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Rate-controlled absorption enhancement of rectally administered cefoxitin in rats by salicylate
Journal
ofControlled Release,
7 (1988) 53-60 B.V., Amsterdam -
Elsevier Science Publishers
53 Printed
in The Netherlands
RATE-CONTROLLED ABSORPTION ENHANCEMENT OF RECTALLY ADMINISTERED CEFOXITIN IN RATS BY SALICYLATE H.J.M.
E.J. van Hoogdalem”, Center for Bio-Pharmaceutical Box 9503,230O (Received
July
Sciences,
van Kan, A.G. de Boer and D.D. Breimer Divisron of Pharmacology,
Sylvius Laboratories,
State University
of Leyden, P.0
RA Leyden (The Netherlands) 1, 1987;
accepted
in rewsed
form
September
24,
1987)
Sodium salicylate has been reported to enhance the rectal absorption of the antibiotic cefoxitin in rats and humans. In the present study some factors influencing the enhancing effect of salicylate on rectal cefoxitin absorption were investigated, in particular in relationship to the rate of delivery and absorption of salicylate itself. Salicylate proved to be rapidly and completely absorbed after bolus delivery as well as after rectal infusion. The absorption of cefoxitin, which was absorbed to a limited extent (8 to 32%) without enhancer, could be promoted by salicylate on bolus delivery, resulting in mean bioavailabilities of 46% and 101% after delivery 2 mm and 1 cm from the anus, respectively. Rectal infusion or delivery of the same amount of cefoxitin and salicylate to a larger mucosa area resulted in poor absorption of the antibiotic. Results indicate a critical influence of the amount of salicylate delivered area-l time-’ on the absorption-enhancing effect.
INTRODUCTION Application of many polar drugs is restricted to parenteral routes of administration because they cross biological membranes poorly. This is a serious limitation with drugs such as insulin, several antibiotics andpeptide drugs. Enhancement of intestinal absorption would in principle make it possible to administer such compounds by the oral or rectal route. Several studies have indicated that enhancement of absorption is feasible by co-administration of absorption promoters. Intestinal uptake of the cephalosporin antibiotic cefoxitin was reported to be enhanced by sodium salicylate and Brij 35 [ 1 ] , acylcarnitines [ 2 1, phosphate derivatives [ 31, sodium 5methoxysalicylate [ 451, epinephrine metabolites [ 61, concanavalin A [ 71, EDTA and polyoxyethylene-23-lauryl ether *To whom correspondence
0168.3659/88/$03,50
should be addressed.
0 1988 Elsevier Science Publishers
[ 81. Among the enhancers studied, sodium salicylate is an interesting agent because of its reported effectiveness and its applicability in man. It has been shown to increase intestinal absorption of [ Asu~‘~] -eel calcitonin [ 91, dextran [lo], insulin [ 11,121, antibiotics [ 4,5,13-151 and phenylalanine [ 161 in rats or dogs. In the studies referred to no attention was paid to the influence of delivery rate on the effect of the absorption promoter. Because the absorption-enhancing action of salicylate decreases with the disappearance of salicylate from the intestinal lumen [ 171, the retention of a minimal effective concentration of salicylate at the mucosal surface seems to be required to exert an adequate absorption-enhancing action. The influence of delivery rate on rectal spreading behaviour of delivered bromophenol blue solution was investigated. The enhancing effect of salicylate on cefoxitin absorption was evaluated in relationship to its rate of delivery.
B.V.
54
MATERIALS
AND METHODS
Chemicals
Cefoxitin sodium (Mefoxin@ ) was a gift from Merck, Sharp & Dohme, Haarlem, The Netherlands. Cefazolin sodium ( KefzolO ) was a gift from Eli Lilly & Co., Utrecht, The Netherlands. Sodium salicylate (Brocacef, Maarssen, The Netherlands) was of pharmaceutical grade. Ethyl acetate (Baker Chemicals, Deventer, The Netherlands) was distilled before use. Bromophenol blue was obtained from Janssen Chimica, Beerse, Belgium. All other reagents used were of analytical grade. Animals
of cefoxitin
For i.v. administration a solution of cefoxitin sodium 15 mg/ml, made isotonic by addition of sodium chloride, and a solution containing cefoxitin sodium 15 mg/ml and sodium salicylate 60 mg/ml were used. For rectal administration solutions containing cefoxitin sodium 15 mg/ml were used, with or without sodium salicylate 60 mg/ml, in 0.067 A4 phosphate buffer pH 7.4 or in water. In one experiment less-concentrated solutions were used, containing cefoxitin sodium 3 mg/ml with or without sodium salicylate 12 mg/ml in 0.067 M phosphate buffer pH 7.4. The ionic strength of all solutions used rectally was adjusted to 0.75 by adding sodium chloride. Drug administration
The animal experiments performed were registered according to the Dutch Experimental Animals Act. For the assessment of rectal spreading male Wistar rats of laboratory breed, weighing 185-235 g, were used. For experiments concerning rectal absorption of cefoxitin female Wistar rats of laboratory breed, weighing 175-200 g, were used. From 16 h prior to the experiments until their completion only water intake was allowed. Drug solutions Rectal spreading
To assess rectal spreading a solution was used containing bromophenol blue 10 mg/ml in water. Bioavailability
Bioavailability
of salicylate
For i.v. administration a solution of sodium salicylate 5 mg/ml with cefoxitin sodium 15 mg/ml was used, made isotonic by addition of sodium chloride. For rectal administration a solution of sodium salicylate 5 mg/ml and cefoxitin sodium 15 mg/ml in 0.067 M phosphate buffer pH 7.4 was used. The ionic strength was adjusted to 0.75 using sodium chloride.
