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Transdermal delivery of a melanotropic peptide hormone analogue
Life Sciences, Vol. 43, pp. 1111-1117 Printed in the U.S.A.
TRAWDERMALDELI~OPA
Pergamon Press
MELAN(ILaopIC PEPI'IDEHDRMONE AN?WXE
Brenda V. Dawsonl, Mac E. Hpleylf2, Kristie Kreutzfeldl, Robert T. Dorr3, Victor J. Hruby , Fahad Al-Obeidi4,and Scott Don1 Departmentsof Anatcxnyl and Molecular & Cellular Biology2 nternal +4 Medicine (Pharmacologyand Dncology3),and Chemistry University of Arizona, Tucson, Arizona 85724 (Received in final form August 11, 1988) Sunmary We reviously reported that topical application of [Nle4,DPhe $ Ialpha-MSH, a superpotent analogue of alpha-melanocyte stimulating hormone, to mice induces a darkening of follicular melanocytes throughout the skin. We now report that the melanotropin analogue can be delivered across mouse but not rat skin in an in vitro model system. Passage of the analogue from the topically-&l vehicle (polyethyleneglycol) across the skin into a subcutaneousreceiving vessel was demonstratedby both bioassay as well as by radioimmunoassay. The bioassay data demonstrate that percutaneous absorption of the melanotropin did not result in loss of biological activity of the peptide. The differential penetration of the peptide across rodent skin reveals that one cannot predict percutaneousabsorptionof a substance across the stratum corneum from studies on a single species. The present results are the first to demonstrate, by direct quantitative measurements, that a bioactive peptide can be delivered across the vertebrate integument in vitro. These studies point out the potential of a topically zpliedmelanotropin for tanning of the skin and possibly for treatment of certain hypopigmentarydisorders. We previously demonstratedthat an analogue of alpha-MSH (alpha-melanocyte stimulatinghormone, alpha-malanotropin)when applied topically to the skin of a yellow strain (C57BL/6JAy)of mouse resulted in a darkening of hair follicles witpin 24 hrs of application (1). Although the melanotropin, [Nle4,DPhe lalpha-MSH, was applied to a small area of the dorsum, all growing hai& throughoutthe body became dark in color, suggesting that the peptide had been delivered systemically following percutaneousabsorption. We also demonstrated that two fragmentanalogues of the parenttridecapeptide could be even more efficaciously delivered by the transdermal route (2). These results demonstratedthat a topically applied melanotropin could activate cellular processes within follicular melanocytes leading to new eumelanin formation. These results suggested to us that melanotropin delivery acrcss the skin might prove to be clinically relevant for the treatment of hypopigmentarydisorders or even for tanning of the skin in the absence of the sun. Melanotropin inducedmelanogenesis of the human integument might even prove an important factor in decreasing the incidence of skin cancer. In this regard it became imperative to directly determine whether a melanotropin can cross the stratum corneum of the epidermis. To our knowledge peptides have not previously been shown to cross the skin. The present results clearly document that a peptide melanotropin analogue can be delivered transdennallyacross mouse skin.
