• YAKHAK HOEJI
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• v.38 no.5
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• pp.530-543
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• 1994

To study the feasibility of transmucosal delivery of leucine enkephalin (Leu-Enk) and $[D-ala^2]$-leucine enkephalinamide (YAGFL), their degradation extents and pathways in various rabbit mucosa extracts were investigated by high performance liquid chromatography. The degradation of Leu-Enk and YAGFL was observed to follow the first-order kinetics. The degradation half-lives of Leu-Enk in the nasal, rectal and vaginal mucosal extracts were 1.62, 0.37 and 1.12 hrs and those of YAGFL were 30.55, 9.70 and 6.82 hrs, respectively, indicating Leu-Enk was degraded in a more extensive and rapid manner than YAGFL. But the mucosal and serosal extracts of the same mucosa showed the similar degradation rates for both pentapeptides. The degradation was most rapid in the neutral pH and increasing concentrations of substrates retarded the degradation rates. The maior hydrolytic fragments of Leu-Enk were Des-Tyr-Leu-Enk and tyrosine, indicating the enzymatic hydrolysis by aminopeptidases. However, the data also suggested endopeptidases such as dipeptidyl carboxypeptidase and dipeptidyl aminopeptidase could play some role in the degradation of Leu-Enk. On the other hand, the hydrolytic fragments of YAGFL in all the mucosa extracts were mainly Tyr-D-Ala-Gly and Phe-Leu-Amide, demonstrating the hydrolytic breakdown by endopeptidases. The degradation pathways were further explored by concomitantly determining the formation of smaller metabolites of primary hydrolytic fragments of Leu-Enk and YAGFL in the mucosa extracts.

• Journal of Pharmaceutical Investigation
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• v.26 no.3
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• pp.175-185
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• 1996

To inhibit the enzymatic degradation of leucine enkephalin (Leu-Enk) and its synthetic analog. $[D-ala^2]$-leucine enkephalinamide (YAGFL), in the nasal, rectal and vaginal mucosal and serosal extracts of rabbits, effects of enzyme inhibitors such as amastatin (AM), puromycin (PM), thiorphan (TP), thimerosal (TM), EDTA, N-carboxymethyl-Phe-Leu (CPL), phenylethyl alcohol (PEA), phenylmercuric acetate (PMA), benzalkonium chloride (BC) and modified cyclodextrins, alone or in combination, were observed by assaying the pentapeptides staying intact during incubation. Mucosa extracts were prepared by exposing freshly-excised mucosal specimens mounted on Valia-Chien cells to isotonic phosphate buffer while stirring. The degradation of Leu-Enk and YAGFL followed the apparent first-order kinetics. The half-lives (mean) in the nasal, rectal and vaginal mucosal extracts were found to be 1.07, 0.33 and 1.14 hr for Leu-Enk, and 16.9, 6.2 and 6.8 hr for YAGFL, respectively. AM or PM, which is an aminopeptidase inhibitor, did not show a sufficient inhibition of Leu-Enk $(50\;{\mu}g/ml)$ degradation in all kinds of extracts. $Dimethyl-{\beta}-cyclodextrin\;(DM-{\beta}-CyD)$ decreased the degradation rate constants of Leu-Enk about 2 or 3 times, comparing with no additive. However, the use of mixed inhibitors of AM $(50\;{\mu}M)$/TM (0.25 mM)/EDTA (5 mM) resulted in a full stabilization of Leu-Enk by decreasing the degradation rate constants 67.3, 161.3 and 113.8 times far the nasal, rectal and vaginal mucosal extracts, respectively, comparing with no inhibitor. With mixed inhibitors, Leu-Enk remained intact more than 90% after 6 hr-incubation. In the stabilization of YAGFL, hM, TP or CPL alone showed little efffct, and some additives demonstrated a considerable inhibition of YAGFL degradation in the rank order of TM > BC > EDTA. However, the addition of mixed inhibitors such as TM (0.5 mM) and EDTA (5 mM) into the extracts protected YAGFL from the degradation by more than 85% even after 24 hr-incubation, suggesting almost complete inhibition of YAGFL degradation in the extract. On the other hand, $DM-{\beta}-CyD\;or\;hydroxypropyl-{\beta}-cyclodextrin$ (10%) were also found to retard enzymatic degradation rates of YAGFL markedly, and resulted in staying intact more than 80% of YAGFL in the nasal and vaginal mucosal extracts, and more than 60% in the rectal mucosal extract after 16 hr-incubation.