For i.v. infusion a poly(viny1 chloride) cannula (length 150 cm; i.d. 0.5 mm; o.d. 1 mm; Talas, Ommen, The Netherlands) filled with 200 ~1 of drug solution was inserted into the right jugular vein under light ether anesthesia. For rectal administration of the drug solutions a poly (vinyl chloride) cannula (id. 0.5 mm; o.d. 1 mm) containing 200 ~1 or 1 ml of’ drug solution was connected to a rectal delivery device. This device consisted of 3 cm of stainless steel tubing (i.d. 0.5 mm; o.d. 1 mm ), introduced into the tip of the plunger of a l-ml disposable syringe (Bect.on-Dickinson, Ireland). The tubing emerging from the plunger tip was elongated using a length of teflon cannula (i.d. 1.15 mm; o.d. 1.75 mm; Talas, Ommen, The Netherlands) to establish rectal delivery at a distance of 2 mm or 1 cm from the anus. Under light ether anesthesia the teflon cannula was introduced into the rat rectum and the device was ligated with the anus closing around the plunger tip. After insertion the intravenous or rectal cannula was pulled s.c., emerging on the nape of the neck, and suspended above the rat, allowing free movement. After a recovery period of 2 h the cannula was connected to an infusion pump
55
(Braun, Melsungen, West-Germany). To achieve bolus administration 200 ~1 or 1 ml were delivered in 24 s. Linear infusion was performed by delivering 200 ~1 in 32 min and 1 ml in 26.7 min.
Blood sampling
Under light ether anesthesia a poly (vinyl chloride) cannula (length 50 cm; i.d. 0.5 mm; o.d. 1 mm) filled with heparin in saline (200 III/ml) was inserted into the right carotid artery. The cannula was pulled s.c., emerging on the nape of the neck. The cannula was suspended above the caged rat, allowing free movement. Blood samples of 100 ~1 were taken at regular intervals after starting the experiment and were haemolysed in 500 ~1 of distilled water. The samples were stored at - 20’ C until analysis. After each sampling the cannula was filled with heparinized saline to prevent clotting. In the study of salicylate kinetics cannulas were removed under light ether anesthesia 10 h after starting the experiment, to prevent destruction by the rat overnight. From 19 h after starting the experiment blood samples were obtained by orbital puncture.
Assessment
of rectal spreading
Drug assays Sodium salicylate
The assay of sodium salicylate in haemolysed blood was based on the method described by Bekersky et al. [ 181. To the blood samples 70 ~1 of an internal standard solution containing 3,4,5trimethoxybenzoic acid 50 yg/ml were added. The samples were acidified with 100 ~1 of 1.0 M HCl and 5 ml of ethyl acetate were added. After whirlmixing for 30 set and centrifugation for 5 min at 2000 g the organic layer was transferred to another test tube. 1 ml of 0.067 Mphosphate buffer pH 7.4 was added and the samples were extracted by whirlmixing for 30 s. The organic layer was discarded after centrifugation for 5 min at 2000 g. Residual ethyl acetate was removed under reduced pressure (Vortex-Evaporator, Buchler Instruments Inc., Fort Lee, NJ, U.S.A.). 25 ~1 of the aqueous extract were analysed using a liquid chromatographic system (Waters Associates, Millford, MA, U.S.A.) consisting of an M-45 pump, a WISP 710B automatic sample injector, a Zmodule containing a Radial-Pak Cl8 cartridge, and a Model 481 LC spectrophotometer (wavelength 237 nm) . The mobile phase consisted of 1.5% v/v acetic acid containing 46% v/v methanol. The flow rate was 2.0 ml/min. Data processing was performed by a Chromatopack CR3A reporting integrator (Shimadzu, Kyoto, Japan ) . Cefoxitin
Under urethane anesthesia (600 mg/lOO g i.p.) the delivery device was ligated in the rat rectum, allowing administration of bromophenol blue solution in the rectum at a distance of 2 mm from the anus. After laparotomy the rectum was exposed. The cannula was connected to the infusion pump and 200~1 of bromophenol blue solution were delivered as a bolus in 24 set or as an infusion in 32 min. Spreading of the dye solution from the delivery site higher into the rectum was investigated for 1 h and was measured at regular intervals using a ruler.
sodium
Cefoxitin sodium was assayed in haemolysed blood by reversed-phase high-performance liquid chromatography, based on the methods described by Brisson and Fourtillan [ 191 and by Charles and Ravenscroft [ 201. To the haemolysed blood samples 70 ~1 of an internal standard solution containing cefazolin sodium 2.4 ,ug/ml were added. After acidification by adding 250 ~1 of 0.4 M HCl the samples were extracted with 5 ml of ethyl acetate for 15 s on a whirlmixer. After centrifugation for 5 min at 2000 g the organic layer was transferred to another test
56
tube. 500 ~1 of 0.067 M phosphate buffer pH 7.4 were added and the samples were extracted by whirlmixing for 15 s. The organic layer, obtained after centrifugation for 5 min at 2000 g, was discarded and residual ethyl acetate was removed under reduced pressure (Vortex-Evaporator, Buchler Instruments Inc., Fort Lee, NJ, U.S.A.). 50 ~1 of the residue were injected on the liquid chromatographic system, at a wavelength of 270 nm. As mobile phase was used 0.05 M KH,PO, solution (pH 2.9 using phosphoric acid) containing 18% acetonitrile. The flow rate was 2.5 ml/min.