0024-3205188$3.00 + .OO Copyright (c) 1988 Pergamon Press plc
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TransdermalDelivery of a Melanotropin
Vol. 43, No. 14, 1988
Materials -and Methods Mouse Skin. Twenty-three full thickness skin samples (approximately1 l/2" in diameter) were removed from the trunk area of 9 either black or yellow adult C57BL/6JAy mice killed by cervical dislocation. Each sample was placed on the LGA (LaboratoryGlass Apparatus)permeationapparatus described below. Rat Skin. Seventeen full thickness skin samples 1 l/2" diameter were removed from the trunks of mature Sprague Dawley rats. They were treated identically to the mouse skins. Permeation Apparatus. Skin to be tested was placed on a special permeation apparatus developed by LGA, based on an established permeation apparatus design (3). This consisted of a water-jacketed glass penetration cell with inlet and outlet port for water flow. The cell was firmly clamped to a glass lid to preclude any leakage of solution, and the apparatus with contained solution was maintained at 37oC. The melanotropin preparation was warmed to 40'C and delivered by micropipetteto the epidermis in a known volume (0.1-0.2 ml). The subcutaneousinner chamber of the permeation apparatus was filled with physiologic saline, ensuring complete contact with the dermis. Magnetic spin bars provided adequate mixing of the subdermal collection fluid. The skins were generally maintained on the permeation apparatus for 24 hrs. This time period was chosen based on other workers' experience. It is long enough to cover the lag period of permeationand short enough to preclude skin autolysis (4,5). Bacterial contaminationwas not a problem since the MSH analogue is not biodegradeable (6). The fluid within the collecting vessel was then removed and inmediately frozen to await assay. was Melanotropin Synthesis and Preparation. The [Nle4,g-Phe7]alpha-MSH synthesizedas prevously described (7). This tridecapeptideanalogue of alphaMSH was selected for our initial studies because of its several unique attributes: A) the analogue is superpotent, being 10-1000 timas more active than the native hormone, alpha-MSH, in several bioassays (8-11);B) the peptide is prolonged acting, that is, it causes a very long lasting stimulation of melanocytes in vitro and _in vivo (12,13); C) the peptide is nonbiodegradeable, to enzymatic inactivationby sera (14),brain enzymes that is, it z xtant (15),or purified proteolytic enzymes (16). In the present experimentsthe analogue was first dissolved at liav3Min a small volume of distilled water and then diluted in a polyethylene glycol vehicle (26% PEG 400 and 74% PEG 3350, by weight) to 10-4M for all studies. This mixture of high and low molecular weight PEG species results in a solid cream base which slowly liquefies at physiologic skin temperatures. The analogue was also dissolved in propylene glycol as a liquid vehicle. This is the classical assay for detecting and Frog Skin Bioassay. determining the biological activity of melanotropins. The frog skin bioassay has been described in detail elsewhere (16). Briefly, the assay is based upon the fact that biologically active melanotropins will stimulate movement (centrifugal dispersion) of melanosomes (melanin granules) within dermal melanocytes of frog skin. This response results in a rapid darkening of the skin that can be quantitativelymeasured by a reflectometer. In this assay the skin samples can usually det ct the parent alpha-MSHmolecule ataminimal concentrationof about 2 x 10-flM. Since parallel 3-point dose response curves are produced in response to amelanotropin, it is possible to quantitate the amount of melanotropin that is transdermally delivered. Most importantly, the
vol.
43, No. 14, 1988
Transderrnal Delivery of a Melanotropin
1113
bioassay can also detect [Nle4,g-Phe'lalpha-MSH at ?- 10-fold lower concentration (e.g.,2 x 10-12M). The importance of this bioassay is that it only detects biologically active melanotropins. In other words, positive bioassayswould indicate that the melanotropin had retained biological activity following transit across the skin. RIA's do not necessarily indicate whether the moiety being measured is biologically intact. To perform the frog skin bioassay, frozen samples of physiological saline containing any transdermallydelivered melanotropin were thawed and 0.2 ml added to each of the skin samples in 20 ml of Ringer solution. This results in a 1/100th dilution of the original collection sample. Nevertheless, as demonstrated in this report, the exquisite sensitivity of the bioassay allowed in most cases the demonstration of biological activity of the analogue following topical application and penetrationthrough the skin. Radioinmunoassay. An alpha-MSH radioimnunoassaykit (InmunoNuclearC ro; Stillwater, MN) was used for the detection and quantitation of [Nle8,DPhe7]alpha-MSH. The assay was performed as prescribed by the kit with the aliquot size maintained at 50 ul for both standards and sali e samples and assayed in duplicate. The analogue was dissolved in H 0 at 10-9M and diluted in BSA-borate buffer supplied in the kit. The usefu 1 range of the standard curve for [Nle4,g-Phe'lalpha-MSH from 10 to 95% binding ranged from 300 to 1 pg analogue per tube, respectively. Application of the alpha-MSH RIA kit to quFy;;zii,o,' at;; ;T#ogue, although a heterologous system using anti-alpha-labelled] alpha-MSH, yields standard curves that are repeatable and linear (17).Only 13 of the 23 samples were tested by RIA, since earlier samples had been collected and used by FSB before development of the RIA. Microscopy. After 24 hrs at room temperature (20’C) on the permeation cells, all samples were examined grossly for loss of anatomical integrity. In addition, each skin sample was sectioned, stained with henatoxylin and eosin, and examined by light microscopy.
