ACKNOWLEDGMENTS AND ADDRESSES
`Received May 19, 1975, from the Pharmaceutics Research Unit,
`University of Nottingham, University Park, Nottingham, NG7 2RD,
`United Kingdom.
`
`Accepted for publication August 5, 1975.
`For the gifts of compounds used in this study, grateful acknowl(cid:173)
`edgment is made to the Boots Co. Ltd.
`• To whom inquiries should be directed.
`
`Absorption and Distribution of Radioactivity from
`Suppositories Containing 3H-Benzocaine in Rats
`
`JAMES W. AYRESx, DUANGCHIT LORSKULSINT, ALBERT LOCK,
`LYNDA KUHL; and PAUL A. LASKAR
`
`Abstract 0 The effects of the suppository vehicle, drug concentra(cid:173)
`tion, and nonionic surfactants on in vitro benzocaine dialysis through
`a cellulose membrane and on rectal absorption in rats of total radio(cid:173)
`activity following administration of 3H-benzocaine were investigated.
`In vitro dialysis correlatea quite well with in vivo absorption, and drug
`release was greater from water-soluble vehicles than from oleaginous
`vehicles. Inclusion of a nonionic hydrophilic or lipophilic surfactant
`in cocoa butter resulted in a statistically significant increase for in
`vitro drug release, while a lipophilic surfactant showed little effect
`in vivo and a hydrophilic surfactant depressed release in vivo. Both
`types of surfactant had small effects on release from polyethylene
`glycol. In vitro release of benzocaine from some commercially avail(cid:173)
`able suppositories was compared with experimental preparations.
`Variation in blood radioactivity following administration of the same
`concentration of3H-benzocaine in the same dosage form in male and
`female rats is reported.
`
`Keyphrases C Absorption-benzocaine from suppositories, effect
`of vehicle, drug concentration, and nonionic surfactants, rats C Dis(cid:173)
`tribution-benzocaine fr-om suppositories, effect of vehicle, drug
`concentration, and nonionic surfactants, rats C Benzocaine-ab(cid:173)
`sorption and distribution from suppositories, rats D Suppositories(cid:173)
`absorption and distribution of benzocaine, rats D Dosage forms(cid:173)
`suppositories, absorption and distribution of benzocaine, rats
`
`It is well recognized that formulation factors can in(cid:173)
`fluence the availability of a drug from a dosage form.
`Surface-active agents included in dosage forms may
`exert their effects on the !lctive ingredient, the dosage
`form itself, or the membrane at the absorption site.
`Surfactants have been reported to increase and to de(cid:173)
`crease the absorption of drugs (1). Moreover, varying
`the concentrations of a surfactant can enhance or retard
`drug absorption, depending on the type of surfactant
`and whether or not micelle formation occurs (1).
`The complex mechanisms of surfactant effects on
`drug absorption were reviewed previously (2). The in
`vitro release of benzocaine from ointment vehicles was
`reported (3) and compared (4) to the rate of absorption
`and resulting total blood level radioactivity following
`rectal administration of 20% 3H-benzocaine (ethyl p(cid:173)
`aminobenzoate) from ointment vehicles in rats. This
`paper reports the effects of suppository vehicles, vari(cid:173)
`ations in drug concentration, and the presence of a
`nonionic hydrophilic or lipophilic surfactant on the in
`vitro dialysis of benzocaine and the absorption of 3H(cid:173)
`benzocaine in rats.
`
`832 I Journal of Pharmaceutical Sciences
`
`EXPERIMENTAL
`
`Dosage Form Preparation-All suppositories were prepared by
`the fusion method, and commercial products were used as received.
`The reagents and equipment used were similar to those reported
`previously (3, 4). Additional materials used in the present experiment
`were: dialysis membrane, available as a 2.54-cm (l-in.) X 30.5-m
`(100-ft) rolF; cocoa butter2; polysorbate 803; and sorbitan monoole(cid:173)
`ate4.
`For in vivo studies, 3H-benzocaine was dissolved in the polyethylene
`glycol vehicle (75% polyethylene glycollOOO and 25% polyethylene
`glycol 4000) or suspended in the cocoa butter vehicle. Suppository
`vehicles containing 3H-benzocaine were poured into plastic, dispos(cid:173)
`able, U-80 insulin syringes, which were refrigerated until completely
`congealed. The tips of the syringes were cut off, and the excess
`semisolid was removed.