Data analysis
According to Chiou [ 211 the areas under the individual blood concentration time curves were calculated from t = 0 to maximal concentration with the linear trapezoidal rule and after the maximal concentration to the last sampling point using the logarithmic trapezoidal rule. For extrapolation to infinity of i.v. curves and curves following rectally delivered salicylate, individual elimination rate constants were used. Because of the irregular pattern of the apparent elimination phase an accurate elimination rate constant of rectally delivered cefoxitin could not be obtained. For this reason curves obtained after rectal cefoxitin administration were extrapolated using the mean elimination rate constant for i.v. administration. Systemic clearance of cefoxitin sodium was calculated as D/AUC, where D is the administered i.v. dose of cefoxitin sodium and AUC is the total area under the curve. Using statistical moment theory [ 221 the mean residence time (MRT) after i.v. and rectal administration of sodium salicylate was calculated and corrected for the rate of delivery. The Wilcoxon rank sum test was used for st,atistical evaluation of the results. A p-value smaller than 0.05 was considered as a significant difference.
I’
.Y
,A
:
‘-
Fig. 1. Spreading distance into the rectum i SD of 200 ~1 of bromophenol blue solution after rectal delivery 2 mm from the anus as bolus in 24 s (0, n =6) or as linear infusionin32min (A,n=5).
RESULTS
The spreading profile of rectally delivered bromophenol blue is shown in Fig. 1. Immediately after bolus delivery 53 ? 10 mm rectum is filled with solution. Spreading continued relatively slowly to 70 29 mm. Rectal infusion, however, resulted in a slow progression of the dye solution in the rectum. It lasted about 40 min before the length of rectum was reached, which is momentarily filled on bolus delivery. After infusion a shorter ultimate spreading distance was reached as compared to bolus delivery. After rectal bolus delivery and rectal infusion of 1 mg of sodium salicylate at a distance of 1 cm from the anus comparable blood concentrations were obtained (Fig. 2). The mean AUC 2 SD after rectal infusion and bolus delivery of 107 & 28 ,ug h/ml and 128 2 25 ,ug h/ml, respectively, did not differ significantly from the value obtained after i.v. infusion (109 t 47 fig h/ml). After i.v. infusion a mean MRT? SD of 4.3 2 1.0 h was determined. Rectal infusion and bolus delivery resulted in comparable values of 5.6 -t 1.9 h and 5.6? 1.0 h, respectively, which did not differ significantly from the i.v. data.
57
Fig. 2. Mean blood levels of sodium salicylate i SD after rectal delivery of 1 mg of sodium salicylate with 3 mg of cefoxitin sodium in phosphate buffer, as a bolus ( 0, n = 6) and as infusion ( A, n=6) at a distance of 1 cm from the anus. Fig. 4. Mean blood levels of cefoxitin sodium &SD after rectal bolus delivery of 3 mg of cefoxitin sodium in phosphate buffer at a distance of 1 cm from the anus, with ( 0, n = 5) or without ( A, n = 7 ) 12 mg of sodium salicylate. AUC,,,lug
mm/ml1
Fig. 3. Mean blood levels of cefoxitin sodium +SD after rectal bolus delivery of 3 mg of cefoxitin sodium in phosphate buffer at a distance of 2 mm from the anus, with ( 0, n = 6) or without ( A, n = 6) 12 mg of sodium salicylate.
These results indicate a rapid and complete absorption of salicylate, irrespective of delivery rate. Intravenous infusion of 3 mg of cefoxitin sodium resulted in a mean AUC of 312? 73 pg min/ml and a systemic clearance value of 9.8? 2.6 ml/min (n = 7). Co-administration of 12 mg of sodium salicylate i.v. resulted in a comparable AUC of 358%42 pg min/ml and a systemic clearance of 8.5 -t 0.9 ml/min ( n = 3 ) , which excludes an interaction of salicylate with cefoxitin elimination kinetics. Bolus delivery of 200 ~1 of cefoxitin solution in phosphate buffer 2 mm (Fig. 3) and 1 cm
D
E
G
H
I
J
Fig. 5. Histogram of the mean AUC &SD of cefoxitin sodium after iv. and rectal administration of 3 mg of cefoxitin sodium with (cross-hatched) and without (blank) 12 mg of sodium salicylate, under various circumstances: A: i.v. infusion; B, C: bolus delivery (B) and infusion (C) in phosphate buffer. 2 mm from the anus; D, E: bolus delivery (D) and infusion (E) in water, 2 mm from the anus; F, G: bolus delivery (F) and infusion ( G ) in phosphate buffer, 1 cm from the anus; H, I: bolus delivery (H) and infusion ( I) in phosphate buffer, 1 cm from the anus; J: infusion in phosphate buffer over a length of 2 mm to 1 cm from the anus; administered volume: 200 ~1 (A-G, J ) or 1 ml (H, I) ; * : significantly different from control value without enhancer (p < 0.05, Wilcoxon rank sum test ) .
58
(Fig. 4) from the anus resulted in relatively low blood concentrations and AUC values ( Fig. 5 ) , which were very substantially enhanced by salicylate. Infusion with or without phosphate buffer also resulted in low AUC values, but interestingly these were not enhanced by salicylate. Infusion of 200 ~1 between 0.2 cm and 1 cm from the anus as well as infusion or bolus administration of 1 ml of the less concentrated cefoxitin solution resulted in low mean AUC values, which could not be influenced by coadministration of salicylate. After bolus delivery and infusion without phosphate buffer low mean AUC values were obtained, which were not enhanced by salicylate (Fig. 5 1.
