`Surfactant
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`None
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`Average
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`Weight
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`for
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`Dialysis
`
`Study“, g
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`—
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`2.35
`—
`2.41
`2.42
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`2.42
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`Total
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`Benzo-
`eaine for
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`Dialysis, mg
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`Total
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`Benzo-
`caine
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`Dialyzed
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`in 5 hr, mg
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`-—
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`235.0
`—
`72.3
`72.6
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`72.6
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`—
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`80.5
`—
`57.0
`30.5
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`45.8
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`Percent
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`Dialyzed
`in 5 hr
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`—
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`
`34.3
`—
`78.8
`83.3
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`Average
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`Weight
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`for In Viuo
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`Releaseb, g
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`0.579
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`0.573
`0.567
`0.569
`0.558
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`0.554
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`Benzo-
`caine, ‘Z:
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`20
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`10
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`5
`3
`3
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`3
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`Total
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`’H-Benzo-
`caine
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`Available
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`for In Viuo
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`Release, mg
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`115.8
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`57.3
`28.4
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`17.1
`16.7
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`16.6
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`Polyethylene
`glycol?
`
`Pollyethlylene
`g yco
`
`Pollyethlylene
`g YCO
`
`Pollyetlilylene
`g yco
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`Polyethylene
`glycol
`
`}’o!yethylene
`glycol
`
`Pollyethlylene
`g yco
`
`Pogllyethlylene
`yea
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`Polyethylene
`giycol
`
`Cocoa butter
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`
`Cocoa butter”
`
`Cocoa butter
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`Cocoa butter
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`
`Cocoa butter
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`Cocoa butter
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`Cocoa butter
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`
`Oleaginoust’-f
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`Cocoa buttei-9:8
`
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`Cocoa buttere-5'
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`
`G
`
`I-I
`I
`.1
`
`K.
`
`L
`
`M
`
`
`N
`
`
`None
`None
`
`None
`Sorbitan
`
`monooleate. 1%
`
`
`Sorbitan
`
`monooleate, 0.5%
`
`
`
`
`
`
`Polysorbate 80. 1%
`
`
`Polysorbate 80. 0.05%
`None
`
`None
`
`None
`
`None
`
`Sorbitan
`
`monooleabe, 1%
`
`
`Sorbitan
`
`monooleate, 0.05%
`
`
`Polysoi-bate 80, 1%
`
`
`
`Polysorbate 80. 0.05%
`
`
`Unknown
`
`Unknown
`
`Unknown
`
`
`
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`
`
`
`
`
`
`
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`
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`
`
`2.41
`2.33
`—
`__
`1.63
`1.74
`1.77
`
`1.76
`
`1.67
`1.60
`2.14
`2.65
`2.40
`
`0
`
`P
`
`Q
`
`R
`
`3
`
`5 Average weight of‘ H1 supposituritu‘-. 5 Avcra :2 weight of six or more suppositories. I‘-‘Polyctliylcne glycol vclaiclc colisistud of polycrhylcm: glycol 1000 (75%) and polyetliylcmt glycol 4000 ['_’S%_}
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`
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`dHL‘rSl)£!}«"‘S. ‘°(‘.onmu-rciaully zlvuilublu product. Also contains hcxaclilompliunt-. cphcdrim: sulfate, and bismutli subgallatc. Xfilso contains oxyquiimlinc sulfate, zinc oxide. menthol. and balsam
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`pcru. “Also contains eplicdrine sulfate. oxyquinoline sulfate. zinc oxide. bismuth subgallatc. and balsam peru.
