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Characterization of the Oral Absorption of β-Lactam Antibiotics II: Competitive Absorption and Peptide Carrier Specificity
Characterization of the Oral Absorption of p-Lactam Antibiotics II: Competitive Absorption and Peptide Carrier Specificity PATRICK J. SINKO** AND GORDONL. AMIDON*"
Received Au ust 30, 1988, from the 'College of Pharmacy, The University of Michi an, Ann Arbor, MI 48109-7065, and 'Therapeutic Systems Accepteffor publication January 24, 1989. Research La%oratories,Inc., 7979 Green Road, Ann Arbor, MI 48705. Abstract 0The p-lactam antibiotic oral absorption pathway is studied
using a single-pass perfusion technique in the rat small intestine. p-Lactam antibiotic absorption iin the presence of amino acids, small peptides, and other p-lactams is modeled using a simple competitive inhibition boundary condition at the intestinal wall, with a corrected value for the intestinal wall concentration, C,, derived from the modified boundary layer (analysis.The model-predicted permeability in the presence of an inhibitor is used tlo characterize the p-lactam antibiotic intestinal carrier system. Several concentrations of cephalexin, coperfused with a constant concentration of cefadroxil (equal to its &), showed that the K, of cephalexin approximately doubled from 7.2 (21 . l ) to 18.8 (24.1) rnM; J f , remained unchanged at 9.2 (r1.2) and 11.1 (e2.1) mM; and the carrier permeability, Pa,was reduced by -50% from 1.11 (kO.10) to1 0.59 (-r-0.04), consistent with competitive absorption kinetics. The predicted in situ wall permeability, < P ;>, of p-lactams perfused in the presence of other p-lactams was calculated and then compared with isxperimentallydetermined values. For cefadroxil, P t, = 0.27 (+0.04), < P > ; = 0.29; for cefatrizine, P ; = 0.67 (+0.09), < P > ; = 0.59; and for cephalexin, P:; = 0.56 (20.05), < P > ; = 0.59. The characteristics of the p-lactam intestinal carrier system were further studied by co-perfusing &lactarns with several di- or tripeptides, which resulted in a significant decrease in p-lactam permeability, whereas co-perfusion with amino acids did not decrease p-lactam permeability. These results strongly suppoll p-lactam absorption by the intestinal peptide transport system in rats. Moreover,these results, consistent with simple competitive inhibition, indicate that macroscopic absorption parameters may be useful for predicting drug-drug and possibly fooddrug absorption interactions in the gastrointestinal tract of humans. _____-
~ _ _ _ ~ _ _ _ _ ~
Inhibition of p-lactam absorption by amino acids, small peptides, and other p-liactam antibiotics14 has been demonstrated using in vitro and i.n situ perfusion techniques in the rat small intestine. P-Lactam absorption in the rat has been attributed to several intestinal carriers and mechanisrns.2.5~6 For example, Miyazak:i et 211.7 reported that cyclacillin shares a common transport mecjhanism with cephradine but not cephalexin. Others2.6 have concluded that cephalexin absorption is independent of intestinal concentration and that it does not have a n absorption pathway in common with some of the other orally absorbed cephalosporins. Based on recently reported findings5 it appears that the latter results were a consequence of the cephalexin concentrations being low compared with its K,, rather than due t o its absorption mechanism. Despit'e the dispariky of these results, the similarity of p-lactam structures would seem to preclude absorption by distinct carriers. Quays and Quay and F~osterswere the first to report the inhibition of cephalexin absorption by the dipeptide Lphenylalanylglycine and the amino acid glycine in excised rat jejunum. They reported t h a t the interaction with Lphenylalanylglycine is due to competition for a carriermediated abisorption site, whereas the interaction with glycine "is not due to tissue .toxicity nor to competition for the imino or monoamino monocarboxylic acid c a r r i e d . 8 Later reports14 generally support these initial findings; however, 0022-3549/89/0900-0723$01 .OO/O 0 1989, American PharmaceLiticaI Association
they are not always in agreement. For example, Nakashima et al.3 demonstrated that the absorption of several p-lactams was inhibited by dipeptides. On the other hand, Kimura et a1.2 reported that the absorption of cephradine, but not cefadroxil, was significantly inhibited by amino acids, whereas the absorption of both aminocephalosporins was not inhibited by a 50 times larger concentration of the dipeptides. Recent reports by Okano et al.4 provide a mechanistic basis for the transport of aminocephalosporins by the dipeptide transport system in the intestinal brush border membrane. Suggestions that the p-lactam antibiotics at least in part share the small peptide absorption pathway are also based on structural similarities to the small peptides. Most of the orally administered p-lactam antibiotics are structurally analogous to tripeptides. The orally absorbed cephalosporins are of the 7-phenylglycyl type, with different substituents a t positions 3 and 7. Cefatrizine, with a bulky heterocyclicthiomethyl group a t position 3 of the thiazine ring, is the most structurally distinct of the series. Furthermore, cefatrizine has a significantly lower intrinsic Michaelis constant (0.7 mM) than the other 7-phenylglycyl cephalosporins studied (3-8 mM) in rats.10 An analogous situation occurs with the dipeptides; that is, substitution of a n amino acid with a bulkier side chain onto the peptide lowers the Michaelis constant of the peptide for membrane absorption sites in humans.11 Specifically, Adibi and Soleimanpour12 demonstrated that the Michaelis constant of glycylglycine is 43.4 mM, whereas the bulkier dipeptide, glycylleucine, has a K, value equal to 26.8 mM in perfused human jejunum. Similarly, Dunn et al.13 reported that the absorption site was even more discriminating to structural differences with para substitution of a hydroxyl group on the 7-phenylglycyl cephalosporins, resulting in a significant increase in absorption over the metu-substituted compound in the mouse. Even though more mechanistic studies are being done with a variety of in vitrolin situ techniques, it is also necessary to attain a macroscopic understanding of the absorption mechanism of amino acids, small peptides, and the p-lactam antibiotics. Therefore, the objectives of this report are: (1) to study the p-lactam intestinal absorption mechanism in a quantitative yet macroscopic fashion, using intrinsic membrane absorption parameters and competitive inhibition studies, using the p-lactams, amino acids, and small peptides as inhibitors; and (2) to establish a basis for predicting drugdrug and possibly food-drug interactions for compounds absorbed by the amino acid or small peptide pathways.