• Journal of Pharmaceutical Investigation
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• v.22 no.3
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• pp.173-183
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• 1992

To study the feasibility of transmucosal delivery of $[D-ala^2]-methionine$ enkephalinamide (YAGFM), its enzymatic degradation and stabilization in various rabbit mucosal extracts were investigated by HPLC method. The degradation of YAGFM was observed to follow the first-order kinetics and the half-lives of YAGFM in the nasal, rectal and vaginal mucosal extracts were found to be 25.7, 3.0 and 7.8 hr, respectively. However, there was no significant difference in degradation rates of YAGFM between the mucosal and serosal extracts obtained from the same mucosal membrane. This finding suggests that even a synthetic enkephalin analog, which is designed to be resistent to aminopeptidases, needs to be fully protected from the enzymatic degradation in mucosal sites for the delivery of the analog through mucosal routes. To inhibit the degradation of YAGFM in various mucosal extracts, effects of enzyme inhibitors such as bestatin (BS), amastatin (AM), thiorphan (TP), thimerosal (TM) and EDTA, alone or in combination, and modified cyclodextrins were observed by assaying YAGFM staying intact during 24 hr-incubation at $37^{\circ}C$. It was found from the results that mixed inhibitors such as TM (0.5 mM)/EDTA (5 mM) or AM $(50{\mu}M)/TM$ (0.5 mM)/EDTA (5 mM) provided very useful means for the stabilization in various mucosal extracts. The latter was found to protect YAGFM from the degradation in the nasal, rectal, and vaginal mucosal extracts by 90.9, 90.4 and 91.3%, respectively, after 24 hr-incubation, suggesting almost complete inhibition of YAGFM-degrading enzymes present in the incubation mixture. However, BS $(50{\mu}M)$, AM 50 $(50{\mu}M)$ or TP$(50{\mu}M)$ alone did not reveal sufficient inhibition except TM (0.5 mM) or EDTA (5 mM). The adddition of $2-hydroxylpropyl-{\beta}-cyclodextrin$(10%) to the nasal mucosal extract, and $dimethyl-{\beta}-cyclodextrin$(10%) to the rectal and vaginal mucosal extracts reduced the first-order rate constants for the degradation of YAGFM by 5.8, 17.3 and 8.9 times, respectively, compared to those with no additive.

• Biomolecules & Therapeutics
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• v.10 no.2
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• pp.104-113
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• 2002

The effects of enzyme inhibitors and penetration enhancers on the permeation of leucine enkephalin (Leu-Enk) and its synthetic analog, [${D-ala}^2$]-leucine enkephalinamide (YAGFL) across the nasal, rectal and vaginal mucosae were evaluated. Enzyme inhibitors and penetration enhancers employed for Leu-Enk permeation study were amastatin(AM), thimerosal(TM) and ethylenediaminetetraacetic acid disodium salt(EDTA), and sodium taurodihydrofusidate (STDHF). Those for YAGFL permeation study were TM, benzalkonium chloride(BC) and EDTA, and STDHF, sodium deoxycholate(SDC), sodium glycholate(SGC), glycyrrhizic acid ammonium salt (GAA), L-$\alpha$-Iysophosphatidylcholine(LPC) and mixed micelle (MM, STDHF: linoleic acid = 15 mM : 5 mM). The addition of TM alone on the donor and receptor solutions for Leu-Enk permeation study across all the three kinds of mucosae failed to inhibit the degradation; it completely degraded in 6 hrs, and no permeation occurred. However, with addition of three kinds of inhibitors together, the fluxes across nasal, rectal and vaginal mucosae were $\20.7{pm}2.5$>/TEX>,$\0.3{pm}0.05$>/TEX> and $\1.4{pm}0.5$ $\mu$\mid$textrm{m}$/$\textrm{cm}^2$/hr, respectively. Moreover, the addition of STDHF in the presence of the above three inhibitors enhanced permeation across nasal, rectal and vaginal mucosae 1.3, 15 and 1.3 times, respectively. YhGFL also degraded in the donor and receptor solutions rapidly as time went. With mixed inhibitors of TM and EDTA, the percents of YAGFL remaining in the donor solutions facing nasal, rectal and vaginal mucosae were 69.7, 69.8 and 79.8%, respectively; the percent permeated increased to 10, 2.1 and 5.7%, respectively. The addition of STDHF in the presence of either BC/EDTA or TW/EDTA increased the permeation 2.2, 11.0 and 2.9 times, and 2.21, 14.0 and 2.7 times for nasal, rectal and vaginal mucosae, respectively. With SDC, SGC, GAA, LPC ud MM in the presence of TM/EDTA increased permeation; especially, they increased permeation across vaginal mucosae effectively, and the enhancement factors were 12.5, 7.6, 8.7, 5.7 and 5.5, respectively. The degradation extent of YAGFL was correlated with protein concentrations in the epidermal and serosal extracts. The flux of YAGFL across nasal mucosa increased dose-dependently.