`A suppository volume of 0.5 ml was used for the experiment. The
`amount of surfactant used was too small to weigh directly, and the
`aliquot method was used for preparation.
`In Vitro Dialysis-Dialysis tubing was cut into 10-cm lengths and
`soaked for at least 24 hr in distilled water. At the time of the test, the
`tubing was closed and weighted at one end by tying with a thin strip
`of the dialysis tubing to a glass stopper. The suppository was intro(cid:173)
`duced into the tubing followed by 2.5 ml of distilled water. The top
`was tied to form a container, which was as nearly full as possible
`without loss of water.
`The sample was then placed in a 600-ml beaker containing 500 ml
`of distilled water maintained at 37.5°. It floated upward, being held
`near the center of the container by the glass stopper weight. At the
`appropriate time periods, 5-ml samples were pipetted from the beaker
`and 5 ml of distilled water (37.5°) was returned to the beaker. Care
`was taken to draw each sample from as close to the same place in the
`beaker as possible and to avoid stirring.
`Analytical Method-The analysis of the benzocaine released
`during the in vitro test was carried out by the method of Matsumoto
`et al. (5). Aliquot portions of a sample solution were pi petted into a
`test tube followed by 2 N HCl (2 ml) and 0.2% NaN02 (0.4 ml), and
`the mixture was shaken for 5 min. Then 0.5% NH4S03NH2 (0.4 ml)
`was added, and the mixture was shaken for 3 min. N -(2-Diethylami(cid:173)
`noethyl}-1-naphthylamine hydrochloride (1.0 ml of 0.5%) was then
`added with shaking.
`After 30 min of intermittent shaking, the percent transmittance
`was measured at 550 nm and the concentration of benzocaine was
`determined from a standard curve.
`In Vivo Studies-Female Sprague-Dawley rats were used for all
`experiments except the male versus female study. Animal weights
`varied between 100 and 280 g. Surgical preparation, cannulation,
`
`1 Seamless regenerated cellulose dialysis tubing, Catalog No. 25225-226, VWR
`Scientific Supplies, Portland, Ore.
`2 Hershey Food Corp., Hershey, Pa.
`3 Tween 80, J. T. Baker, Phillipsburg, N.J.
`• Span 80, J. T. Baker, Phillipsburg, N.J.
`
`Page 1 of 8
`
`SENJU EXHIBIT 2141
`INNOPHARMA v SENJU
`IPR2015-00903
`
`

`
`Table 1-Composition and Benzocaine Concentration of Various Types of Suppository Bases
`
`~-~-~---------------------------
`
`Average
`Weight
`for
`Dialysis
`Studya,g
`-
`
`2.35
`-
`
`2.41
`
`2.42
`
`2.42
`
`2.41
`
`2.33
`-
`-
`1.63
`1.74
`1.77
`
`1.76
`
`Total
`Benzo-
`caine for
`Dialysis, mg
`-
`235.0
`-
`
`72.3
`
`72.6
`
`72.6
`
`72.3
`
`69.9
`-
`-
`163.0
`53.2
`53.1
`
`52.8
`
`Total
`Benzo-
`caine
`Dialyzed
`in 5 hr, mg
`-
`80.5
`-
`57.0
`
`60.5
`
`45.8
`
`57.8
`
`55.5
`-
`-
`22.9
`10.0
`11.9
`
`12.4
`
`Percent
`Dialyzed
`in 5 hr
`-
`34.3
`-
`78.8
`
`83.3
`
`63.0
`
`79.9
`
`79.4
`-
`-
`14.0
`18.8
`22.4
`
`23.5
`
`Benzo-
`caine,%
`
`20
`
`10
`
`5
`
`3
`
`3
`
`3
`
`3
`
`3
`
`1
`
`20
`10
`3
`3
`
`3
`
`Average
`Weight
`for In Viuo
`Releaseb, g
`
`0.579
`
`0.573
`
`0.567
`
`0.569
`
`0.558
`
`0.554
`
`0.552
`
`0.556
`
`0.581
`
`0.510
`0.510
`0.475
`0.438
`
`0.443
`
`Total
`3H-Benzo-
`caine
`Available
`for In Viuo
`Release, mg
`
`115.8
`
`57.3
`
`28.4
`
`17.1
`
`16.7
`
`16.6
`
`16.6
`
`16.7
`
`5.8
`
`102.0
`51.0
`14.2
`13.1
`
`13.3
`
`Sup-
`posi-
`tory
`- - - - -
`A
`
`B
`c
`D
`
`E
`
`F
`
`G
`
`H
`
`J
`K
`L
`M
`
`N
`
`Vehicle
`
`Polyethylene
`glycoJc
`Polyethylene
`glycol
`Polyethylene
`glycol
`Polyethylene
`glycol
`Polyethylene
`glycol
`Polyethylene
`glycol
`Polyethylene
`glycol
`Polyethylene
`glycol
`Polyethylene
`glycol
`Cocoa butter
`Cocoa butterd
`Cocoa butter
`Cocoa butter
`
`Cocoa butter
`
`Cocoa butter
`Cocoa butter
`Oleaginouse,f
`Cocoa buttere,g
`Cocoa buttere,h
`
`Surfactant
`
`None
`
`None
`
`None
`
`None
`
`Sorbitan
`monooleate, 1%
`Sorbitan
`monooleate, 0.5%
`Polysorbate 80, 1%
`
`Polysorbate 80, 0.05%
`
`None
`
`None
`None
`None
`Sorbitan
`monooleate, 1%
`Sorbitan
`monooleate, 0.05%
`Polysorbate 80, 1%
`Polysorbate 80, 0.05%
`Unknown
`Unknown
`Unknown
`
`~
`_g;
`~
`·"' ? ;:,
`"' .....