Experimental Section The following materials were used cefadroxil, cephalexin (potency cephradrine (910 mgig), citric acid, dibasic sodium phosphate, glycine, glycylglycine, glycylalanylalanine, phenylalanine, phenylalanylglycine,monobasic potassium phosphate, polyethylene glycol 4000 (Sigma Chemical, St. Louis, MO), cefaclor (956 mgi g, lot no. 206BV6, Eli Lilly and Company, Indianapolis, IN), cefatrizine 1,3-propylene glycolate (845 mgig, SmithKline Beckman, = 987 mg/g),
Journal of Pharmaceutical Sciences i 723 Vol. 78, No. 9,September 1989
Philadelphia, PA; Cebris, Sermoneta, Italy), [14C]polyethyleneglycol (duPont-New England Nuclear, Boston, MA). All buffer and mobile phase components were analytical (or HPLC) grade and used as received. Perfusion Experiments-Perfusion procedures have been detailed in an earlier report.14 The perfusion solution consisted of a citric aciddibasic sodium phosphate buffer (pH 6.51, 0.01% (wiv) PEG 4000 with a tracer amount of its I4C-labeledisotope, a plactam antibiotic, and an inhibitor. The perfusate (pH6.5,300 2 5 mmoLkgofwater) wasmaintainedat 37 "C by a water bath (Tek-Pro, American Dade, Miami, FL). The osmolality was measured using either a model 5002 automatic osmometer (Precision Systems, Sudsbury, MA) or a model 5500 vapor pressure osmometer (Wescor, Logan, UT). All experiments were performed on male Charles River rats, 250400 g, age 60 to 80 d. The rats were fasted overnight, 12-18 h before each experiment. Water was given ad libitum. Anesthesia was induced by a 50% w/v intramuscular injection of urethane (1.5 g/kg). The jejunum was located and a 10-15-cm segment was perfused. The jejunum was cannulated at 2 4 cm below the ligament of Treitz and 6 1 5 cm distal to the first incision. The intestinal segment was perfused using a constant infusion pump (Harvard Apparatus, model 931, S. Natick, MA) for 90 to 105 min. The perfusion flow rate was -0.25 mlimin. Steady-state was achieved in -30 min, after which four to six samples were taken at 10-15-min intervals. All samples were simultaneously quenched at the experiments completion with 6% (wh) citric acid solution. After the last sample was taken, the length was measured by placing a piece of string along the intestine and measuring the string with a ruler. Analytical Method-The samples were analyzed by liquid scintillation counting and by high-performance liquid chromatography (HPLC). Samples were counted using a Beckman LS-9000 counter (Beckman Instruments, Fullerton, CA) with automatic quench correction. A 0.5-mL sample was mixed with 15 mL of scintillation fluid (Ready-Solv, Hph, Beckman Instruments) before counting. Samples were counted using a single-channel technique. The HPLC instrumentation consisted of a pump (Beckman 112 solvent delivery module, Beckman Instruments, Berkeley, CA), automated sampler (WISP, model 710B, Waters Associates, Milford, MA), ultraviolet detector (Kratos Spectroflow 783 absorbance detedor, Kratos Analytical Instruments, Ramsey, NJ), and a reversed-phase column (pBondapak CIS, Waters Associates).The cephalosporin mobile phase consisted of methanol and pH 5 citric aciddibasic sodium phosphate buffer (22:78). Samples were eluted at a flow rate of 1.5 mlimin.
Theoretical Section
dimensionless aqueous permeability, P* = PRID, R is the radius of the intestinal lumen, and D is the estimated aqueous diffusion coefficient. The mean intestinal wall concentration, C,, used is derived from the boundary layer analysis.15 A more general boundary condition for the wall permeability in the presence of an inhibitor than eq 2 is:
c = c,, 2 = 0, t 2 0 aC -arr
-0
where r is the tube radius, z is the axial coordinate, v, is the axial velocity profile, D is the solute aqueous diffusivity, P , is the membrane (wall) permeability, and C, = C(R,z)is the concentration along the tube wall. The solution to the partial differential equation, in dimensionless form, is a function of three parameters, Gz, C, and P*,, where Gz = ?rDLl2Q,C, is equal to C,(l-P*,dP*,,), C, is the inlet perfusate concentration, P:, is the dimensionless effective permeability, P:, is the 724 I Journal of Pharmaceutical Sciences Vol. 78,No. 9,September 1989
+Pk
(3)
where JLaXis the maximal carrier flux, K"," is the apparent Michaelis constant, and P& is the passive membrane permeability. Dividing the numerator and denominator of the nonlinear term by the apparent Michaelis constant results in an alternate form of the boundary condition in terms of another fundamental parameter, the apparent carrier permeability:
p,*.aPP
~
P,
=
1 + - c,
+ Pk
(4)
KaPP m
where: K",pp =
(I
+g)Km
(5)
(1
g)
(6)
-1
P,**app
=
+
Pc*
where P:vaPP is the apparent carrier permeability, Pz is the dimensionless intrinsic carrier permeability, J L J K , is the apparent carrier permeability, PzpaPPis equal to J L a x / K g P , and the dissociation constants K , and Ki are defined in the usual manner:
Recently, a boundary layer approach (MBL) to analyzing the results of the perfused intestinal segment was presented.15 Briefly, the fundamental transport equation for the flow of a solution through a tube is:
The boundary conditions utilized in the MBL analysis were:
+
= KgP J*,=C,
P;
Km = K.