`
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`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`3
`
`3
`1
`
`20
`10
`3
`3
`
`
`
`
`
`
`3
`
`3
`
`3
`
`11
`
`4.9
`5.4
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`72.3
`69.9
`-—
`—
`163.0
`53.2
`53.1
`
`52.8
`
`
`
`
`
`
`
`
`
`
`
`
`
`57.8
`55.5
`—
`--
`22.9
`10.0
`11.9
`
`12.4
`
`63.0
`
`
`79.9
`79.4
`—
`—
`14.0
`18.8
`22.4
`
`23.5
`
`
`
`
`
`
`
`
`
`
`
`0.552
`0.556
`0.581
`
`0.510
`0.510
`0.475
`0.438
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`16.6
`16.7
`5.8
`102.0
`
`51.0
`
`14.2
`
`13.1
`
`
`0.443
`
`
`
`13.3
`
`
`
`50.1
`48.0
`235.0
`130.0
`130.0
`
`
`
`
`
`
`
`13.9
`11.7
`17.5
`15.5
`13.2
`
`
`
`
`
`
`
`27.6
`24.4
`7.5
`12.0
`10.1
`
`
`
`
`
`
`
`
`
`
`0.468
`0.460
`—
`—
`‘—
`
`
`
`
`14.0
`13.8
`—
`—
`—
`
`
`
`
`
`
`
`
`
`
`
`seeI9161‘atm1"9‘UN'5970A
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`Page 2 of 8
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`Page 2 of 8
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`

`
`1 70
`160
`150
`I40
`
`#9/ml
`. ‘I30
`1 20
`1 10
`‘I00
`90
`80
`70
`60
`50
`
`BENZOCAINERELEASED
`TOTAL
`
`40
`30
`20
`10
`
`Figure l—Eflect of suppository vehicle composition on release of
`drug from preparations containing benzocaine. Key: see Table I.
`
`blood sample collection, and blood analysis methodology were fol-
`lowed as previously reported (4).
`
`RESULTS AND DISCUSSION
`
`Table I shows the composition of suppositories selected for the
`investigation of variations in dialysis and rate of release for absorption
`of benzocaine. Figure 1 illustrates the variation in drug release of some
`experimental suppository fonnuletions and the commercial products
`investigated. Since the active ingredient content of many commercial
`products is reported as a percentage. rather than an amount. Products
`A-P were prepared on a percentage weight per weight basis. The
`specific gravity of the polyethylene glycol vehicle used was about 1.4
`
`mg/100ml
`TOTALBENZOCAINERELEASED,
`
`
`
`7
`
`8
`
`9
`
`10
`
`11
`
`13
`12
`14 ‘I5
`MINUTES‘/'
`
`16
`
`‘I7
`
`‘I8
`
`Figure 2—Relulions-hip belween lntal mass 0/ drug dialyzed and
`timem. Key: see Table I.
`
`834 / Journal of Pharmaceutical Sciences
`
`Page 3 of 8
`
`Table ll—Summary of Calculated Statistical Parameters
`
`Prod-
`uct
`
`Intercept
`(bu)
`
`SE of
`Intercept
`
`SE of
`Regression
`Coefficient Regression
`((7,)
`Coefficient
`
`B
`D
`E
`F
`G
`I-l
`K
`L
`M
`N
`0
`P
`Q
`1?.
`S
`
`-88.9583
`-62.6965
`--90.9342
`-69.2085
`-76.7483
`-81.1662
`-21.1737
`-6.8433
`—8.5915
`"'10.7434
`--10.6500
`—8.6347
`-18.4070
`-11.9034
`-13.0224
`
`4.2313
`7.8624
`8.1531
`5.8930
`5.6375
`4.4574
`1.3366
`0.5277
`0.3772
`0.5166
`1.3357
`0.6929
`1.0351
`0.5641
`0.8578
`
`113.5101
`76.0611
`96.8973
`74.6488
`87.5097
`86.9697
`27.8889
`11.6569
`14.3776
`15.6600
`17.8333
`14.5876
`23.6619
`16.9296
`18.6843
`
`2.4429
`4.2507
`4.5225
`3.2689
`3.0717
`2.4725
`0.7717
`0.3047
`0.2178
`0.2982
`0.7712
`0.4001
`0.5976
`0.3257
`0.4952
`
`times that of the cocoa butter, and this difference was reflected in the
`variation in the total amount of benzocoine present in suppositories
`containing the same percent of benzocaine but different vehicles.