Results By both frog skin bioassay (FSB) and RIA it was determined that [Nle4 DPhe'lalpha-MSH had been delivered transdermally. The results between the & very different assays were in consistent agreement (Fig. 1). For example, it was determined that 15/23 (65%)and 11/13 (85%),respectively, of the samples were positive for the presence of the analogue by FSB and in the RIA. There was a one to one correlation between the two methods with respect to positivity and magnitude of response. What is evident is that low values for the FSB correlated with low values for the RIA, and high FSB readings correlated with high RIA values. These results suggest, therefore, that both assays measure a common entity. Our calculations indicate that approximately 0.05% of the analogue was transdermally delivered through mouse skin exposed to the melanotropin in the PM; cream base vehicle. Using propylene glycol as an alternate vehicle, it appeared that the peptide was delivered to an even greater extent (0.08%) (Fig.2). DMSO, used in a few samples also showed good permeation (Fig. 2).
To our surprise the analogue failed to penetrate to any great extent through the skin of the rat (Fig. 1). Again, there was a close agreement between the two assays. Only one of the 15 FSB's (6.6%) and two of the 17 RIA's (11.8%)were positive for the analogue. These samples were derived from experiments using DMSO as a vehicle. Histologic examination of the stratum corneum, epidermis, and dermis, of all samples (mouse and rat) showed
1114
TransdermalDelivery of a Malanotropin
l Frog Skin Bioassay q Radioimmunoassay
Delivery l
70
Fig. 1. Relative deliv ry of a f ]a lphamelanotropin, [Nle4,D_Phe MSH, through full-thickness skin ofthemouse (C57BL/6JAy) and rat (Sprague-Dawley), using an in vitro permeation cell apparatus, -the relative refractoriness of rat skin compared to mouse skin. Positive samples are those skin samples through which the melanotropincould be delivered as determined by frog skin bioassay (> 10% darkening response)and RIA (greater than 20 pg/50 ul). Note the very good correlation between the two assays employed. The importance of the frog skin bioassay is that it demonstrates the presence of a biologicallyactive peptide.
Rat
Mouse
Transdermol
Vol. 43, No. 14, 1988
of [NleqQ-Phe7]rMSH
Polyethylene
A
Propylene
o
DMSO
Across Mouse Skin
Glycol / Alcohol Glycol
A
60 50
A
A t
l
A
0
0
A
Sample across full-thickness Fig. 2. Transdermal delivery of [Nle4,g-Phe7]alpha-MSH mouse skin _in vitro as determined by the frog skin bioassay. The melanotropin was applied to the skin for 24 hrs in the vehicles indicated. Each point represents the degree of skin darkening obtained from an individual experiment with melanotropinapplied to different mouse skin samples.