`"' ~ -....
`00 ... ...
`
`0
`p
`Q
`R
`s
`a Average weight of 10 suppositories. b Avcrafe weight of six or more suppositories. C Polyethylene glycol vehicle consisted of polyethylene glycol I 000 ( 75%) and polyethylene glycol 4000 ( 25%)
`dHcrshcy's. ecommt•rcially available product .. Also contains hexachlorophene, ephedrine sulfate, and bismuth subgallate. g Also contains oxyquinolinc sulfate, zinc oxide, menthol, and balsam
`peru, hAJso contains ephedrine sulfate, oxyquinoline sulfate, zinc oxide, bismuth subgallate, and balsam peru.
`
`1.67
`1.60
`2.14
`2.65
`2.40
`
`3
`3
`11
`4.9
`5.4
`
`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
`
`Page 2 of 8
`
`

`
`170
`
`160
`
`150
`
`1-
`
`10
`
`•
`
`/~
`•
`. I
`I
`!.
`. 0
`;·
`.,.,.--
`/.
`//
`
`/ .
`
`]_ 140
`"'
`":: 130
`a
`w 120
`Vl
`<(
`w
`110
`...J
`w 100
`a:
`w 90
`2
`~ 80
`() a 70
`N
`2
`60
`w
`co 50
`__,K
`...J
`<( 40
`/
`/6"":- a
`1-a 30
`£>~·--c:::: ~
`_,~~:::::~~l
`./.
`20 ~~0
`•
`o-"
`·....:::::~
`o--
`
`1.0
`
`2.0
`
`3.0
`HOURS
`
`4.0
`
`5.0
`
`Figure 1-Effect 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(cid:173)
`lowed as previously reported (4).
`
`RESULTS AND DISCUSSION
`
`Table I shows the composition or' 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 formulations 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 tbe polyethylene glycol vehicle used was about 1.4
`
`i' •
`
`14
`
`13
`E 12
`
`0
`0
`a, 11
`E
`610
`w
`Vl
`<(
`w
`...J
`w
`a:
`w
`2 ;;:
`() a
`N z
`w
`<0
`...J
`<(
`
`9
`
`8
`
`7
`
`6
`
`5
`
`4
`
`3
`
`2
`
`1-a
`1-
`
`oL-~~--~~~~~~--._~~~
`7
`8
`9
`10 11 12 13 14 15 16 17 18
`MINUTESy,
`
`Figure 2-Relotionship between total mas., of drug dialyzed and
`time 112• Key: see Table I.
`
`834 I Journal of Pharmaceutical Sciences
`
`Table II-Summary of Calculated Statistical Parameters
`
`Prod-
`uct
`
`Intercept
`(bo)
`
`SE of
`Intercept
`
`SE of
`Regression
`Coefficient Regression
`Coefficient
`(b,)
`
`B
`D
`E
`F
`G
`H
`K
`L
`M
`N
`0
`p
`Q
`R
`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
`7.3624
`8.1531
`5.8930
`5.5375
`4.4574
`1.3366
`0.5277
`0.3772
`0.5166
`1.3357
`0.6929
`1.0351
`0.5641
`0.8578
`
`113.5101
`76.061l
`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 benzocaine 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 vitro 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 II) 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 II and III).