`The total mass of drug transferred from semisolids under the
`conditions of the in uitro experiment was nearly linear with respect
`to the square root of time for the times investigated (Fig. 2). The linear
`portions of such benzocaine release curves were used in generating
`a linear least-squares regression line, and comparisons among the
`estimated parameters (Table ll) were made (Table Ill) using the usual
`null hypothesis. The data points for Product D were approximated
`with two linear portions (Fig. 2), and the line for the early time period
`was arbitrarily chosen for statistical comparisons (Tables 11 and 111).
`In uilro testing of suppositories involves many considerations and
`some compromises in simulating conditions operating during rectal
`absorption. The conditions (6) that should be emulated are: (a) an
`average temperature of 36.9‘; (b) water not present in the liquid state
`but present in the semisolid feces, which are 77-82% water: (1') rectal
`mucosa acting as a semipermeable membrane, allowing passage of
`water both away from and into the blood, depending on the osmotic
`gradient; (11) practically no peristaltic movement; (9) pressure on
`rectal contents varying from 0 to 50 cm of water, according to posture;
`and (I) possible presence of feces.
`in normal people, fecal material is present in the rectum just prior
`to defecation only (7). Most of the time. this organ is free of solid
`matter which could physically interfere with absorption. Therefore,
`it is not necessary to introduce a material for in uitro testing that
`would simulate the presence of feces. It is necessary, however. to ex-
`pose the dosage form to some fluid so that the drug has an opportunity
`to dissolve. While this exposure may seem to violate Condition b. a
`positive correlation between in uilru testing and in viva results would
`indicate that such exposure to fluids is acceptable for testing purposes.
`Testing at body temperature is critical, especially for products that
`melt in the rectum. Conditionsa and c are readily satisfied by using
`a temperature-controlled water bath and placing the suppository
`inside a commercially available, semipermeable, dialysis membrane
`tubing. Condition d can be met by placing the dosage form in an un-
`stirred medium. Although this procedure may allow a buildup of drug
`around the dosage form. which can slow drug release. such a static
`dialysis method may have a closer relationship to the absorption of
`drugs through a biological membrane than dialysis when the bulk
`phase is stirred.
`The data reported here were obtained using a simple dialysis pro-
`cedure, without stirring of the bulk receptor phase. which exposed
`the suppositories tested to a single, uniform pressure and approxi-
`mated to some degree Conditions a. c. and d.
`Examination of the results for the commercial products (Fig. 1)
`reveals less release from the 11% preparation than the 10% preparation
`and somewhat greater release from the 4.9% preparation than the 5.4%
`preparation, although the latter difference is not significant. For-
`mulation factors other than concentration that could be playing a role
`include the presence of other ingredients that might interact with the
`benzocaine as well as different vehicle effects. The experimental cocoa
`butter formulations were completely melted within 10 min and the
`polyethylene glycol vehicles were dissolved within 1 hr. There was.
`however. no visible change in any commercial product during the 5-hr
`dialysis period. Each commercial suppository retained its shape, al-
`
`

`
` Table lll—-Individual Comparisons Made“
` Products
`
`3
`
`R.
`
`Q
`
`P
`
`O
`
`N
`
`M
`
`L
`
`K
`
`H
`
`G
`
`F
`
`E
`
`D
`
`B
`
`_
`_
`Prgducts
`—
`—
`D
`—
`—
`E
`_
`_
`F
`—
`—
`Q
`—
`*‘
`H
`_
`_.
`K
`I
`I
`L
`a
`a
`M
`ns
`‘
`N
`na
`na
`0
`a
`s
`p
`3
`0
`—
`na
`3
`
`S .-
`
`1.