Vol. 43, No. 14, 1988
TransdermalDelivery of a Melanotropin
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structural integrity, and intercellular bridges could be seen in the rat epidermis. Discussion We have presented evidence that a peptide analogue of alpha+nelanotropin can be delivered transdennally through mouse skin in vitro. To our knowledge, this is the first such demonstration that a paptidehze can be delivered systemically by this method. We hat demon,stratedpreviously that topical application of the analogue, [Nle ,g-Phe Ialpha-MSH to the skin of mice resulted in follicular melanogenesis (1,2). The response occurred within 24 hrs of a single application of the analogue, depending upon the concentration employed (1,2). In the yellow strain of mouse (C57BL/6JAy)the analogue caused a shift within follicular melanocytes from pheomelanogenesis (yellow-brown melanin) toeumelanogenesis (dark brown-blackmelanin) (18). Although the vehicle was applied to the posterior dorsum, eLmElanogenesisoccurred in all hair follicles. Presumably the melanotropin was delivered by percutaneous absorption to all follicular melanooytes throughout the skin. These present results demonstrate that the analogue was transportedacross the skin in vitro, as determined by both bioassay (FSB) and by radioimmunoassay (RIA). Other investigators have found great discrepancies in percutaneous absorption of several compounds in different animal species (19). Sane investigators argue that many substancescan pass throughmouse skin since so many hairs are present. Previous studies have ranked skin permeability of different species as determined in vitro. In all cases rodent skin was most permeable (rabbit, mouse, rat, oFg=a pig, 20-22). Other investigators, however, refute this claim (20),and it is noteworthy that in the current studies the analogue was not transported across hairy rat skin. Penetrationmay vary with dose and exposure time. Further studies are needed to evaluate these factors. These results suggest that one can not readily extrapolate results from one experimental animal model to another. The reason for the difference between penetrability of the analogue across the mouse and rat skin is unclear at this stage. The rat stratum corneum is thicker than that of themouse, but may be of similar thickness to that of some areas of human skin. However, measurements of skin thickness do not always correlate with species differences in percutaneous absorption (23). Furthermore, there is evidence that [Nle4@Phe7]alpha-MSH can be delivered across same human skin samples -in vitro as determinedby both bioassay and RIA (24). Although relatively low molecular weight substancesof a lipophilicnature can cross the stratum cornea as do steroid hormones (25),we know of no report of a peptide hormone being delivered by such a route. For example, the percutaneousabsorption of hydrocortisonefrom the forearm is limited to about 1% of the dose applied (21). Compared to themelanotropin described herein, (M-W. 1,833), hydrocortisone is both smaller (M-W. 362) and much more lipophilic. Thus the percutaneous absorption of 0.05 to 0.08% of the melanotropin dose applied is highly significant. As we have discussed elsewhere, the ability of the melanotropin analogue to traverse the skin may relate to some unique three-dimensionalconformation of the peptide analogue (8). Possibly, the analcgue is more amphipathicallowing for the development of a lipophilic surface. The lipid nature of the stratum corneum might therefore favor the movement of the analogue across the barrier. Our studies with other related but structurally different alpha-MSH analogues may provide us with more information on the proc sses of transport across the skin. For 7 Ialpha-MSH was 1000 times more effective example we determined that [Nle4,g-Phe in stimulating eumelanogenesis in mouse skin than the native hormone, alphaMSH, when topically applied to the mouse (1). The shorter fragment analogues,
