hydrolysis of leucine enkephalin in the nasal cavity of the rat — a possible factor in the low...

Vol. 133, No. 3, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 3 1, 1985 Pages 923-928

HYDROLYSIS OF LRUCINE ENKEPHALIN IN THE NASAL CAVITY OF THJ3 RAT - A POSSIBLE FACTOR IN THE LOW BIOAVAILABILITY

OF NASALLY ADM1N1STER.D PEPTIDES

Anwar Hussain*, Jabar Faraj, Yukihiko Aramaki and James E. Truelove

College of Pharmacy University of Kentucky

Lexington, KY 40536-0053

Received October 4, 1985

Summary: In order to investigate the utility of intranasal administration of peptides for systemic medication, the nasal absorption of the model peptide, leucine enkephalin (Tyr-Gly-Gly- Phe-Leu), was studied in the rat. At a concentration of 60 ug/ml in Ringer's buffer the pentapeptide was found to undergo,extensive hydrolysis in the nasal cavity. The hydrolysis rather than the polarity of the pentapeptide appears responsible for limiting the nasal absorption of this model compound. In the presence of dipeptides, the hydrolysis of leucine enkephalin was significantly inhibited. These results suggest that the nasal administration of peptides may become an important route for drug administration provided that the peptidases in the nasal mucosa can be transiently inhibited via coadministration of pharmacologically inactive peptidase substrates. B 1985 Academic Press, Inc.

The intranasal administration of peptides for systemic medication

has been used for the antidiuretic agent desmopressin acetate (DDAVP)

and for oxytocin (1,2). Moreover, this route of administration has

recently been considered as an alternative to the parenteral for other

peptides. Although many drugs are absorbed rapidly and quantitatively

following nasal administration (3-8), the peptides have generally

shown low nasal bioavailabilities. For example, studies with

insulin and the LHRH analog, nafarelin acetate, have shown that

considerably greater nasal than parenteral doses are required to

produce similar effects (9-12). In monkeys, studies comparing

270 ug nasal doses and 5 ug subcutaneous doses indicated that the

nasal bioavailability of nafarelin acetate is only = 2%. The

*To whom all correspondence should be addressed.

0006-291X/85 $1.50

923 Copyright D 1985 by Academic Press, Inc.

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same study indicated that the area-under-the-plasma-drug-level

versus time curve (AK) after nasal administration increased in a

nonlinear fashion as a function of dose administered. The

authors suggested saturable metabolic or absorption pathways as

possible explanations for the data (12). Another recent study

employing rats claimed that the pentapeptide enkephalin,

Tyr-IJ-Ala-Gly-&-Phe-g-Leu-OH, was efficiently and completely

absorbed from the nasal cavity (13). It should be noted,

however, that the enkephalin was administered in unrealistically

large doses.

In order to gain some insight into the mechanism of nasal

absorption of peptides, we examined the nasal absorption of

leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) in rats. Using an

in-situ perfusion technique (14), a 60 ug/ml solution of the

enkephalin in Ringer's buffer was circulated through the nasal

cavity of anesthetized rats. The concentrations of the parent

enkephalin and its metabolite (Gly-Gly-Phe-Leu) in the perfusing

solution were determined as a function of time employing a

specific high-pressure liquid chromatographic (HPLC) assay.

MATERIALS AND METHODS

Materials - Leucine enkephalin (L-Tyrosylglycylglycyl-L- phenylalanyl-L-leucine), L-Tyrosylglycine, L-Tyrosylglycyl- glycine, L-Tyrosyl-L-tyrosine, L-Phenylalanyl-L-leucine and Glycylglycine (Sigma Chemical Co., St. Louis, MO); sodium pentobarbital injection (Nembutal, Abbott Laboratories, North Chicago, IL); polyethylene tubing (PE 260, Clay Adams Co., New York, NY); and cyanoacrylate adhesive (Super Glue 3, Woodhill Permatex, Cleveland, OH) were used. All other materials were reagent or analytical grade and used as received.

Analytical Method - Aliquots (100 ~1) of the perfusing solution were periodically removed, diluted immediately with 50 ul of 0.2M citrate buffer (pH 2.3) to quench hydrolysis and subjected to analysis. Chromatography was completed on a 4.6 x 25Omm stainless steelcolumn packed with an octylsilane reverse-phase support (Ultrasphere-Octyl, Su, Beckman Instruments, Irvine, CA) by eluting at 1.5 ml/min with a mobile phase of O.lM NaH2P04-H3P04-CH3CN(916:5:275). Detection was by W at 205nm.

Nasal Perfusion Method - The in situ nasal absorption studies -- were carried out according to the method of Hussain and coworkers

924


(14). Male, Sprague-Dawley rats weighing = 300 g each were anesthetized with sodium pentobarbital (50 mg/kg). An incision was made in the neck and the trachea cannulated with a polyethylene tube (PE 260). A second tube, which served to introduce the perfusing solution, was inserted through the esophagus to the posterior part of the nasal cavity. The nasopalatine was closed with an adhesive to prevent drainage of perfusing solution from the nasal to the oral cavity.

A four-ml portion of the drug solution was placed in a water-jacketed beaker, maintained at 37OC and circulated through the nasal cavity of the rat using a peristaltic pump. The perfusing solution passed through the nasal cavity, out the nostrils and, through a funnel, returned to .the water-jacketed beaker.