`—
`-
`_
`—
`-
`a
`—
`3
`‘
`*
`at
`—
`
`_
`—
`—
`_
`—
`’
`a
`8
`_
`~ ’
`*
`_
`
`_
`—
`-
`_
`8
`-'
`s
`1
`a
`'
`-
`
`_
`—
`—
`a
`—
`"
`a
`t
`an
`’
`
`_
`-
`0
`a
`—
`‘
`a
`1
`—
`
`_
`’
`—
`.—
`—
`"
`0
`—-
`
`a
`"
`—
`—
`—
`"
`—
`
`a
`na
`-
`.. a
`ns
`’
`
`0
`na
`ns
`_
`—
`
`t
`ns
`‘
`._
`
`as
`—
`
`_
`
`“
`-
`
`“Cornparisons among rates of henzocaine release from the semisolid as measured by the regression coefficient of the total henzocaine re-
`leased with respect to the square root of time were made. All of these com arisons are not statistically independent. Therefore. when a com-
`parison is noted as sign iflcant at the 95% confidence level. it is meant that t e rates ofrelcase afbenzocaine for the two products under com-
`parison are likely to be different but at s confidence level slightly less than 95%. The result is that some of the differences in release rate noted
`as ~‘ might not prove to be significantly different under more rigorous testing: ns = not significant. ~‘ = barely significant, ‘ = significant.
`and — - not tested for significance.
`
`though they all became somewhat more pliable at the and of the ex-
`periment than at the beginning.
`The dialysis of drug from saturated benzocaino solutions was
`studied, and the ratio of total henwoaine to free hemocaine increased
`proportionately as the concentration of a nonionic hydrophilic sur-
`factant’ was increased from 0 to 196. Addition of the surfactant to a
`solution containing a fixed amount of beneocaine increased the di-
`alysis rate compared to a solution without surfactant (5, 8). Therefore.
`0.05 and 1.096 of sorbitan monooleate or polysorbate 80 were incor-
`porated into both the polyethylene glycol and the cocoa butter vehicle
`containing 3% henwcaine.
`Figure 3 shows the effect on the cocoa butter suppositories and the
`increase in the amount and rate of benzocaine released The greatest
`increase in release was due to the presence of 196 polyaorbate 3). Since
`the membrane was not controlling the rate of diffusion (as evidenced
`by increasing diffusion with an increased concentration). the sur-
`factant must have been increasing the rate of dissolution of the dmg.
`This finding is consistent with worlr showing that an increase in
`benzocaine dialysis from surfactant-containing solutions iadue to the
`increased aolubilization of the drug because of surfactant—drug in-
`teractiona. followed by a rapid release of free drug as dialyais takes
`place (5. 8).
`
`Figure 4 shows the results of dialysis from polyethylene glycol
`suppositories containing surfactants. Between 63 and 80960! the active
`ingredient was released (Table I).
`It is not poaaibla to use the actual amount of drug released from the
`different products (Table I) as a measure of the effect of the vehicle
`on dialysis of drug. since the amount present varies with the product
`considered. The percent of drug released. however. can be used for
`this purpose. The total released from the polyethylene zlycol vehicle
`containing 1095 bensocaine was almost 2.5 times the total released
`from the corresponding cocoa butter preparation when the percent
`benzocaina dialyud was considered. The ratio was 2.9 to 4.2 when
`comparing the correapondim preparations containing 3% benzocaine.
`When considering the release from the 3 versus 1096 preparations of
`a single vehicle type. it can be aeen that the 1096 formulation released
`a larger amount of drug but a smaller percent of drug during the di-
`alysis period (Table l).
`Various concentrations of ‘H-benzocaine in suppository dosage
`forms with and without surfactant (Table l) were selected for in viva
`testing and were inserted into the rectum of female Sprague-Dawley
`rats. Blood samples (0.l ml) were taken from the inferior vena cava
`at 5, 10, 20, 30, 40. G0, 90, 120, 130. 240. and 300 min. and the total
`
`N 0
`
`Is)0
`
`—l U
`
`at
`
`
`
` 6TOTALBENZOCAINEIRELEASED.I-I9/ml
`
`1.0
`
`3.0
`2.0
`HOURS
`
`4.0
`
`5 .0
`
`pg/ml 3
`TOTALaenzocmusRELEASED,
`
`
`
`1.0
`
`3.0
`2.0
`HOURS
`
`4.0
`
`5.0
`
`figure 3-3//rct 0/ sur/actants on drug release from cocoa butter
`suppositories. Key: see Table I.