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TransdermalDelivery of a Helanotropin
Vol. 43; No. 14, 1988
AC-[Nle4 D-Phe71alpha-MSH even mori;ffective in "i"$\i-NHPand Ac-[Nle4,D-Phe71 alPha+q_l@-q were 00, 00 times) than the larger tridecapeptide -analogue (2). The incorporation of lipophilic amino acids into melanotropin analogues may provide even better transdermal transport molecules. We have also synthesized several melanotropins that were conjugated to relatively large ligands such as biotin (26) and fluorescein, (27). It will be interestingto determine whether melanotropins acting as transport vehicles will be able to deliver these conjugates to the systemic circulation. If so, then it might be possible to provide for the slow continuous.delivery of a melanotropin-drug conjugate to specific target cells (e.g.,melanoma). Acknowledgments This research was supported by grants from the U.S. Public Health Service (AM-17420, V.J.H.;and AM-36021, M.E.H.). References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
M.E. HADLEY, S.H. WOOD, A.M. LEMUS-WILSON, B.V. DAWSON, N. LEVINE, R.T. DORR and V.J.HRUBY, Life Sci.40, 1889-1895 (1987). N. LEVINE, A. LEMUS-WILSON, S.H. WOOD, Z.A. ABDEL MALEK, F. AL-OBEIDI, V.J.HRUBY and M.E.HADLEY, J. Invest.Dermatol.89, 270-273 (1987). T.J. FRANZ, in: Current Problems in Dermatology 7, p. 58-68; G.A. Simon, Paster, M.A. Klingberg & M. Kaye (eds)Karger, Easel (1978). J.E. SHAW, M.E. PREVO and A.A. AMKRAUT, Arch. Dermatol. 123, 1548-1556 (1987). E.R. COOPER, J. Pharm. Sci., 67, 1469 (1979). A.M.L.CASTRUCCI, M.E. HADELY, T.K. SAWYER and V.J. HRUBY, Comp. Biochem. Physiol. E, 519-524 (1984). T.K. SAWYER, P.J. SANFILIPPO, V.J. HRUBY, M.H. ENGEL, C.B. HEWARD, J.B. BURNETT and M.E. HADLEY, Proc. Natl. Acad. Sci. U.S.A.2, 1432-5758 (1980). V.J. HRUBY, B.C. WILKES, W.L. CODY, T.K. SAWYER and M.E. HADLEY, Pept. Protein Rev. 3, l-64, M.T.W.Hearn, ed. Marcel Dekker, Inc. (1984). M.M. MARWAN, Z.A.ABDEL MALEK, K.L.KREUTZFELD, M.E. HADLEY, B.C.WILKES, V.J. HRUBY and A.M.L.CASTRUCXX, Mol. Cell. Endocrinol.4& 171-177. M.E. HADLEY, Z.A. ABDEL MALEK, M.M. MARWAN, K.L. KREUTZFELD and V.J. HRUBY, Endocrinol. Res. Conmun. 2, 157-170 (1986). Z.A. ABDEL MALEK, K.L. KREUTZFELD, M.M. MARWAN, M.E. HADLEY, V.J. HRUBY and B.C.WILKES,Cancer Res.45, 4735-4740 (1985). M.E. HADLEY, B. ANDERSON, C.B. HEWARD, T.K. SAWYER and V.J. HRUBY, Science 213, 1025-1027 (2981). T.K. SAWYER, V.J. HRUBY, M.E. HADLEY and M.H. ENGEL, Amer. Zool.23, 529540 (1983). K. AKIYAMA, H.I. YAMAMURA, B.C. WILKES, W.L. CODY, V.J. HRUBY, A.M.L. CASTRUCCI and M.E. HADLEY, Peptides 2, 1191-1195 (1984). M.E. HADLEY, J.H. MIEYR, B.E. MARTIN and A.M.L.CASTRUCCI, Comp. Biochem. Physiol.e, l-6 (1985). A.M.L.CASTRUCCI, M.E. HADLEY and V.J. HRUBY, Gen. Comp. Endocrinol. 55, 104-111 (1984). K.L. KREUTZFELD, S.T. WILSON and J.T. BAGNARA, J. Invest. Dermatol. 87, 384-448 (1987). J.L. UPTON, R. WOODS, G.L. JESSEN, A.M.L. CASTRUCCI, M.E. HADLEY, B.C. WILKES and V.J. HRUBY, in Pigment Cell 1985, p. 139-143, J.T. Bagnara, S.N.Klaus and M. Schartl (eds.)Univ.Tokyo Press, Tokyo (1984).
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19.
20. 21. 22. 23. 24. 25. 26. 27.
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M.J. BARLETE, J.A. La BUDDE, in: Animal Models in Dermatology, p. 103120, H.I. Maibach (ed)Churchill Livingstone, New York (1975). R.T. TREGEAR, Physical Function of Skin. Academic Press, New York (1966). H.I. MAIBACH, and R.B. STOUGHTON, Med. Clin. N. Amer. -57: 1253-1260 (1973). A.H. McGREESH, Toxicol. Appl. Pharmacol. Suppl. 2, 20-26 (1965). R.L.BRONAUGH, R.F. STEWART, and E.R.COUGDON, Toxicol. Appl. Pharmacol. 62, 481-488 (1982). B.V. DAWSON, M.E. HADLEY, S. DON and V.J. HRUBY, J. Invest. Dennatol. 87, p. 416 (1987). 0-J.WEPIERRE and M.P. MARTY, Trends Pharmacol. Sci.1, 23-26 (1979). D.N. CHATURVEDI, J.J. KNITTEL, V.J. HRUBY, A.M.L. CASTRUCCI and M.E. HADLEY, J. Med.Chemz, 1406-1410 (1984). D.N. CHATURVEDI, V.J. HRUBY, A.M.L.CASTRUCCI, and M.E. HADLEY, J. Pharm. Sci.2, 237-240 (1985).