DISCUSSION
In order to evaluate the luminal distribution of rectally delivered solutions in relationship to rate of delivery the spreading profile of a dye solution was investigated in laparotomized rats. The observed rectal spreading of bromophenol blue demonstrates that a relatively small volume of solution spreads over a considerable length of rectum. After bolus administration a larger section of the rectum is covered as compared with infusion. The rate of spreading proves to be considerably higher after bolus delivery than after infusion. Bromophenol blue is a poorly absorbed compound [ 231 and it will therefore experience maximal spreading; it is conceivable that compounds with a higher absorption rate will be distributed over a shorter length. On bolus administration in phosphate buffer, salicylate significantly enhanced the extent of cefoxitin absorption (Fig. 5)) in contrast with rectal infusion. This difference is not caused by a prolonged presence of the enhancer after bolus delivery, considering the rapid and complete absorption of salicylate (Fig. 2 ) . The difference in spreading distance higher into the rectum after infusion and bolus delivery is too small to explain the difference in enhancing effects
by characteristics of the absorption site. Infusion close to the anus and 1 cm deep into the rectum resulted in comparable effects, excluding important differences in absorbing capacity along the first l-cm section of the rectum. The results of the bolus experiments versus the infusion experiments indicate that rate of delivery is essential in optimizing the effects of the absorption promoter. Optimal absorption enhancement requires the presence of an effective luminal salicylate concentration until cefoxitin absorption is complete. Rectal administration of the dye showed that the amount delivered area- ’ time- ’ after infusion is substantially lower compared with bolus delivery. The slow delivery of rectally infused salicylate and its rapid absorption prevent that effective salicylate concentrations are reached, whereas after bolus input an effective concentration is achieved instantaneously, but only for a very short period of time. The reported steep sigmoidal relationship between effectiveness of salicylate and its concentration [ 81 illustrates that a relatively small decrease in salicylate concent.ration will result in a loss of absorpsolution tion-enhancing action of the administered. The effect of salicylate, which was pronounced after bolus delivery of 200 1~1of solution, was absent after bolus delivery of the same amount of cefoxitin and salicylate in 1 ml (Fig. 5). Delivery in a larger volume lowered the amount of salicylate area- ’ time -’ and probably thereby reduced the effect of the enhancer. The lack of absorption-enhancing effect of salicylate after infusion at 2 mm or 1 cm from the anus was also determined after delivery to a larger mucosa area by infusion between 2 mm and 1 cm from the anus (Fig. 5). Although the area available for absorption was increased, absorption remained poor, which may again be explained by an ineffective amount enhancer area-‘timee’. After rectal delivery without phosphate buffer absorption enhancement could not be achieved and considerable variability in the data was ob-
59
served (Fig. 5). The presence of phosphate buffer seems to reduce variability by standardizing circumstances for absorption. Our results show a striking similarity with those of other authors. Liversidge et al. [ 11,121 observed an increasing enhancing effect of salicylate on rectal insulin absorption from suppositories on decreasing the surface area. Although the authors suggest that this difference is caused by differences in characteristics of the absorption site or in extent of first-pass metabolism, our data indicate that a larger amount of enhancer area- ’ time- ’ after administration in small or short suppositories promotes the effect of salicylate. Nishihata et al. [ 51 observed a better absorption-enhancing effect of 5-methoxysalicylate in gelatin formulation compared with delivery in phosphate buffer. It is likely that administration in the viscous gelatin solution will reduce spreading and will raise the amount of 5-methoxysalicylate area- I time-‘, thus enhancing its effect. The results of our study demonstrate that rate-controlled delivery, fitted to the absorption behaviour of the enhancer, may be decisive for the effectiveness of the absorption-promoting action.
REFERENCES
S.S. Davis, W.R. Burnham, P. Wilson and J. O’Brien, Use of adjuvants for enhancement of rectal absorption of cefoxitin in humans, Antimicrob. Agents Chemother.. 28 (1985) 211-215. J.A. Fix, K. Engle, P.A. Porter, P.S. Leppert, S.J. Selk, C.R. Gardner and J. Alexander, Acylcarnitines: Drug absorption-enhancing agents in the gastrointestinal tract, Amer. J. Physiol., 251 (1986) G332-G340. T. Nishihata, C.-S. Lee, B.T. Nghiem and T. Higuchi, Possible mechanism behind the adjuvant action of phosphate derivatives on rectal absorption of cefoxitin in rats and dogs, J. Pharm. Sci., 73 (1984) 1523-1525. T. Nishihata, H. Takahagi and T. Higuchi, Enhanced small intestinal absorption of cefmetazole and cefoxitin in rats in the presence of non-surfactant adjuvants, J. Pharm. Pharmacol., 35 (1983) 124-125.
5
6
7
8
9
10
11
12
13
14
15
16
T. Nishihata, J.H. Rytting, T. Higuchi, L.J. Caldwell and S.J. Selk, Enhancement of rectal absorption of water-soluble antibiotics in dogs, Int. J. Pharm., 21 (1984) 239-248. T. Nishihata, H. Takahagi, M. Yamamoto, H. Tomida, J.H. Rytting and T. Higuchi, Enhanced rectal absorption of cefmetazole and cefoxitin in the presence of epinephrine metabolites in rats and a high-performance liquid chromatographic assay for cephamytin antibiotics, J. Pharm. Sci., 73 (1984) 109~112. T. Nishihata and T. Higuchi, Promoting effect of concanavalin A on transport of sodium cefoxitin and phenol red from rat rectal compartment, Life Sci., 34 (1984) 419-426. T. Nishihata, H. Tomida, G. Frederick, J.H. Rytting and T. Higuchi, Comparison of the effects of sodium salicylate, disodium ethylenediaminetetraacetic acid and polyoxyethylene-23-lauryl ether as adjuvants for the rectal absorption of sodium cefoxitin, J. Pharm. Pharmacol., 37 (1985) 159-163. M. Miyake, T. Nishihata, A. Nagano, Y. Kyobashi and A. Kamada, Rectal absorption of [ Asu’,‘] -eel calcitonin in rats, Chem. Pharm. Bull., 33 (1985) 740-745. G.B. West, Rectal absorption of dextran in rats in the presence of salicylates, Int. Arch. Allergy Appl. Immunol., 68 (1982) 283-285. G.G. Liversidge, T. Nishihata, K.K. Engle andT. Higuchi, Effect of rectal suppository formulation on the release of insulin and on the glucose plasma levels in dogs, Int. J. Pharm., 23 (1985) 87-95. G.G. Liversidge, T. Nishihata, K.K. Engle andT. Higuchi, Effect of suppository shape on the systemic availability of rectally administered insulin and sodium salicylate, Int. J. Pharm., 30 (1986) 247-250. J.A. Fix, P.S. Leppert, P.A. Porter and L.J. Caldwell, Influence of ionic strength on rectal absorption of gentamicin sulfate in the presence and absence of sodium salicylate, J. Pharm. Sci., 72 (1983) 1134-1137. T. Nishihata, S. Kawabe, M. Miyake, S. Kim and A. Kamada, Effect of sodium ion on the adjuvant action of sodium salicylate in enhancing rectal ampicillin absorption in rats, Int. J. Pharm., 22 (1984) 147-157. H. Yaginuma, T. Nakata, H. Toya, T. Murakami, M. Yamazaki and A. Kamada, Rectal delivery of antiinflammatory drugs. I. The influence of antiinflammatory drugs on rectal absorption of j3-lactam antibiotics, Chem. Pharm. Bull., 29 (1981) 2974-2982. T. Nishihata, C.-S. Lee, M. Yamamoto, J.H. Rytting and T. Higuchi, The effects of salicylate on the rectal absorption of phenylalanine and some peptides, and the effects of these peptides on the rectal absorption of cefoxitin and cefmetazole, J. Pharm. Sci., 73 (1984) 1326-1328.