=
~
[Dl[Cl [C - Dl [Il[CI [C-I]
(7a)
(7b)
where [Cl is the concentration of free carrier, [Dl is equal to the Concentration of drug at intestinal wall (C,), [rJ is the concentration of inhibitor, [C], is the total concentration of carrier, [C - 03 is the concentration of the drug:carrier complex, and [C - r] is the concentration of the inhibitor: carrier complex. The values K , and Kiare macroscopic dissociation constants describing the "affinity" of the drug or inhibitor for an intestinal carrier system that is probably complex in nature. These two parameters and their value in relation to the intestinal drug concentration will be the principal determinants of the absorption behavior of the drug. Substituting eq 5 into eq 3, the expression for the wall permeability used in the experimental data analysis is: &ax
P; = (1
+ g ) K m + C,
+ P;
(8)
In order to study the proposed competitive absorption inter-
action, the experimental results analyzed by eq 8 are compared with the calculated wall permeability, assuming classical competitive inhibition. Therefore, the predicted carrier permeability in the presence of an inhibitor is now defined as:
= (1 -
f)P:
Table I-Summary of Intrinsic Membrane Parameters for Competitive Inhibition Studiese
Parameter
(9)
I11
(10)
i 2)
[I1+ Ki 1 + -
since P*,is a constant, eq 9 can be written as:
W
=
<:p;> +
(11)
where cP*,>is the model-predicted wall permeability in the presence of an inhibitor. With the appropriate substitutions, eq 11can be rewritten in final form as:
= 1 - --[I1 W
Pc* + P;
i 21
[ I ] + Kl 1 +
(12)
-
Using eq 12 and the intrinsic membrane absorption parameters from1 single drug perfusion experiments, the competitive interaction of drugs absorbed by carrier mediated processes is characterized.
Results and Discussion Competitive absorption experiments were performed with cephalexin pe rfused over approximately a lo4 concentration range in the rat small intestine. The inhibitor, cefadroxil, was perfused at a constant conoentration of 7 mM ( C , = 6 mM), which is approximately equal to its K , in rats.10 The data was fit to eqs 3 and 4 using weighted nonlinear regression. A plot of the wall permeability versus the wall concentration of cephalexin, with and without the inhibitor, is shown in Figure
Cefadroxil
7.2 ( k l . 1 ) 18.8 (?4.1)
5.9 (20.8)
-
.-
9.1 ( t l . 2 ) 11.1 (52.1) 1.11 (20.1) 0.59 (k0.04)
where the frac1;ional inhibition, f, is defined as: f =
Cephalexin
4.6 (tO.9) 8.4 (20.8)
-
1.43 (20.1)
-
The concentration of cephalexin ranged from 0.01 to 30 mM; the concentration of cefadroxil remained constant at 5 mM; parameters reported are fitted values i standard deviation.
1.The corresponding intrinsic membrane absorption parameters are summarized in Table I. As seen in Table I, JL,,of cephalexin was unchanged, while the apparent K , and P*, differed. The shift in KZP is not due to a decrease in the affinity of cephalexin for the absorption site, but rather to a portion of the carriers becoming inaccessible to the drug once the inhibitor binds to them since the drug and inhibitor are mutually exclusive at the absorption site. This is manifest as an increase in the apparent or measured Michaelis constant. The inhibitor, cefadroxil, was perfused at its K ,; therefore, from eq 5 the predicted value of KZp should be twice the intrinsic K , of the substrate, cephalexin. This is confirmed in Table I with values of K , and KZpequal to 7.2and 18.8 mM, respectively. For a single carrier system it is expected that the K, ofthe inhibitor should equal its&. This was the case since the K, of cefadroxil in the inhibitor study approximately equaled the K , of cefadroxil determined previously.6 The maximal flux, J*,,,, of cephalexin remained unchanged (Table I) in the presence of cefadroxil; however, it should be noted that much higher cephalexin concentrations are needed PMw
PRw
Na I n h i b i t o r - Inhibitor
m]C a l c u l a t e 0 P e r m e a b l l i t v
1 5
T
$ 1
0
.Ti
T. .d
n
m a,
E L (u
a
T.
T.
m 0 5 3
3 0
Figure 1-Plot of the wall permeability of cephalexin perfused alone and in the presence of a competitive inhibitor, cefadroxil (7mM). The results are reported as the mean wall permeability t SEM.
Csfadroxil
Cefatrizine
Cephalexin
Figure 2-Plot of the mutual inhibition and predicted in situ wall permeability of several p-lactam antibiotics in the presence of other
plactam antibiotics. Experimental values are reported as the mean
2
SEM.
Journal of Pharmaceutical Sciences I725 Vol. 78, No. 9, September 1989
in order to attain any given fraction of J*,,,. In the presence of the inhibitor, the ratio J*,,Xm was reduced by -50% when compared with the carrier permeability in the absence of an inhibitor. Since J*,, remained constant with an increasing K",pp,the apparent carrier permeability, P;saPP, was reduced by a factor of (Kz*/K,) or 50%. These results demonstrate mutual inhibition of j3-lactam antibiotic absorption that is consistent with simple competitive absorption kinetics in the rat small intestine. Recently, Amidon et a1.16 demonstrated an excellent correlation between the intestinal wall permeability in rats and the fraction of dose absorbed in humans. Extending this correlation to predict drug-drug or food-drug interactions in humans is possible once a macroscopic, yet mechanistic basis for these interactions has been established. Therefore, the feasibility of using the macroscopic absorption parameters to predict the apparent Michaelis constant and the apparent carrier permeability of the p-lactam antibiotics in situ was investigated. Several cephalosporins were perfused in the presence of other cephalosporins at concentrations above and below their respective K , values. The individual data were analyzed using eq 12 for the predicted permeability of a compound in the presence of a competitive inhibitor. As seen in Figure 2 and Table I1 there is no significant difference between the predicted and measured wall permeability. This clearly demonstrates that the macroscopic absorption parameters can be used to predict the extent of mutual inhibition in situ. Based on the previously reported correlation of the
fraction dose absorbed in humans and the intestinal wall permeability,le it is expected that in vivo drug-drug absorption interactions and their effect on the pharmacokinetic profile of the drug will be predicted equally well for the p-lactam antibiotics. Inhibition experiments were also performed with amino acids and several small peptides. Co-perfusion of cefatrizine (0.1mM) and the amino acids phenylalanine and glycine (100 mM) did not result in a significant change (p=0.40 and p = 0.27, respectively) in cefatrizine wall permeability (Figure 3). However, co-perfusion of cefatrizine and the dipeptides GlyGly (100 mM) or Phe-Gly (60 mM) or a tripeptide analogue of cephalexin, Gly-Ala-Ala (23 mM), resulted in a significant decrease (p < 0.03, p < 0.035, and p < 0.025, respectively) in wall permeability of cefatrizine (Figure 4, Table 111). However, calculating the degree of inhibition in the small peptide absorption studies using eq 12 is complicated by the luminal hydrolysis of the peptide and the subsequent competition of the amino acids and peptide fragments for the absorption site. Until these "peptide effects" can be adequately modeled and investigated, it is difficult to predict the magnitude of the food-drug absorption interaction. The results in this paper demonstrate that drug-drug interactions observed at the absorption site can be predicted Table Ill-Measured Permeabilities of Cefatrizine (0.1 mM) in the Presence of Various Inhibitors Inhibitor
Table Il-Calculation of the Predicted Permeability of PLactams in the Presence of a &Lactam Inhibitor'
p:
Compound
(no inhibitor)
p:, (inhibitor)
Cefadroxil Cefatrizine Cephalexin
0.75 (50.05) 1.27 (50.10) 1.27 (kO.10)
0.27 (T0.04) 0.67 (20.09) 0.56 (k0.05)
(inhibitor) 0.29 0.59 0.59
Glycine Phenylalanine GLY-GLY PHE-GLY G LY-ALA-ALA Cefatrizine (control)
* Parameters are fitted values t SEM.