`In vitro 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; (c) rectal
`mucosa acting as a semipermeable membrane, allowing passage of
`water both away from and into the blood, depending on the osmotic
`gradient; (d) practically no peristaltic movement; (e) pressure on
`rectal contents varying from 0 to 50 em of water, according to posture;
`and ({) 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 vitro testing that
`would simulate the presence of feces. It is necessary, however, to ex(cid:173)
`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 vitro testing and in vivo 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. Conditions a and care 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 on(cid:173)
`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(cid:173)
`cedure, without stirring of the bulk receptor phase, which exposed
`the suppositories tested to a single, uniform pressure and approxi(cid:173)
`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(cid:173)
`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-
`
`Page 3 of 8
`
`

`
`Table III-Individual Comparisons Made 0
`
`s
`
`R
`
`*
`*
`*
`ns
`*
`*
`ns
`
`*
`*
`ns
`ns
`*
`*
`
`Q
`
`*
`
`*
`*
`*
`*
`*
`
`Products
`B
`D
`E
`F
`G
`H
`K
`L
`M
`N
`0
`p
`Q
`R
`s
`
`p
`
`0
`
`N
`
`M
`
`L
`
`Products
`
`*
`
`*
`
`*
`*
`
`*
`*
`
`*
`*
`*
`*
`
`*
`
`*
`*
`*
`
`*
`*
`*
`-*
`*
`
`K
`
`*
`*
`
`H
`
`*
`us
`-*
`ns
`
`G
`
`*
`ns
`ns
`
`F
`
`*
`ns
`*
`
`E
`
`*
`*
`
`B
`
`D
`
`*
`
`a Comparisons among rates of benzocain~ release from the semisolid as mea~ured by the reg~eS;Sion c_oefficient of the total benzocaine re(cid:173)
`leased with respect to the square root of t1me were made. All of these compansons are not statiStically mdependent. Therefore, when a com(cid:173)
`parison is noted as significant at the 95% confidence level, it is meant that the rates of release of benzocaine for the two products under com(cid:173)
`parison ate likely to be different but at a 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 end of the ex(cid:173)
`periment than at the beginning.
`The dialysis of drug from saturated benzocaine solutions was
`studied, and the ratio of total benzocaine to free benzocaine increased
`proportionately as the concentration of a nonionic hydrophilic sur(cid:173)
`factant3 was increased from 0 to 7%. Addition of the surfactant to a
`solution containing a fixed amount of benzocaine increased the di(cid:173)
`alysis rate compared to a solution without surfactant (5, 8}. Therefore,
`0.05 and 1.0% of sorbitan monooleate or polysorbate 80 were incor(cid:173)
`porated into both the polyethylene glycol and the cocoa butter vehicle
`containing 3% benzocaine.
`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 1% polysorbate 80. Since
`the membrane was not controlling the rate of diffusion (as evidenced
`by increasing diffusion with an increased concentration), the sur(cid:173)
`factant must have been increasing the rate of dissolution of the drug.
`This finding is consistent with work showing that an increase in
`benzocaine dialysis from surfactant-containing solutions is due to the
`increased solubilization of the drug because of surfactant-drug in(cid:173)
`teractions, followed by a rapid release of free drug as dialysis takes
`place (5, 8}.
`
`Figure 4 shows the results of dialysis from polyethylene glycol
`suppositories containing surfactants. Between 63 and 80% of the active
`ingredient was released (Table I).
`It is not possible 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 glycol vehicle
`containing 10% benzocaine was almost 2.5 times the total released
`from the corresponding cocoa butter preparation when the percent
`benzocaine dialyzed was considered. The ratio was 2.9 to 4.2 when
`comparing the corresponding preparations containing 3% benzocaine.
`When considering the release from the 3 versus 10% preparations of
`a single vehicle type, it can be seen that the 10% formulation released
`a larger amount of drug but a smaller percent of drug during the di(cid:173)
`alysis period (Table I).
`Various concentrations of 3H-benzocaine in suppository dosage
`forms with and without surfactant {Table 1} were selected for in uiuo
`testing and were inserted into the rectum of female Sprague-Dawley
`rats. Blood samples (0.1 ml} were taken from the inferior vena cava
`at 5, 10, 20, 30, 40, 60, 90, 120, 180, 240, and 300 min, and the total
`
`E
`0, 25
`:t
`0
`LU
`(/)
`<t: 20
`w
`_J
`w
`a:
`~ 15
`::i:
`u
`0
`N
`2 10
`w
`co
`..J
`
`<t: b 5
`....