TRAWDERMALDELI~OPA
Pergamon Press
MELAN(ILaopIC PEPI'IDEHDRMONE AN?WXE
Brenda V. Dawsonl, Mac E. Hpleylf2, Kristie Kreutzfeldl, Robert T. Dorr3, Victor J. Hruby , Fahad Al-Obeidi4,and Scott Don1 Departmentsof Anatcxnyl and Molecular & Cellular Biology2 nternal +4 Medicine (Pharmacologyand Dncology3),and Chemistry University of Arizona, Tucson, Arizona 85724 (Received in final form August 11, 1988) Sunmary We reviously reported that topical application of [Nle4,DPhe $ Ialpha-MSH, a superpotent analogue of alpha-melanocyte stimulating hormone, to mice induces a darkening of follicular melanocytes throughout the skin. We now report that the melanotropin analogue can be delivered across mouse but not rat skin in an in vitro model system. Passage of the analogue from the topically-&l vehicle (polyethyleneglycol) across the skin into a subcutaneousreceiving vessel was demonstratedby both bioassay as well as by radioimmunoassay. The bioassay data demonstrate that percutaneous absorption of the melanotropin did not result in loss of biological activity of the peptide. The differential penetration of the peptide across rodent skin reveals that one cannot predict percutaneousabsorptionof a substance across the stratum corneum from studies on a single species. The present results are the first to demonstrate, by direct quantitative measurements, that a bioactive peptide can be delivered across the vertebrate integument in vitro. These studies point out the potential of a topically zpliedmelanotropin for tanning of the skin and possibly for treatment of certain hypopigmentarydisorders. We previously demonstratedthat an analogue of alpha-MSH (alpha-melanocyte stimulatinghormone, alpha-malanotropin)when applied topically to the skin of a yellow strain (C57BL/6JAy)of mouse resulted in a darkening of hair follicles witpin 24 hrs of application (1). Although the melanotropin, [Nle4,DPhe lalpha-MSH, was applied to a small area of the dorsum, all growing hai& throughoutthe body became dark in color, suggesting that the peptide had been delivered systemically following percutaneousabsorption. We also demonstrated that two fragmentanalogues of the parenttridecapeptide could be even more efficaciously delivered by the transdermal route (2). These results demonstratedthat a topically applied melanotropin could activate cellular processes within follicular melanocytes leading to new eumelanin formation. These results suggested to us that melanotropin delivery acrcss the skin might prove to be clinically relevant for the treatment of hypopigmentarydisorders or even for tanning of the skin in the absence of the sun. Melanotropin inducedmelanogenesis of the human integument might even prove an important factor in decreasing the incidence of skin cancer. In this regard it became imperative to directly determine whether a melanotropin can cross the stratum corneum of the epidermis. To our knowledge peptides have not previously been shown to cross the skin. The present results clearly document that a peptide melanotropin analogue can be delivered transdennallyacross mouse skin.