Concentration, mM
pk
SEM
Significance
100 100 100 60 23 0.1
1.48 1.44 0.91 1.09 0.99 1.28
0.17 0.17 0.13 0.39 0.12 0.10
ns ns p < 0.03 p < 0.035 p < 0.025
Cefatrizine
-
NO
I c e f a t r i z i n e
-
I n h = pept!des
-
Inhibitor
2.0 Cefatrizine
-
NO
m c e f a t r i z i n e
-
Inninitor
T
inhlnitor
-
Phe o r G I ~
T 1 .F z
u
-ri
rl .rl
c l lo 0 E
1.0
a, R r-l
c1
10
3
0.5
PHE
GI-Y
Figure +Plot of the wall permeability of cefatrizine (0.1 mM) in the presence of the amino acids L-phenylalanine (100 mM) or glycine (1 00 mM). Experimental values are reported as the mean 2 SEM. 726 I Journal of Pharmaceutical Sciences Vol. 78, No. 9, September 7989
GLY-GLY
PHE-GLY
Gt~Y - A L A - ALP.
Figure &Plot of the wall permeability of cefatrizine (0.1 mM) in the presence of Gly-Gly (100 mM), Phe-Gly (60 mM), Gly-Ala-Ala (23 mM). Experimental values are reported as the mean t SEM.
based on simplie competitive absorption kinetics. On the other hand, the effects of dietary peptides and amino acids on drug absorption require further study before the effects on Dlactam antibiotic absorption can be predicted.
Conclusions Using the results of the boundary layer analysis on the perfused intestinal segment;, with a modified boundary condition accounting for competitive inhibition, allowed the determination of the apparent and intrinsic membrane absorption parameters for several cephalosporins in the presence of competitive inhibitors. Mutual inhibition of the p-lactams was characterized using a derived expression for the predicted :in situ wall permeability. The observed inhibition is consistent with simple competitive absorption kinetics. Mutual inhibition among the &lactam antibiotics was observed with the Ki values equal to previously determined K , values. While di- and tripeptides inhibited p-lactam absorption, various amino acids did not; however, due to the complexities i.n the intestinal lumen, the magnitude of the “peptide effects” is difficult to predict and requires further analysis. The results of thlese studies begin to provide a n alternative mechanistic basis for plactam-p-lactam and possibly food--&lactam absorption interactions.
References and Notes 1. Kimura, ‘IEndo, ’.;H.; Yoshikawa, M.; Muranishi, S.;Sezaki, H. J. Pharm. Dyn. 1978, 1, 262-267. 2. Kimura, T.; Yamamoto, T.;Mizuno, M.; Suga, Y.; Kitade, S.;
Sezaki, H. J . Pharm. Dyn. 1983,6, 246-253. 3. Nakashima, E.; Tsuji, A,; Kagatani, S.; Yamana, T. J. Pharm. Dyn. 1984, 7, 452-464. 4. Okano, T.; Inui, K.; Maegawa, H.; Takano, M.; Hori, R. J . Biol. Chen. 1986.30.14130-14134. 5. Miyazaki, K.; Ogino, 0.; Nakano, M.; Arita, T. Chem. Pharm. Bull. 1977,25, 246-252. 6. Umeniwa, K.; Ogino, 0.;Miyazaki, K.; Arita, T. Chem. Pharm. Bull. 1979,27, 2177-2182. 7. Miyazaki, K.; Obtani, K.; Umeniwa, K.; Arita, T. J . .Pharm. Dyn. 1982,5, 555-563. 8. Quay, J. F. Physiologist 1972, 15, 241. 9. Quay, J. F.; Foster, L. Physiologist 1970,13, 287. 10. Sinko, P. J.; Amidon, G. L. Pharm. Res. 1988, 5, 645-650. 11. Adibi, S. A.; Kim, Y. S. In Physiology of the Gastrointestinul Tract; Johnson, L. R., Ed.; Raven: New York, 1981; pp 10731095. 12. Adibi, S. A.; Soleimanpour, M. R. J . Clzn. Znuest. 1974,53,13681374. 13. Dunn, G. L.; Hoover, J. R. E .; Berges, D. A.;Taggart, J. J.; Davis, L. D.; Dietz, E. M.; Jakas, D. R.; Yim, N.; Actor, P.; Uri, J . V.; Weisbach, J . A. J . Antibiot. 1976,29, 65-80. 14. Hu, M.; Sinko, P. J.; dehleere, A. L. J.; Johnson, D. A.; Amidon, G. L. J . Theor. Biol. 1988,131, 107-114. 15. Johnson, D. A.; Amidon, G. L. J . Theor. Biol. 1988,131,93-106. 16. Amidon, G. L.; Sinko, P. J.; Fleisher, D. Pharm. Res. 1988, 5, 651-654.
Acknowledgments Support by SmithKline Beckman and NIGMS Grant GM7188-01is r t e f u l l y acknowledged. The authors would also like to thank Mrs. oyce Durcanin-Robbins for her technical assistance with the perfusion studies.