`
`0 ~----~----~----~----~----~
`1.0
`2.0
`3.0
`4.0
`5.0
`HOURS
`Figure 3-Effect of surfactants on drug release from cocoa butter
`suppositories. Key: see Table I.
`
`120
`
`110
`
`E 100
`"' ::l. 90
`a'
`LU 80
`II)
`<t:
`LU
`..J 70
`
`LU a: 60
`w
`2
`~ 50
`u
`0
`40
`N
`2
`w 30
`10
`..J
`<t: 20
`
`... 0
`...
`
`10
`
`0
`
`1.0
`
`4.0
`
`5.0
`
`3.0
`2.0
`HOURS
`Figure 4-Effect of surfactants on drug release from polyethylene
`glycol suppositories. Key: see Table I.
`
`Vol. 65, No.6, June 1976/835
`
`Page 4 of 8
`
`

`
`4501
`400
`
`350
`
`3\JO
`
`280
`E
`'E 26o
`a.
`"0
`240
`' 0
`
`220
`
`X
`
`I
`I V\I
`
`120
`
`i: 200
`> 180
`i=
`u
`<( 160
`0
`~ 140
`a:
`0
`0
`0
`_J 100
`a:l
`
`A)-L,"I
`!Ji.
`20 "'i/
`
`80
`
`60
`
`40
`
`/r 0- &....1-r ~r_ ___ J c
`
`~T
`0 --00
`
`a ...... -•- .. -
`
`0
`
`2
`
`3
`HOURS
`
`4
`
`5
`
`Figure 5-B/ood radioactivity after the application of 3H-ben(cid:173)
`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 Bat 5 and 10 min; curve C versus
`Bat 5, 10, 20, and 300 min; and curve B versus A at 180 min. (One side
`of the standard error of the mean is shciwn.)
`
`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 due 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(cid:173)
`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
`3H-benzocaine in polyethylene glycol suppositories (1, 3, 5, 10, and
`20%) and cocoa butter suppositories (3, 10, and 20%) are shown in Figs.
`5 and 6. Increasing the concentration of 3H-benzocaine in both
`polyethylene glycol and cocoa butter suppository bases resulted in
`a higher total radioactivity in the blood. Since the volume of sup(cid:173)
`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(cid:173)
`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 I Journal of Pharmaceutical Sciences
`
`100
`
`>--
`f-
`>
`i=E 80
`u-
`<( E
`0 a. 60
`-" o~
`~6 40
`a:~
`ox 20
`0
`0
`_J
`a:l
`
`0
`
`2
`
`4
`
`5
`
`3
`HOURS
`Figure 6-Blood radioactivity after the application of 3H-ben(cid:173)
`zocaine in a cocoa butter suppository vehicle. Key: see Table I. A
`t-value larger than the critical t-value was obtained for all point
`comparison.< except curve L versus Kat 180 min, curve L versus J
`at 240 min, and curve K versus J at 180 and 240 min. (One side of the
`standard error of the mean is shown.)
`
`for the radioactivity to appear, under the conditions of this experi(cid:173)
`ment the total radioactivity represents several metabolites rather than
`intact drug (4). Therefore, no pharmacokinetic analysis using the
`blood level radioactivity versus time curves was done in the present
`experiment.
`A previous in vitro 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 benzo(cid:173)
`caine in a polyethylene glycol-water solution, which formed under
`the in vitro conditions. This effect was not observed during the cur(cid:173)
`rent dialysis experiments and is apparently not occurring in vivo, 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 rate 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(cid:173)
`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 importance, since
`melting occurs readily at body temperature. After liquefaction, the
`
`70
`
`E
`'E 6o
`a.
`'0
`' ~ 50
`
`X
`>-
`1:: 40
`>
`i=
`~ 30
`0
`0
`~ 20
`0
`0 g 10
`
`a:l
`
`0
`
`40
`
`80 120 160 200 240 280
`MINUTES
`Figure 7-Blood radioactivity after the application of 3"< 3H(cid:173)
`benzocaine and surfactants in a polyethylene glycol vehicle. Key:
`see Table I. A t-value larger than the critical t-value was only ob(cid:173)
`tained for point comparisons on curveD versus E after 240 min, curue
`D versus G after 240 min, curve E versus F after 240 min, and curve
`F versus Hat 90 min.