60 17
18
19
T. Suzuka, T. Nishihata, M. Yamazaki and A. Kamada, Effect of salicylate and disodium ethylenediaminetetraacetic acid on the rat intestinal absorption of cefmetazole, Chem. Pharm. Bull., 33 (1985) 4600-4605. I. Bekersky, H.G. Boxenbaum, M.H. Whitson, C.V. Puglisi, R. Pocelinko and S.A. Kaplan, Simultaneous determination of salicylic acid and salicyluric acid in plasma and urine by high-pressure liquid chromatography, Anal. Lett., 10 (1977) 539-550. A.M. Brisson and J.B. Fourtillan, Determination of cephalosporins in biological material by reversed phase liquid column chromatography, J. Chromatogr., 223 (1981) 393-399.
20
21
22 23
B.G. Charles and P.J. Ravenscroft, Rapid HPLC analysis of cefoxitin in plasma and urine, J. Antimicrab. Chemother., 13 (1984) 291-294. W.L. Chiou, Critical evaluation of the potential error in pharmacokinetic studies of using the linear trapezoidal rule method for the calculation of the area under the plasma level-time curve, J. Pharmacokin. Biopharm., 6 (1978) 539-546. M. Gibaldi and D. Perrier, Pharmacokinetics, 2nd edn., Marcel Dekker, New York, NY, 1982. J. Nakamura, K. Shima, T. Kimura, S. Muranishi and H. Sezaki, Role of intestinal mucus in the absorption of quinine and water-soluble dyes from the rat small intestine, Chem. Pharm. Bull., 26 (1978) 857-863.
ofControlled Release,
7 (1988) 53-60 B.V., Amsterdam -
Elsevier Science Publishers
53 Printed
in The Netherlands
RATE-CONTROLLED ABSORPTION ENHANCEMENT OF RECTALLY ADMINISTERED CEFOXITIN IN RATS BY SALICYLATE H.J.M.
E.J. van Hoogdalem”, Center for Bio-Pharmaceutical Box 9503,230O (Received
July
Sciences,
van Kan, A.G. de Boer and D.D. Breimer Divisron of Pharmacology,
Sylvius Laboratories,
State University
of Leyden, P.0
RA Leyden (The Netherlands) 1, 1987;
accepted
in rewsed
form
September
24,
1987)
Sodium salicylate has been reported to enhance the rectal absorption of the antibiotic cefoxitin in rats and humans. In the present study some factors influencing the enhancing effect of salicylate on rectal cefoxitin absorption were investigated, in particular in relationship to the rate of delivery and absorption of salicylate itself. Salicylate proved to be rapidly and completely absorbed after bolus delivery as well as after rectal infusion. The absorption of cefoxitin, which was absorbed to a limited extent (8 to 32%) without enhancer, could be promoted by salicylate on bolus delivery, resulting in mean bioavailabilities of 46% and 101% after delivery 2 mm and 1 cm from the anus, respectively. Rectal infusion or delivery of the same amount of cefoxitin and salicylate to a larger mucosa area resulted in poor absorption of the antibiotic. Results indicate a critical influence of the amount of salicylate delivered area-l time-’ on the absorption-enhancing effect.
INTRODUCTION Application of many polar drugs is restricted to parenteral routes of administration because they cross biological membranes poorly. This is a serious limitation with drugs such as insulin, several antibiotics andpeptide drugs. Enhancement of intestinal absorption would in principle make it possible to administer such compounds by the oral or rectal route. Several studies have indicated that enhancement of absorption is feasible by co-administration of absorption promoters. Intestinal uptake of the cephalosporin antibiotic cefoxitin was reported to be enhanced by sodium salicylate and Brij 35 [ 1 ] , acylcarnitines [ 2 1, phosphate derivatives [ 31, sodium 5methoxysalicylate [ 451, epinephrine metabolites [ 61, concanavalin A [ 71, EDTA and polyoxyethylene-23-lauryl ether *To whom correspondence
0168.3659/88/$03,50
should be addressed.