Journal of Pharmaceutical Sciences I 727 Vol. 78,No. 9,September 1989
Received Au ust 30, 1988, from the 'College of Pharmacy, The University of Michi an, Ann Arbor, MI 48109-7065, and 'Therapeutic Systems Accepteffor publication January 24, 1989. Research La%oratories,Inc., 7979 Green Road, Ann Arbor, MI 48705. Abstract 0The p-lactam antibiotic oral absorption pathway is studied
using a single-pass perfusion technique in the rat small intestine. p-Lactam antibiotic absorption iin the presence of amino acids, small peptides, and other p-lactams is modeled using a simple competitive inhibition boundary condition at the intestinal wall, with a corrected value for the intestinal wall concentration, C,, derived from the modified boundary layer (analysis.The model-predicted permeability in the presence of an inhibitor is used tlo characterize the p-lactam antibiotic intestinal carrier system. Several concentrations of cephalexin, coperfused with a constant concentration of cefadroxil (equal to its &), showed that the K, of cephalexin approximately doubled from 7.2 (21 . l ) to 18.8 (24.1) rnM; J f , remained unchanged at 9.2 (r1.2) and 11.1 (e2.1) mM; and the carrier permeability, Pa,was reduced by -50% from 1.11 (kO.10) to1 0.59 (-r-0.04), consistent with competitive absorption kinetics. The predicted in situ wall permeability, < P ;>, of p-lactams perfused in the presence of other p-lactams was calculated and then compared with isxperimentallydetermined values. For cefadroxil, P t, = 0.27 (+0.04), < P > ; = 0.29; for cefatrizine, P ; = 0.67 (+0.09), < P > ; = 0.59; and for cephalexin, P:; = 0.56 (20.05), < P > ; = 0.59. The characteristics of the p-lactam intestinal carrier system were further studied by co-perfusing &lactarns with several di- or tripeptides, which resulted in a significant decrease in p-lactam permeability, whereas co-perfusion with amino acids did not decrease p-lactam permeability. These results strongly suppoll p-lactam absorption by the intestinal peptide transport system in rats. Moreover,these results, consistent with simple competitive inhibition, indicate that macroscopic absorption parameters may be useful for predicting drug-drug and possibly fooddrug absorption interactions in the gastrointestinal tract of humans. _____-
~ _ _ _ ~ _ _ _ _ ~
Inhibition of p-lactam absorption by amino acids, small peptides, and other p-liactam antibiotics14 has been demonstrated using in vitro and i.n situ perfusion techniques in the rat small intestine. P-Lactam absorption in the rat has been attributed to several intestinal carriers and mechanisrns.2.5~6 For example, Miyazak:i et 211.7 reported that cyclacillin shares a common transport mecjhanism with cephradine but not cephalexin. Others2.6 have concluded that cephalexin absorption is independent of intestinal concentration and that it does not have a n absorption pathway in common with some of the other orally absorbed cephalosporins. Based on recently reported findings5 it appears that the latter results were a consequence of the cephalexin concentrations being low compared with its K,, rather than due t o its absorption mechanism. Despit'e the dispariky of these results, the similarity of p-lactam structures would seem to preclude absorption by distinct carriers. Quays and Quay and F~osterswere the first to report the inhibition of cephalexin absorption by the dipeptide Lphenylalanylglycine and the amino acid glycine in excised rat jejunum. They reported t h a t the interaction with Lphenylalanylglycine is due to competition for a carriermediated abisorption site, whereas the interaction with glycine "is not due to tissue .toxicity nor to competition for the imino or monoamino monocarboxylic acid c a r r i e d . 8 Later reports14 generally support these initial findings; however, 0022-3549/89/0900-0723$01 .OO/O 0 1989, American PharmaceLiticaI Association
they are not always in agreement. For example, Nakashima et al.3 demonstrated that the absorption of several p-lactams was inhibited by dipeptides. On the other hand, Kimura et a1.2 reported that the absorption of cephradine, but not cefadroxil, was significantly inhibited by amino acids, whereas the absorption of both aminocephalosporins was not inhibited by a 50 times larger concentration of the dipeptides. Recent reports by Okano et al.4 provide a mechanistic basis for the transport of aminocephalosporins by the dipeptide transport system in the intestinal brush border membrane. Suggestions that the p-lactam antibiotics at least in part share the small peptide absorption pathway are also based on structural similarities to the small peptides. Most of the orally administered p-lactam antibiotics are structurally analogous to tripeptides. The orally absorbed cephalosporins are of the 7-phenylglycyl type, with different substituents a t positions 3 and 7. Cefatrizine, with a bulky heterocyclicthiomethyl group a t position 3 of the thiazine ring, is the most structurally distinct of the series. Furthermore, cefatrizine has a significantly lower intrinsic Michaelis constant (0.7 mM) than the other 7-phenylglycyl cephalosporins studied (3-8 mM) in rats.10 An analogous situation occurs with the dipeptides; that is, substitution of a n amino acid with a bulkier side chain onto the peptide lowers the Michaelis constant of the peptide for membrane absorption sites in humans.11 Specifically, Adibi and Soleimanpour12 demonstrated that the Michaelis constant of glycylglycine is 43.4 mM, whereas the bulkier dipeptide, glycylleucine, has a K, value equal to 26.8 mM in perfused human jejunum. Similarly, Dunn et al.13 reported that the absorption site was even more discriminating to structural differences with para substitution of a hydroxyl group on the 7-phenylglycyl cephalosporins, resulting in a significant increase in absorption over the metu-substituted compound in the mouse. Even though more mechanistic studies are being done with a variety of in vitrolin situ techniques, it is also necessary to attain a macroscopic understanding of the absorption mechanism of amino acids, small peptides, and the p-lactam antibiotics. Therefore, the objectives of this report are: (1) to study the p-lactam intestinal absorption mechanism in a quantitative yet macroscopic fashion, using intrinsic membrane absorption parameters and competitive inhibition studies, using the p-lactams, amino acids, and small peptides as inhibitors; and (2) to establish a basis for predicting drugdrug and possibly food-drug interactions for compounds absorbed by the amino acid or small peptide pathways.