`
`Page 5 of 8
`
`

`
`4.0
`
`X
`>-'
`t:
`>
`i= u 2.0
`<l:
`0
`0
`<(
`a:
`0 g 1.0
`
`..J
`to
`
`0
`
`70
`
`E
`E' so
`a.
`'0
`7
`0 50
`X
`
`>-t: 40
`>
`i=
`~ 30
`0
`0
`~ 20
`0
`0 g 10
`co
`
`0
`
`o
`
`4o
`
`80 120 1so 2oo 240 280
`MINUTES
`Figure 8-Blood radioactivity after the application of 3~;, 3H(cid:173)
`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 most drug would still have an oleaginous coating in which the drug
`has a very low solubility. The drug would have to diffuse 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 non ionic 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 sign if(cid:173)
`icant at most times.
`With 3% 3H-benzocaine in cocoa butter base, the lipophilic sur(cid:173)
`factant in both concentrations studied (1 and 0.05%) showed no sig(cid:173)
`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(cid:173)
`tigation.
`Table III shows that for the products in Fig. 4 the release rates of
`only Formulations D and E were significantly different in vitro. All
`products were essentially the same in vivo with respect to the rate of
`drug release. All products with surfactant in cocoa butter, however,
`demonstrated a significant increase in the r'l.te of drug release in vitro,
`but the only significant effect in vivo was a decrease in release with
`1% polysorbate 80. In some cases the in vitro method did not accu(cid:173)
`rately predict the in vivo effect. In these cases there were relatively
`small actual differences for the in vitro system, although the differ(cid:173)
`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
`using 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(cid:173)
`sorption across rectal membranes is usually considered to be a passive
`diffusion process. In passive diffusion, either the release of drug from
`
`40
`
`80
`
`120 160 200 240 280
`MINUTES
`Figure 9-Comparison of blood radioactivity following 3% 3H(cid:173)
`benzocaine in a polyethylene glycol vehicle in male and female rats.
`Key: see Table I. At-value larger than the critical t-value was found
`for point comparisons of male versus female rats receiving the same
`formulation except for the first 60 min for Ep versus EM and Gp
`versus GM. (The F subscript is for female rats, and theM subscript
`is for male rats.)
`
`the vehicle or drug dissolution in rectal fluids would be the rate-lim(cid:173)
`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%
`3H-benzocaine in polyethylene glycol indicates that the rate of ab(cid:173)
`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(cid:173)
`ferent rates and if different 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. Furth~r 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 ben(cid:173)
`zocaine from various experimental and commercially available sup(cid:173)
`positories. Wide variations were found in the amount of benzocaine
`dialyzed. Small differences, which were detectable in vitro, were not
`seen in vivo in rats, although substantial differences in vitro were
`correlated well with experimental results obtained in vivo.
`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 same vehicle to
`male and female rats results in lower blood radioactivity versus time
`curves for the male rats.
`Some formulation f{lctor other than the concentration of benzocaine
`affected the relative amounts of benzocaine released in vitro from the
`commercially available products examined. Although the dialysis
`method is useful for evaluating the effects of formulation on drug
`
`Vol. 65, No.6, June 1976/837
`
`Page 6 of 8
`
`

`
`release from suppositories, the desirable release rate for the specific
`drug investigated has not been determined since its minimum effec(cid:173)
`tive concentration is not known. Considerably more research is needed
`in this area.
`
`(7) E. Nasset, in "Medical Physiology," 12th ed., P. Bard, Ed., C.
`V. Mosby, St. Louis, Mo., 1968, p. 559.
`(8) H. Matsumoto, H. Matsumura, and S. Iguchi, Chern. Pharm.
`Bull., 14, 391(1966).
`·
`
`REFERENCES
`
`(1) W. W. Davis and W. E. Wright, in "Pharmacology and the
`Skin," W. Montagna, E. J. Vanscott, and R. B. Stoughton, Eds., Ap(cid:173)
`pleton-Century Crofts, New York, N.Y., 1972, pp. 37-39.
`(2) M. Gibaldi and S. Feldman, J. Pharm. Sci., 59, 579(1970).
`(3) J. W. Ayres and P. A. Laskar, ibid., 63, 1402(1974).
`(4) J. W. Ayres, D. Lorskulsint, and A. Lock, ibi