`
`Figure 4—Ef/oer ol surfactants on drug release from polyethylene
`glycol suppositories. Key: see Table I.
`
`Vol. 65. No. 6, June 1976 / 835
`
`Page 4 of 8
`
`

`
`
`
`.
`
`>'
`I; mo
`2.l-
`80
`DE(5
`9% 6°
`3?
`:19 “°
`Ox
`0
`20
`
`oJ
`
`°°
`
`o
`
`1
`
`2
`
`3
`HOURS
`
`4
`
`5
`
`Figure 6——l'lloorI radioactivity after the application of 3H-ben-
`zocaine in a cocoa butter suppository vehicle. Key: see Table I. A
`t-ualue larger than the critical t-value was obtained /or all point
`cornparisons except curve L versus K at I80 min, curue L versus J
`at 240 min, and cum: K versusJ at I80and 240 min. (One side of the
`standard error a/ the mean is shown.)
`
`for the radioactivity to appear, under the conditions of this experi-
`ment the total radioactivity represents several metabolites rather than
`intact drug (4). Therefore, no pharmacolrinetic analysis using the
`blood level radioactivity versus time curves was done in the present
`experiment.
`A previous in uitro study (3) found that an increased concentration
`of benzocaine in polyethylene glycol ointment caused a decrease in
`release through a dialysis membrane. That decrease was explained
`on the basis of a decreased solubility and precipitation of the heme-
`caine in a polyethylene glycol-water solution, which formed under
`the in uitro conditions. This effect was not observed during the cur-
`rent dialysis experiments and is apparently not occurring in viva. as
`evidenced by increasing absorption from an increased concentration
`of drug in this water-soluble vehicle.
`The absorption from a cocoa butter suspension of drug is much less
`in rnte and amount when compared to equal concentrations of drug
`in polyethylene glycol (Fig. 6). The latter vehicle is water soluble and
`can dissolve in the rectum. Benzocaine was dissolved in the polyeth-
`ylene glycol vehicle and. therefore. was available to partition into the
`rectal fluids and the rectal mucosa during liquefaction (dissolution)
`of the polyethylene glycol. Cocoa butter does not dissolve but melts
`in the rectum.
`Before liquefaction, dissolution of drug in rectal fluids is limited
`to the drug located at the surface of the suppository. Diffusion through
`the semisolid suppository is probably of little inlpurtance. since
`melting occurs readily at body temperature. After liquefaction, the
`Si O
`
`01O
`
`0’!O
`
`RADIOACTIVITY,x1o"apm/mi 3
`BLOOD
`
`D-O
`
`1.)O
`
`NO
`
`0
`
`40
`
`80
`
`120
`160
`MINUTES
`
`200 240 280
`
`Figure 7—Blood radioactivity after the application 0/ 3"; “H-
`benzocaine and surfactants in a polyethylene glycol vehicle. Key:
`see Table I. A t-value larger than the critical t-value was only ob-
`tained for point comparisons on curve D versus Ea/(er 240 min, curve
`1) versus (7 after 240 min, curve E versus Falter 240 min. and curve
`Fversus H at 90 min.
`
`450
`400
`
`350
`
`300
`
`280
`
`260
`
`240
`
`220
`
`200
`1 80
`
`160
`
`‘I40
`
`1 20
`
`100
`
`80
`
`60
`
`40
`
`20
`
`
`
`BLOODRADIOACTIVITY,X10"dpm/ml
`
`Figure 5-—Blood radioactivity after the application of 3H-ben-
`zocaine in a polyethylene glycol suppository vehicle. Key: see Table
`I. A t-value larger than the critical t-value was obtained for all point
`comparisons except curve I versus B at 5 and 10 min; curve C‘ versus
`84115.10, 20, and 300 min:and curve B versus A at I80 min. (One side
`0/ the standard error of the mean is shown.)