Experimental Section The following materials were used cefadroxil, cephalexin (potency cephradrine (910 mgig), citric acid, dibasic sodium phosphate, glycine, glycylglycine, glycylalanylalanine, phenylalanine, phenylalanylglycine,monobasic potassium phosphate, polyethylene glycol 4000 (Sigma Chemical, St. Louis, MO), cefaclor (956 mgi g, lot no. 206BV6, Eli Lilly and Company, Indianapolis, IN), cefatrizine 1,3-propylene glycolate (845 mgig, SmithKline Beckman, = 987 mg/g),
Journal of Pharmaceutical Sciences i 723 Vol. 78, No. 9,September 1989
Philadelphia, PA; Cebris, Sermoneta, Italy), [14C]polyethyleneglycol (duPont-New England Nuclear, Boston, MA). All buffer and mobile phase components were analytical (or HPLC) grade and used as received. Perfusion Experiments-Perfusion procedures have been detailed in an earlier report.14 The perfusion solution consisted of a citric aciddibasic sodium phosphate buffer (pH 6.51, 0.01% (wiv) PEG 4000 with a tracer amount of its I4C-labeledisotope, a plactam antibiotic, and an inhibitor. The perfusate (pH6.5,300 2 5 mmoLkgofwater) wasmaintainedat 37 "C by a water bath (Tek-Pro, American Dade, Miami, FL). The osmolality was measured using either a model 5002 automatic osmometer (Precision Systems, Sudsbury, MA) or a model 5500 vapor pressure osmometer (Wescor, Logan, UT). All experiments were performed on male Charles River rats, 250400 g, age 60 to 80 d. The rats were fasted overnight, 12-18 h before each experiment. Water was given ad libitum. Anesthesia was induced by a 50% w/v intramuscular injection of urethane (1.5 g/kg). The jejunum was located and a 10-15-cm segment was perfused. The jejunum was cannulated at 2 4 cm below the ligament of Treitz and 6 1 5 cm distal to the first incision. The intestinal segment was perfused using a constant infusion pump (Harvard Apparatus, model 931, S. Natick, MA) for 90 to 105 min. The perfusion flow rate was -0.25 mlimin. Steady-state was achieved in -30 min, after which four to six samples were taken at 10-15-min intervals. All samples were simultaneously quenched at the experiments completion with 6% (wh) citric acid solution. After the last sample was taken, the length was measured by placing a piece of string along the intestine and measuring the string with a ruler. Analytical Method-The samples were analyzed by liquid scintillation counting and by high-performance liquid chromatography (HPLC). Samples were counted using a Beckman LS-9000 counter (Beckman Instruments, Fullerton, CA) with automatic quench correction. A 0.5-mL sample was mixed with 15 mL of scintillation fluid (Ready-Solv, Hph, Beckman Instruments) before counting. Samples were counted using a single-channel technique. The HPLC instrumentation consisted of a pump (Beckman 112 solvent delivery module, Beckman Instruments, Berkeley, CA), automated sampler (WISP, model 710B, Waters Associates, Milford, MA), ultraviolet detector (Kratos Spectroflow 783 absorbance detedor, Kratos Analytical Instruments, Ramsey, NJ), and a reversed-phase column (pBondapak CIS, Waters Associates).The cephalosporin mobile phase consisted of methanol and pH 5 citric aciddibasic sodium phosphate buffer (22:78). Samples were eluted at a flow rate of 1.5 mlimin.
Theoretical Section
dimensionless aqueous permeability, P* = PRID, R is the radius of the intestinal lumen, and D is the estimated aqueous diffusion coefficient. The mean intestinal wall concentration, C,, used is derived from the boundary layer analysis.15 A more general boundary condition for the wall permeability in the presence of an inhibitor than eq 2 is:
c = c,, 2 = 0, t 2 0 aC -arr
-0
where r is the tube radius, z is the axial coordinate, v, is the axial velocity profile, D is the solute aqueous diffusivity, P , is the membrane (wall) permeability, and C, = C(R,z)is the concentration along the tube wall. The solution to the partial differential equation, in dimensionless form, is a function of three parameters, Gz, C, and P*,, where Gz = ?rDLl2Q,C, is equal to C,(l-P*,dP*,,), C, is the inlet perfusate concentration, P:, is the dimensionless effective permeability, P:, is the 724 I Journal of Pharmaceutical Sciences Vol. 78,No. 9,September 1989
+Pk
(3)
where JLaXis the maximal carrier flux, K"," is the apparent Michaelis constant, and P& is the passive membrane permeability. Dividing the numerator and denominator of the nonlinear term by the apparent Michaelis constant results in an alternate form of the boundary condition in terms of another fundamental parameter, the apparent carrier permeability:
p,*.aPP
~
P,
=
1 + - c,
+ Pk
(4)
KaPP m
where: K",pp =
(I
+g)Km
(5)
(1
g)
(6)
-1
P,**app
=
+
Pc*
where P:vaPP is the apparent carrier permeability, Pz is the dimensionless intrinsic carrier permeability, J L J K , is the apparent carrier permeability, PzpaPPis equal to J L a x / K g P , and the dissociation constants K , and Ki are defined in the usual manner:
Recently, a boundary layer approach (MBL) to analyzing the results of the perfused intestinal segment was presented.15 Briefly, the fundamental transport equation for the flow of a solution through a tube is:
The boundary conditions utilized in the MBL analysis were:
+
= KgP J*,=C,
P;
Km = K.