0 1988 Elsevier Science Publishers
[ 81. Among the enhancers studied, sodium salicylate is an interesting agent because of its reported effectiveness and its applicability in man. It has been shown to increase intestinal absorption of [ Asu~‘~] -eel calcitonin [ 91, dextran [lo], insulin [ 11,121, antibiotics [ 4,5,13-151 and phenylalanine [ 161 in rats or dogs. In the studies referred to no attention was paid to the influence of delivery rate on the effect of the absorption promoter. Because the absorption-enhancing action of salicylate decreases with the disappearance of salicylate from the intestinal lumen [ 171, the retention of a minimal effective concentration of salicylate at the mucosal surface seems to be required to exert an adequate absorption-enhancing action. The influence of delivery rate on rectal spreading behaviour of delivered bromophenol blue solution was investigated. The enhancing effect of salicylate on cefoxitin absorption was evaluated in relationship to its rate of delivery.
B.V.
54
MATERIALS
AND METHODS
Chemicals
Cefoxitin sodium (Mefoxin@ ) was a gift from Merck, Sharp & Dohme, Haarlem, The Netherlands. Cefazolin sodium ( KefzolO ) was a gift from Eli Lilly & Co., Utrecht, The Netherlands. Sodium salicylate (Brocacef, Maarssen, The Netherlands) was of pharmaceutical grade. Ethyl acetate (Baker Chemicals, Deventer, The Netherlands) was distilled before use. Bromophenol blue was obtained from Janssen Chimica, Beerse, Belgium. All other reagents used were of analytical grade. Animals
of cefoxitin
For i.v. administration a solution of cefoxitin sodium 15 mg/ml, made isotonic by addition of sodium chloride, and a solution containing cefoxitin sodium 15 mg/ml and sodium salicylate 60 mg/ml were used. For rectal administration solutions containing cefoxitin sodium 15 mg/ml were used, with or without sodium salicylate 60 mg/ml, in 0.067 A4 phosphate buffer pH 7.4 or in water. In one experiment less-concentrated solutions were used, containing cefoxitin sodium 3 mg/ml with or without sodium salicylate 12 mg/ml in 0.067 M phosphate buffer pH 7.4. The ionic strength of all solutions used rectally was adjusted to 0.75 by adding sodium chloride. Drug administration
The animal experiments performed were registered according to the Dutch Experimental Animals Act. For the assessment of rectal spreading male Wistar rats of laboratory breed, weighing 185-235 g, were used. For experiments concerning rectal absorption of cefoxitin female Wistar rats of laboratory breed, weighing 175-200 g, were used. From 16 h prior to the experiments until their completion only water intake was allowed. Drug solutions Rectal spreading
To assess rectal spreading a solution was used containing bromophenol blue 10 mg/ml in water. Bioavailability
Bioavailability
of salicylate
For i.v. administration a solution of sodium salicylate 5 mg/ml with cefoxitin sodium 15 mg/ml was used, made isotonic by addition of sodium chloride. For rectal administration a solution of sodium salicylate 5 mg/ml and cefoxitin sodium 15 mg/ml in 0.067 M phosphate buffer pH 7.4 was used. The ionic strength was adjusted to 0.75 using sodium chloride.
For i.v. infusion a poly(viny1 chloride) cannula (length 150 cm; i.d. 0.5 mm; o.d. 1 mm; Talas, Ommen, The Netherlands) filled with 200 ~1 of drug solution was inserted into the right jugular vein under light ether anesthesia. For rectal administration of the drug solutions a poly (vinyl chloride) cannula (id. 0.5 mm; o.d. 1 mm) containing 200 ~1 or 1 ml of’ drug solution was connected to a rectal delivery device. This device consisted of 3 cm of stainless steel tubing (i.d. 0.5 mm; o.d. 1 mm ), introduced into the tip of the plunger of a l-ml disposable syringe (Bect.on-Dickinson, Ireland). The tubing emerging from the plunger tip was elongated using a length of teflon cannula (i.d. 1.15 mm; o.d. 1.75 mm; Talas, Ommen, The Netherlands) to establish rectal delivery at a distance of 2 mm or 1 cm from the anus. Under light ether anesthesia the teflon cannula was introduced into the rat rectum and the device was ligated with the anus closing around the plunger tip. After insertion the intravenous or rectal cannula was pulled s.c., emerging on the nape of the neck, and suspended above the rat, allowing free movement. After a recovery period of 2 h the cannula was connected to an infusion pump
55
(Braun, Melsungen, West-Germany). To achieve bolus administration 200 ~1 or 1 ml were delivered in 24 s. Linear infusion was performed by delivering 200 ~1 in 32 min and 1 ml in 26.7 min.
Blood sampling
Under light ether anesthesia a poly (vinyl chloride) cannula (length 50 cm; i.d. 0.5 mm; o.d. 1 mm) filled with heparin in saline (200 III/ml) was inserted into the right carotid artery. The cannula was pulled s.c., emerging on the nape of the neck. The cannula was suspended above the caged rat, allowing free movement. Blood samples of 100 ~1 were taken at regular intervals after starting the experiment and were haemolysed in 500 ~1 of distilled water. The samples were stored at - 20’ C until analysis. After each sampling the cannula was filled with heparinized saline to prevent clotting. In the study of salicylate kinetics cannulas were removed under light ether anesthesia 10 h after starting the experiment, to prevent destruction by the rat overnight. From 19 h after starting the experiment blood samples were obtained by orbital puncture.