`
`radioactivity present was determined. The means of the radioactivity
`detected are shown in Figs. 5-9.
`Statistical analysis using unequal variance techniques indicated
`that weight variation among animals accounted for less than 5% of
`the variation following different dosage formulations. The standard
`error of the mean is included in some figures but not in others clue to
`crowding. A point-by-point comparison of the means obtained at each
`sample time for the nonlinear curves was made using the Student t
`test (95% confidence level). and the results are summarized in the
`figure legends.
`Due to the relatively large dispersion of experimental values. the
`ability to distinguish between mean values. which appear quite dis-
`tinct, is compromised. An example of this situation can be seen when
`comparing the results from Formulation A versus Formulation B in
`Fig. 5 for the 180-min sample. The mean values for A and B were
`significantly different for each sample time (95% confidence level)
`except at 180 min due to the relatively large variances at that time.
`Increasing the number of animals in the study may have resulted in
`a significant difference in this case.
`The blood level radioactivities from different concentrations of
`31-I-benzocaine in polyethylene glycol suppositories (1. 3. 5. 10, and
`2096) and cocoa butter suppositories (3, 10, and 20%) are shown in Figs.
`5 and 6.
`lncreasing the concentration of -‘H-benzocaine in both
`polyethylene glycol and cocoa butter suppository bases resulted in
`a higher total radioactivity in the blood. Since the volume or sup—
`positories was equal with every concentration of drug administered,
`the total dose was increased by increasing the drug concentration.
`Both concentration and variation in total dose may he causes for the
`difference in the shape of the blood level curves using the same sup-
`pository vehicle.
`it is clear from Fig. 5 that an increase in the 3H-benzocaine in
`polyethylene glycol increased the area under the curve up to 5 hr.
`Although this finding indicates an increase in the amount of drug
`being absorbed from the rectum, since the drug had to be absorbed
`
`836 / Journal of Pharmaceutical Sciences
`
`Page 5 of 8
`
`

`
`P ca
`
`5"o
`
`-1N"oo
`
`
`
`BLOODRADIOACTIVITY,xio"°dpm/mi
`
`O
`
`40
`
`80
`
`120 160 200 240 230
`MINUTES
`
`Figure 8—Blood radioactivity after the application of 3% “H-
`benzocaine and surfactants in a cocoa butter suppository vehicle.
`Key: see Table I. A t-value larger than the critical t-value was found
`for at least half of the points when comparing the curves of L versus
`0, L versus P, M versus P, N versus 0, and N versus P.
`
`suspended drug would be coated with melted cocoa butter, which is
`much less viscous than the original semisolid.
`Some drug may be exposed to the rectal fluids for rapid dissolution,
`but mostdrug would still have an oleaginous coating in which the drug
`has a very low solubility. The drug would have todiffuse through this
`coating before absorption could occur. Therefore, the slower drug
`absorption from the cocoa butter is not surprising, since the drug
`particles would be coated by a hydrophobic substance in which the
`drug has a low solubility.
`Surfactants were included in the 3% 3H-benzocaine in polyethylene
`glycol and cocoa butter suppositories to determine if the presence of
`a nonianic hydrophilic or lipophilic surfactant would affect the rate
`or amount of benzocaine absorption. Incorporation of a 1% lipophilic
`or 0.05 or 1% hydrophilic surfactant in the polyethylene glycol vehicle
`resulted in an apparent increase in total blood radioactivity (Fig. 7)
`for times beyond 180 min, although the range of values was wide
`enough for the differences of the means to be not statistically signif-
`icant at most times
`With 3% 3H-henzocaine in cocoa butter base, the lipophilic sur-
`factant in both concentrations studied (1 and 0.05%) showed no sig-
`nificant influence on the amount absorbed (Fig. 8). However, the
`hydrophilic surfactant in both concentrations studied decreased the
`total counts in the blood significantly with most times under inves-
`tigation.