=
~
[Dl[Cl [C - Dl [Il[CI [C-I]
(7a)
(7b)
where [Cl is the concentration of free carrier, [Dl is equal to the Concentration of drug at intestinal wall (C,), [rJ is the concentration of inhibitor, [C], is the total concentration of carrier, [C - 03 is the concentration of the drug:carrier complex, and [C - r] is the concentration of the inhibitor: carrier complex. The values K , and Kiare macroscopic dissociation constants describing the "affinity" of the drug or inhibitor for an intestinal carrier system that is probably complex in nature. These two parameters and their value in relation to the intestinal drug concentration will be the principal determinants of the absorption behavior of the drug. Substituting eq 5 into eq 3, the expression for the wall permeability used in the experimental data analysis is: &ax
P; = (1
+ g ) K m + C,
+ P;
(8)
In order to study the proposed competitive absorption inter-
action, the experimental results analyzed by eq 8 are compared with the calculated wall permeability, assuming classical competitive inhibition. Therefore, the predicted carrier permeability in the presence of an inhibitor is now defined as:
= (1 -
f)P:
Table I-Summary of Intrinsic Membrane Parameters for Competitive Inhibition Studiese
Parameter
(9)
I11
(10)
i 2)
[I1+ Ki 1 + -
since P*,is a constant, eq 9 can be written as:
=
<:p;> +
(11)
where cP*,>is the model-predicted wall permeability in the presence of an inhibitor. With the appropriate substitutions, eq 11can be rewritten in final form as:
Pc* + P;
i 21
[ I ] + Kl 1 +
(12)
-
Using eq 12 and the intrinsic membrane absorption parameters from1 single drug perfusion experiments, the competitive interaction of drugs absorbed by carrier mediated processes is characterized.
Results and Discussion Competitive absorption experiments were performed with cephalexin pe rfused over approximately a lo4 concentration range in the rat small intestine. The inhibitor, cefadroxil, was perfused at a constant conoentration of 7 mM ( C , = 6 mM), which is approximately equal to its K , in rats.10 The data was fit to eqs 3 and 4 using weighted nonlinear regression. A plot of the wall permeability versus the wall concentration of cephalexin, with and without the inhibitor, is shown in Figure
Cefadroxil
7.2 ( k l . 1 ) 18.8 (?4.1)
5.9 (20.8)
-
.-
9.1 ( t l . 2 ) 11.1 (52.1) 1.11 (20.1) 0.59 (k0.04)
where the frac1;ional inhibition, f, is defined as: f =
Cephalexin
4.6 (tO.9) 8.4 (20.8)
-
1.43 (20.1)
-
The concentration of cephalexin ranged from 0.01 to 30 mM; the concentration of cefadroxil remained constant at 5 mM; parameters reported are fitted values i standard deviation.
1.The corresponding intrinsic membrane absorption parameters are summarized in Table I. As seen in Table I, JL,,of cephalexin was unchanged, while the apparent K , and P*, differed. The shift in KZP is not due to a decrease in the affinity of cephalexin for the absorption site, but rather to a portion of the carriers becoming inaccessible to the drug once the inhibitor binds to them since the drug and inhibitor are mutually exclusive at the absorption site. This is manifest as an increase in the apparent or measured Michaelis constant. The inhibitor, cefadroxil, was perfused at its K ,; therefore, from eq 5 the predicted value of KZp should be twice the intrinsic K , of the substrate, cephalexin. This is confirmed in Table I with values of K , and KZpequal to 7.2and 18.8 mM, respectively. For a single carrier system it is expected that the K, ofthe inhibitor should equal its&. This was the case since the K, of cefadroxil in the inhibitor study approximately equaled the K , of cefadroxil determined previously.6 The maximal flux, J*,,,, of cephalexin remained unchanged (Table I) in the presence of cefadroxil; however, it should be noted that much higher cephalexin concentrations are needed PMw
PRw
Na I n h i b i t o r - Inhibitor
m]C a l c u l a t e 0 P e r m e a b l l i t v
1 5
T
$ 1
0
.Ti
T. .d
n
m a,
E L (u
a
T.
T.
m 0 5 3
3 0
Figure 1-Plot of the wall permeability of cephalexin perfused alone and in the presence of a competitive inhibitor, cefadroxil (7mM). The results are reported as the mean wall permeability t SEM.
Csfadroxil
Cefatrizine
Cephalexin
Figure 2-Plot of the mutual inhibition and predicted in situ wall permeability of several p-lactam antibiotics in the presence of other
plactam antibiotics. Experimental values are reported as the mean
2
SEM.
Journal of Pharmaceutical Sciences I725 Vol. 78, No. 9, September 1989
in order to attain any given fraction of J*,,,. In the presence of the inhibitor, the ratio J*,,Xm was reduced by -50% when compared with the carrier permeability in the absence of an inhibitor. Since J*,, remained constant with an increasing K",pp,the apparent carrier permeability, P;saPP, was reduced by a factor of (Kz*/K,) or 50%. These results demonstrate mutual inhibition of j3-lactam antibiotic absorption that is consistent with simple competitive absorption kinetics in the rat small intestine. Recently, Amidon et a1.16 demonstrated an excellent correlation between the intestinal wall permeability in rats and the fraction of dose absorbed in humans. Extending this correlation to predict drug-drug or food-drug interactions in humans is possible once a macroscopic, yet mechanistic basis for these interactions has been established. Therefore, the feasibility of using the macroscopic absorption parameters to predict the apparent Michaelis constant and the apparent carrier permeability of the p-lactam antibiotics in situ was investigated. Several cephalosporins were perfused in the presence of other cephalosporins at concentrations above and below their respective K , values. The individual data were analyzed using eq 12 for the predicted permeability of a compound in the presence of a competitive inhibitor. As seen in Figure 2 and Table I1 there is no significant difference between the predicted and measured wall permeability. This clearly demonstrates that the macroscopic absorption parameters can be used to predict the extent of mutual inhibition in situ. Based on the previously reported correlation of the
fraction dose absorbed in humans and the intestinal wall permeability,le it is expected that in vivo drug-drug absorption interactions and their effect on the pharmacokinetic profile of the drug will be predicted equally well for the p-lactam antibiotics. Inhibition experiments were also performed with amino acids and several small peptides. Co-perfusion of cefatrizine (0.1mM) and the amino acids phenylalanine and glycine (100 mM) did not result in a significant change (p=0.40 and p = 0.27, respectively) in cefatrizine wall permeability (Figure 3). However, co-perfusion of cefatrizine and the dipeptides GlyGly (100 mM) or Phe-Gly (60 mM) or a tripeptide analogue of cephalexin, Gly-Ala-Ala (23 mM), resulted in a significant decrease (p < 0.03, p < 0.035, and p < 0.025, respectively) in wall permeability of cefatrizine (Figure 4, Table 111). However, calculating the degree of inhibition in the small peptide absorption studies using eq 12 is complicated by the luminal hydrolysis of the peptide and the subsequent competition of the amino acids and peptide fragments for the absorption site. Until these "peptide effects" can be adequately modeled and investigated, it is difficult to predict the magnitude of the food-drug absorption interaction. The results in this paper demonstrate that drug-drug interactions observed at the absorption site can be predicted Table Ill-Measured Permeabilities of Cefatrizine (0.1 mM) in the Presence of Various Inhibitors Inhibitor
Table Il-Calculation of the Predicted Permeability of PLactams in the Presence of a &Lactam Inhibitor'
p:
Compound
(no inhibitor)
p:, (inhibitor)
Cefadroxil Cefatrizine Cephalexin
0.75 (50.05) 1.27 (50.10) 1.27 (kO.10)
0.27 (T0.04) 0.67 (20.09) 0.56 (k0.05)
(inhibitor) 0.29 0.59 0.59
Glycine Phenylalanine GLY-GLY PHE-GLY G LY-ALA-ALA Cefatrizine (control)
* Parameters are fitted values t SEM.