Assessment
of rectal spreading
Drug assays Sodium salicylate
The assay of sodium salicylate in haemolysed blood was based on the method described by Bekersky et al. [ 181. To the blood samples 70 ~1 of an internal standard solution containing 3,4,5trimethoxybenzoic acid 50 yg/ml were added. The samples were acidified with 100 ~1 of 1.0 M HCl and 5 ml of ethyl acetate were added. After whirlmixing for 30 set and centrifugation for 5 min at 2000 g the organic layer was transferred to another test tube. 1 ml of 0.067 Mphosphate buffer pH 7.4 was added and the samples were extracted by whirlmixing for 30 s. The organic layer was discarded after centrifugation for 5 min at 2000 g. Residual ethyl acetate was removed under reduced pressure (Vortex-Evaporator, Buchler Instruments Inc., Fort Lee, NJ, U.S.A.). 25 ~1 of the aqueous extract were analysed using a liquid chromatographic system (Waters Associates, Millford, MA, U.S.A.) consisting of an M-45 pump, a WISP 710B automatic sample injector, a Zmodule containing a Radial-Pak Cl8 cartridge, and a Model 481 LC spectrophotometer (wavelength 237 nm) . The mobile phase consisted of 1.5% v/v acetic acid containing 46% v/v methanol. The flow rate was 2.0 ml/min. Data processing was performed by a Chromatopack CR3A reporting integrator (Shimadzu, Kyoto, Japan ) . Cefoxitin
Under urethane anesthesia (600 mg/lOO g i.p.) the delivery device was ligated in the rat rectum, allowing administration of bromophenol blue solution in the rectum at a distance of 2 mm from the anus. After laparotomy the rectum was exposed. The cannula was connected to the infusion pump and 200~1 of bromophenol blue solution were delivered as a bolus in 24 set or as an infusion in 32 min. Spreading of the dye solution from the delivery site higher into the rectum was investigated for 1 h and was measured at regular intervals using a ruler.
sodium
Cefoxitin sodium was assayed in haemolysed blood by reversed-phase high-performance liquid chromatography, based on the methods described by Brisson and Fourtillan [ 191 and by Charles and Ravenscroft [ 201. To the haemolysed blood samples 70 ~1 of an internal standard solution containing cefazolin sodium 2.4 ,ug/ml were added. After acidification by adding 250 ~1 of 0.4 M HCl the samples were extracted with 5 ml of ethyl acetate for 15 s on a whirlmixer. After centrifugation for 5 min at 2000 g the organic layer was transferred to another test
56
tube. 500 ~1 of 0.067 M phosphate buffer pH 7.4 were added and the samples were extracted by whirlmixing for 15 s. The organic layer, obtained after centrifugation for 5 min at 2000 g, was discarded and residual ethyl acetate was removed under reduced pressure (Vortex-Evaporator, Buchler Instruments Inc., Fort Lee, NJ, U.S.A.). 50 ~1 of the residue were injected on the liquid chromatographic system, at a wavelength of 270 nm. As mobile phase was used 0.05 M KH,PO, solution (pH 2.9 using phosphoric acid) containing 18% acetonitrile. The flow rate was 2.5 ml/min.
Data analysis
According to Chiou [ 211 the areas under the individual blood concentration time curves were calculated from t = 0 to maximal concentration with the linear trapezoidal rule and after the maximal concentration to the last sampling point using the logarithmic trapezoidal rule. For extrapolation to infinity of i.v. curves and curves following rectally delivered salicylate, individual elimination rate constants were used. Because of the irregular pattern of the apparent elimination phase an accurate elimination rate constant of rectally delivered cefoxitin could not be obtained. For this reason curves obtained after rectal cefoxitin administration were extrapolated using the mean elimination rate constant for i.v. administration. Systemic clearance of cefoxitin sodium was calculated as D/AUC, where D is the administered i.v. dose of cefoxitin sodium and AUC is the total area under the curve. Using statistical moment theory [ 221 the mean residence time (MRT) after i.v. and rectal administration of sodium salicylate was calculated and corrected for the rate of delivery. The Wilcoxon rank sum test was used for st,atistical evaluation of the results. A p-value smaller than 0.05 was considered as a significant difference.
I’
.Y
,A
:
‘-
Fig. 1. Spreading distance into the rectum i SD of 200 ~1 of bromophenol blue solution after rectal delivery 2 mm from the anus as bolus in 24 s (0, n =6) or as linear infusionin32min (A,n=5).
RESULTS
The spreading profile of rectally delivered bromophenol blue is shown in Fig. 1. Immediately after bolus delivery 53 ? 10 mm rectum is filled with solution. Spreading continued relatively slowly to 70 29 mm. Rectal infusion, however, resulted in a slow progression of the dye solution in the rectum. It lasted about 40 min before the length of rectum was reached, which is momentarily filled on bolus delivery. After infusion a shorter ultimate spreading distance was reached as compared to bolus delivery. After rectal bolus delivery and rectal infusion of 1 mg of sodium salicylate at a distance of 1 cm from the anus comparable blood concentrations were obtained (Fig. 2). The mean AUC 2 SD after rectal infusion and bolus delivery of 107 & 28 ,ug h/ml and 128 2 25 ,ug h/ml, respectively, did not differ significantly from the value obtained after i.v. infusion (109 t 47 fig h/ml). After i.v. infusion a mean MRT? SD of 4.3 2 1.0 h was determined. Rectal infusion and bolus delivery resulted in comparable values of 5.6 -t 1.9 h and 5.6? 1.0 h, respectively, which did not differ significantly from the i.v. data.
57
Fig. 2. Mean blood levels of sodium salicylate i SD after rectal delivery of 1 mg of sodium salicylate with 3 mg of cefoxitin sodium in phosphate buffer, as a bolus ( 0, n = 6) and as infusion ( A, n=6) at a distance of 1 cm from the anus. Fig. 4. Mean blood levels of cefoxitin sodium &SD after rectal bolus delivery of 3 mg of cefoxitin sodium in phosphate buffer at a distance of 1 cm from the anus, with ( 0, n = 5) or without ( A, n = 7 ) 12 mg of sodium salicylate. AUC,,,lug
mm/ml1
Fig. 3. Mean blood levels of cefoxitin sodium +SD after rectal bolus delivery of 3 mg of cefoxitin sodium in phosphate buffer at a distance of 2 mm from the anus, with ( 0, n = 6) or without ( A, n = 6) 12 mg of sodium salicylate.