RESULTS AND DISCUSSION

The disappearance of the enkephalin and the appearance of

the metabolite in the perfusing solution are shown in Figure 1.

Preliminary mass-balance analysis of the data indicated that 30%

of the initial concentration of enkephalin could not be accounted

for. When a 60 ug/ml solution of the metabolite alone was

circulated through the nasal cavity, however, a continuous

decline in concentration and the appearance of new peaks in the

HPLC chromatograms was observed, indicating that the metabolite

is subject to further hydrolysis.

100

0

Minutes

Fig. 1 The disappearance of leucine enkephalin, 8; and the appearance of Gly-Gly-Phe-Leu, 0; in the nasal perfusate. The symbols represent the mean and standard error of 4 animals.

925


Assuming that the overall rate of disappearance of the

pentapeptide is due to scission of the tyrosyl-glycine bond

followed by further hydrolysis of the Gly-Gly-Phe-Leu metabolite,

a concentration-time profile for the metabolite can be calculated

using the following relationship:

[Metabolitelt = ktlBA& (eBkit - eBkzt) Eqn. 1

where kl and k2 are the first-order rate constants for the

disappearance of the pentapeptide and its metabolite,

respectively, and A0 is the initial molar concentration of

pentapeptide.

The rate constants kl and k2 were calculated from

first-order plots of the disappearance of the enkephalin and that

of the metabolite, respectively. The values found were kl =

2.25 x 10e2 min -I and k2 = 4.61 x low3 min-'. Using these rate

constants, the metabolite concentration-time profile shown as the

line in Figure 2 was constructed. The general agreement of the

OL 0 6 10 15 20 25 30 35 40

Minutes

Fig. 2 The concentration-time profile for Gly-Gly-Phe-Leu. The symbols represent the mean and standard error for 4 animals and the line is that generated by Eqn. 1.

926


-

Fig. 3. The influerlce of competing peptidase substrates on the hydrolysis of leucine enkephalin in Ringer's buffer pre-circulated through the nasa,l cavity.

experimental points and the calculated line strongly suggests

that the overall disappearance of leucine enkephalin from the

nasal perfusate is due to hydrolysis and that the extent of

absorption, if any, is less than about 10%.

To confirm that the enkephalin is indeed hydrolyzed by

enzymes in the nasal mucosa, Ringer's buffer was circulated

through the nasal cavity for thirty minutes and the resulting

mixture incubated with leucine enkephalin for 1 hour at 37°C.

Following incubation, 50% of the pentapeptide had been

hydrolyzed, primarily to Gly-Gly-Phe-Leu. As shown in Figure 3,

the extent of hydrolysis of the enkephalin in perfused buffer was

considerably reduced by the addition of a ZO-fold molar excess of

other peptides such as L-tyrosyl-L-tyrosine.

The conclusions that can be drawn from the literature cited

and our results are:

a) Polar compounds such as peptides can penetrate the nasal

mucosa:

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b) When administered in low concentrations, peptides undergo

extensive hydrolysis in the nasal mucosa; and

cl The hydrolysis of leucine enkephalin can be inhibited by the

addition of peptidase labile peptides to the nasal

perfusate. The coadministration of a competing,

pharmacologically inactive peptide, therefore, may be a

useful approach to improving the bioavailability of

nasally administered peptides.

REFERENCES

1. The United States Pharmacopeia; 20th rev., U.S. Pharmacopeial Convention, Inc., Rockville, Maryland, 597 (1980).

2. Physician's Desk Reference; 37th ed., Medical Economics Company, Inc., Oradell, New Jersey, 592 (1983).

3. Hussain, A., Hirai, S., and Bawarshi, R. (1979) J. Pharm. &, 68, 1196.

4. Hussain, A., Hirai, S., and Bawarshi, R. (1980) J. Pharm. &, 69, 1411-1413.

5. Hussain, A., Foster, T., Hirai, S., Kashihara, T., Batenhorst, R., and Jones, M. (1980) J. Pharm. Sci., 69, 1240.

6. Hussain, A., Hirai, S., and Bawarshi, R. (1981) J. Pharm. Sci., 70, 466-467.

7. Hussain, A., Kimura, R., Huang, C., and Kashihara, T. (1984) Int. J. Pharm., 21, 233-237.

8. Hussain, A., Kimura, R., Huang, C., and Mustafa, R. (1984) Int. J. Pharm., 21, 289-294.

9. Hirai, S., Ikenaga, T., and Matsuzawa, T. (1978) Diabetes, 27, 296-299.

10. Hirai, S., Yashiki, T., and Mima, H. (1981) Int. J. Pharm., 9, 173-184.

11. Hirai, S., Yashiki, T., and Mima, H. (1981) Int. J. Pharm., 9, 165-172.

12. Anik, S., McRae, G., Nerenberg, C., Worden, A., Foreman, J., Yu, H. J., Kushinsky, S., Jones, R., and Vickery, B. (1984) J. Pharm. Sci., 73, 684-685.

13. Su, K., Campanale, K., Mendelsohn, L., Kerchner, G., and Gries, C. (1985) J. Pharm. Sci., 74, 394-398.

14. Huang, C.H., Kimura, R., Bawarshi-Nassar, R. and Hussain, A. (1985) J. Pharm. Sci., 74, 608-611.

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hydrolysis of leucine enkephalin in the nasal cavity of the rat — a possible factor in the low...

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