Concentration, mM
pk
SEM
Significance
100 100 100 60 23 0.1
1.48 1.44 0.91 1.09 0.99 1.28
0.17 0.17 0.13 0.39 0.12 0.10
ns ns p < 0.03 p < 0.035 p < 0.025
Cefatrizine
-
NO
I c e f a t r i z i n e
-
I n h = pept!des
-
Inhibitor
2.0 Cefatrizine
-
NO
m c e f a t r i z i n e
-
Inninitor
T
inhlnitor
-
Phe o r G I ~
T 1 .F z
u
-ri
rl .rl
c l lo 0 E
1.0
a, R r-l
c1
10
3
0.5
PHE
GI-Y
Figure +Plot of the wall permeability of cefatrizine (0.1 mM) in the presence of the amino acids L-phenylalanine (100 mM) or glycine (1 00 mM). Experimental values are reported as the mean 2 SEM. 726 I Journal of Pharmaceutical Sciences Vol. 78, No. 9, September 7989
GLY-GLY
PHE-GLY
Gt~Y - A L A - ALP.
Figure &Plot of the wall permeability of cefatrizine (0.1 mM) in the presence of Gly-Gly (100 mM), Phe-Gly (60 mM), Gly-Ala-Ala (23 mM). Experimental values are reported as the mean t SEM.
based on simplie competitive absorption kinetics. On the other hand, the effects of dietary peptides and amino acids on drug absorption require further study before the effects on Dlactam antibiotic absorption can be predicted.
Conclusions Using the results of the boundary layer analysis on the perfused intestinal segment;, with a modified boundary condition accounting for competitive inhibition, allowed the determination of the apparent and intrinsic membrane absorption parameters for several cephalosporins in the presence of competitive inhibitors. Mutual inhibition of the p-lactams was characterized using a derived expression for the predicted :in situ wall permeability. The observed inhibition is consistent with simple competitive absorption kinetics. Mutual inhibition among the &lactam antibiotics was observed with the Ki values equal to previously determined K , values. While di- and tripeptides inhibited p-lactam absorption, various amino acids did not; however, due to the complexities i.n the intestinal lumen, the magnitude of the “peptide effects” is difficult to predict and requires further analysis. The results of thlese studies begin to provide a n alternative mechanistic basis for plactam-p-lactam and possibly food--&lactam absorption interactions.
References and Notes 1. Kimura, ‘IEndo, ’.;H.; Yoshikawa, M.; Muranishi, S.;Sezaki, H. J. Pharm. Dyn. 1978, 1, 262-267. 2. Kimura, T.; Yamamoto, T.;Mizuno, M.; Suga, Y.; Kitade, S.;
Sezaki, H. J . Pharm. Dyn. 1983,6, 246-253. 3. Nakashima, E.; Tsuji, A,; Kagatani, S.; Yamana, T. J. Pharm. Dyn. 1984, 7, 452-464. 4. Okano, T.; Inui, K.; Maegawa, H.; Takano, M.; Hori, R. J . Biol. Chen. 1986.30.14130-14134. 5. Miyazaki, K.; Ogino, 0.; Nakano, M.; Arita, T. Chem. Pharm. Bull. 1977,25, 246-252. 6. Umeniwa, K.; Ogino, 0.;Miyazaki, K.; Arita, T. Chem. Pharm. Bull. 1979,27, 2177-2182. 7. Miyazaki, K.; Obtani, K.; Umeniwa, K.; Arita, T. J . .Pharm. Dyn. 1982,5, 555-563. 8. Quay, J. F. Physiologist 1972, 15, 241. 9. Quay, J. F.; Foster, L. Physiologist 1970,13, 287. 10. Sinko, P. J.; Amidon, G. L. Pharm. Res. 1988, 5, 645-650. 11. Adibi, S. A.; Kim, Y. S. In Physiology of the Gastrointestinul Tract; Johnson, L. R., Ed.; Raven: New York, 1981; pp 10731095. 12. Adibi, S. A.; Soleimanpour, M. R. J . Clzn. Znuest. 1974,53,13681374. 13. Dunn, G. L.; Hoover, J. R. E .; Berges, D. A.;Taggart, J. J.; Davis, L. D.; Dietz, E. M.; Jakas, D. R.; Yim, N.; Actor, P.; Uri, J . V.; Weisbach, J . A. J . Antibiot. 1976,29, 65-80. 14. Hu, M.; Sinko, P. J.; dehleere, A. L. J.; Johnson, D. A.; Amidon, G. L. J . Theor. Biol. 1988,131, 107-114. 15. Johnson, D. A.; Amidon, G. L. J . Theor. Biol. 1988,131,93-106. 16. Amidon, G. L.; Sinko, P. J.; Fleisher, D. Pharm. Res. 1988, 5, 651-654.
Acknowledgments Support by SmithKline Beckman and NIGMS Grant GM7188-01is r t e f u l l y acknowledged. The authors would also like to thank Mrs. oyce Durcanin-Robbins for her technical assistance with the perfusion studies.
Journal of Pharmaceutical Sciences I 727 Vol. 78,No. 9,September 1989