These results indicate a rapid and complete absorption of salicylate, irrespective of delivery rate. Intravenous infusion of 3 mg of cefoxitin sodium resulted in a mean AUC of 312? 73 pg min/ml and a systemic clearance value of 9.8? 2.6 ml/min (n = 7). Co-administration of 12 mg of sodium salicylate i.v. resulted in a comparable AUC of 358%42 pg min/ml and a systemic clearance of 8.5 -t 0.9 ml/min ( n = 3 ) , which excludes an interaction of salicylate with cefoxitin elimination kinetics. Bolus delivery of 200 ~1 of cefoxitin solution in phosphate buffer 2 mm (Fig. 3) and 1 cm
D
E
G
H
I
J
Fig. 5. Histogram of the mean AUC &SD of cefoxitin sodium after iv. and rectal administration of 3 mg of cefoxitin sodium with (cross-hatched) and without (blank) 12 mg of sodium salicylate, under various circumstances: A: i.v. infusion; B, C: bolus delivery (B) and infusion (C) in phosphate buffer. 2 mm from the anus; D, E: bolus delivery (D) and infusion (E) in water, 2 mm from the anus; F, G: bolus delivery (F) and infusion ( G ) in phosphate buffer, 1 cm from the anus; H, I: bolus delivery (H) and infusion ( I) in phosphate buffer, 1 cm from the anus; J: infusion in phosphate buffer over a length of 2 mm to 1 cm from the anus; administered volume: 200 ~1 (A-G, J ) or 1 ml (H, I) ; * : significantly different from control value without enhancer (p < 0.05, Wilcoxon rank sum test ) .
58
(Fig. 4) from the anus resulted in relatively low blood concentrations and AUC values ( Fig. 5 ) , which were very substantially enhanced by salicylate. Infusion with or without phosphate buffer also resulted in low AUC values, but interestingly these were not enhanced by salicylate. Infusion of 200 ~1 between 0.2 cm and 1 cm from the anus as well as infusion or bolus administration of 1 ml of the less concentrated cefoxitin solution resulted in low mean AUC values, which could not be influenced by coadministration of salicylate. After bolus delivery and infusion without phosphate buffer low mean AUC values were obtained, which were not enhanced by salicylate (Fig. 5 1.
DISCUSSION
In order to evaluate the luminal distribution of rectally delivered solutions in relationship to rate of delivery the spreading profile of a dye solution was investigated in laparotomized rats. The observed rectal spreading of bromophenol blue demonstrates that a relatively small volume of solution spreads over a considerable length of rectum. After bolus administration a larger section of the rectum is covered as compared with infusion. The rate of spreading proves to be considerably higher after bolus delivery than after infusion. Bromophenol blue is a poorly absorbed compound [ 231 and it will therefore experience maximal spreading; it is conceivable that compounds with a higher absorption rate will be distributed over a shorter length. On bolus administration in phosphate buffer, salicylate significantly enhanced the extent of cefoxitin absorption (Fig. 5)) in contrast with rectal infusion. This difference is not caused by a prolonged presence of the enhancer after bolus delivery, considering the rapid and complete absorption of salicylate (Fig. 2 ) . The difference in spreading distance higher into the rectum after infusion and bolus delivery is too small to explain the difference in enhancing effects
by characteristics of the absorption site. Infusion close to the anus and 1 cm deep into the rectum resulted in comparable effects, excluding important differences in absorbing capacity along the first l-cm section of the rectum. The results of the bolus experiments versus the infusion experiments indicate that rate of delivery is essential in optimizing the effects of the absorption promoter. Optimal absorption enhancement requires the presence of an effective luminal salicylate concentration until cefoxitin absorption is complete. Rectal administration of the dye showed that the amount delivered area- ’ time- ’ after infusion is substantially lower compared with bolus delivery. The slow delivery of rectally infused salicylate and its rapid absorption prevent that effective salicylate concentrations are reached, whereas after bolus input an effective concentration is achieved instantaneously, but only for a very short period of time. The reported steep sigmoidal relationship between effectiveness of salicylate and its concentration [ 81 illustrates that a relatively small decrease in salicylate concent.ration will result in a loss of absorpsolution tion-enhancing action of the administered. The effect of salicylate, which was pronounced after bolus delivery of 200 1~1of solution, was absent after bolus delivery of the same amount of cefoxitin and salicylate in 1 ml (Fig. 5). Delivery in a larger volume lowered the amount of salicylate area- ’ time -’ and probably thereby reduced the effect of the enhancer. The lack of absorption-enhancing effect of salicylate after infusion at 2 mm or 1 cm from the anus was also determined after delivery to a larger mucosa area by infusion between 2 mm and 1 cm from the anus (Fig. 5). Although the area available for absorption was increased, absorption remained poor, which may again be explained by an ineffective amount enhancer area-‘timee’. After rectal delivery without phosphate buffer absorption enhancement could not be achieved and considerable variability in the data was ob-
59
served (Fig. 5). The presence of phosphate buffer seems to reduce variability by standardizing circumstances for absorption. Our results show a striking similarity with those of other authors. Liversidge et al. [ 11,121 observed an increasing enhancing effect of salicylate on rectal insulin absorption from suppositories on decreasing the surface area. Although the authors suggest that this difference is caused by differences in characteristics of the absorption site or in extent of first-pass metabolism, our data indicate that a larger amount of enhancer area- ’ time- ’ after administration in small or short suppositories promotes the effect of salicylate. Nishihata et al. [ 51 observed a better absorption-enhancing effect of 5-methoxysalicylate in gelatin formulation compared with delivery in phosphate buffer. It is likely that administration in the viscous gelatin solution will reduce spreading and will raise the amount of 5-methoxysalicylate area- I time-‘, thus enhancing its effect. The results of our study demonstrate that rate-controlled delivery, fitted to the absorption behaviour of the enhancer, may be decisive for the effectiveness of the absorption-promoting action.
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