`Table II I shows that for the products in Fig. 4 the release rates of
`only Fonnulations D and E were significantly different in ultra. All
`products were essentially the same in viva with respect to the rate of
`drug release. All products with surfactant in cocoa butter, however,
`demonstrated a significant increase in the rate of drug release in uitra,
`but the only significant effect in viva was a decrease in release with
`1% polysorhate 80. in some cases the in vitro method did not accu-
`rately predict the in vivo effect. in these cases there were relatively
`small actual differences for the in oifro system, although the differ-
`ences were statistically significant.
`In the earlier part of the study, some experiments were run to
`measure the blood level concentration of radioactivity versus time
`Lsing male rats. Female rats showed a total radioactivity in the blood
`about twice that of male rats (Fig. 9) when the same dosage form was
`administered. These differences could be due to differences in the rate
`of absorption, biotransformation, distribution, or excretion. Ab-
`sorption across rectal membranes is usually considered to be a passive
`diffusion process. ln passive diffusion, either the release of drug from
`
`Page 6 of 8
`
`V0
`
`UIO
`
`atO
`
`apm/ml 3
`BLOODRADIOACTIVITY,x10*’
`
`
`OO
`
`(JO
`
`NO
`
`0
`
`40
`
`BO
`
`200 240 280
`120 160
`MINUTES
`
`Figure 9—Comparison of blood radioactivity following 3% “H-
`benzocaine in a polyethylene glycol vehicle in male and female rats.
`Key: see Table I. A t-ualue larger than the critical L-value was found
`for point comparisons of male versus female rats receiving the some
`formulation except for the first 60 min for E; versus EM and G‘;-
`versus GM. (The F subscript is for female rats, and the M subscript
`is /or male rats.)
`
`the vehicle or drug dissolution in rectal fluids would be the rate~lirn-
`iting step for absorption, especially with a drug like benzocaine which
`has a low water solubility.
`The decreased rate of appearance of radioactivity in the blood from
`20% 3H-benzocaine in the cocoa butter vehicle compared to 20%
`“H-benzocaine in polyethylene glycol indicates that the rate of ab-
`sorption is dependent on the amount of drug released from the vehicle
`and presented to the rectal mucosa, at least for the concentrations of
`drug studied here. Since the same vehicle and drug concentration were
`administered to males and females, it is unlikely that the observed
`differences were due to differences in absorption. Furthermore, the
`differences probably were not due to differences in excretion half-life
`of the parent drug or of identical amounts of the same metabolites in
`males or females because most drugs are excreted by a first-order
`process in either sex.
`Excretion of total radioactivity and loss from the bloodstream may
`be different in males and females if metabolism is occurring at dif-
`ferent rates and ifdifferent amounts of metabolites are available for
`excretion. One possible explanation for the results could be that males
`were metabolizing the drug to more polar products faster than the
`females and the more polar products were being cleared from the
`bloodstream more rapidly than the parent drug. Another possible
`explanation could be that distribution of the molecules containing
`radioactivity was different for male and female rats. Further work
`involving tissue distribution and metabolism is underway in these
`laboratories to determine which possibility is correct.
`
`SUMMARY AND CONCLUSIONS
`
`A simple dialysis method was used to measure the release of hen-
`zocaine from various experimental and commercially available sup-
`positories Wide variations were found in the amount of benzocaine
`dialyzed. Small differences. which were detectable in uitro, were not
`seen in viva in rats, although substantial differences in uitro were
`correlated well with experimental results obtained in viva.
`Benzocaine was dialyzed and absorbed rectally in rats more rapidly
`from a polyethylene glycol vehicle than from cocoa butter. and the
`effects of the surfactants‘ tested were variable. Rectal administration
`of the same concentration of 3H—benzocaine in the aame vehicle to
`male and female rats results in lower blood radioactivity versus time
`curves for the male rats,
`Some formulation factor other than the concentration of benzocaine
`affected the relative amounts of benuwaine released in uitm from the
`commercially available products examined. Although the dialysis
`method is useful for evaluating the effects of formulat