0024-3205188$3.00 + .OO Copyright (c) 1988 Pergamon Press plc
1112
TransdermalDelivery of a Melanotropin
Vol. 43, No. 14, 1988
Materials -and Methods Mouse Skin. Twenty-three full thickness skin samples (approximately1 l/2" in diameter) were removed from the trunk area of 9 either black or yellow adult C57BL/6JAy mice killed by cervical dislocation. Each sample was placed on the LGA (LaboratoryGlass Apparatus)permeationapparatus described below. Rat Skin. Seventeen full thickness skin samples 1 l/2" diameter were removed from the trunks of mature Sprague Dawley rats. They were treated identically to the mouse skins. Permeation Apparatus. Skin to be tested was placed on a special permeation apparatus developed by LGA, based on an established permeation apparatus design (3). This consisted of a water-jacketed glass penetration cell with inlet and outlet port for water flow. The cell was firmly clamped to a glass lid to preclude any leakage of solution, and the apparatus with contained solution was maintained at 37oC. The melanotropin preparation was warmed to 40'C and delivered by micropipetteto the epidermis in a known volume (0.1-0.2 ml). The subcutaneousinner chamber of the permeation apparatus was filled with physiologic saline, ensuring complete contact with the dermis. Magnetic spin bars provided adequate mixing of the subdermal collection fluid. The skins were generally maintained on the permeation apparatus for 24 hrs. This time period was chosen based on other workers' experience. It is long enough to cover the lag period of permeationand short enough to preclude skin autolysis (4,5). Bacterial contaminationwas not a problem since the MSH analogue is not biodegradeable (6). The fluid within the collecting vessel was then removed and inmediately frozen to await assay. was Melanotropin Synthesis and Preparation. The [Nle4,g-Phe7]alpha-MSH synthesizedas prevously described (7). This tridecapeptideanalogue of alphaMSH was selected for our initial studies because of its several unique attributes: A) the analogue is superpotent, being 10-1000 timas more active than the native hormone, alpha-MSH, in several bioassays (8-11);B) the peptide is prolonged acting, that is, it causes a very long lasting stimulation of melanocytes in vitro and _in vivo (12,13); C) the peptide is nonbiodegradeable, to enzymatic inactivationby sera (14),brain enzymes that is, it z xtant (15),or purified proteolytic enzymes (16). In the present experimentsthe analogue was first dissolved at liav3Min a small volume of distilled water and then diluted in a polyethylene glycol vehicle (26% PEG 400 and 74% PEG 3350, by weight) to 10-4M for all studies. This mixture of high and low molecular weight PEG species results in a solid cream base which slowly liquefies at physiologic skin temperatures. The analogue was also dissolved in propylene glycol as a liquid vehicle. This is the classical assay for detecting and Frog Skin Bioassay. determining the biological activity of melanotropins. The frog skin bioassay has been described in detail elsewhere (16). Briefly, the assay is based upon the fact that biologically active melanotropins will stimulate movement (centrifugal dispersion) of melanosomes (melanin granules) within dermal melanocytes of frog skin. This response results in a rapid darkening of the skin that can be quantitativelymeasured by a reflectometer. In this assay the skin samples can usually det ct the parent alpha-MSHmolecule ataminimal concentrationof about 2 x 10-flM. Since parallel 3-point dose response curves are produced in response to amelanotropin, it is possible to quantitate the amount of melanotropin that is transdermally delivered. Most importantly, the
vol.
43, No. 14, 1988
Transderrnal Delivery of a Melanotropin
1113
bioassay can also detect [Nle4,g-Phe'lalpha-MSH at ?- 10-fold lower concentration (e.g.,2 x 10-12M). The importance of this bioassay is that it only detects biologically active melanotropins. In other words, positive bioassayswould indicate that the melanotropin had retained biological activity following transit across the skin. RIA's do not necessarily indicate whether the moiety being measured is biologically intact. To perform the frog skin bioassay, frozen samples of physiological saline containing any transdermallydelivered melanotropin were thawed and 0.2 ml added to each of the skin samples in 20 ml of Ringer solution. This results in a 1/100th dilution of the original collection sample. Nevertheless, as demonstrated in this report, the exquisite sensitivity of the bioassay allowed in most cases the demonstration of biological activity of the analogue following topical application and penetrationthrough the skin. Radioinmunoassay. An alpha-MSH radioimnunoassaykit (InmunoNuclearC ro; Stillwater, MN) was used for the detection and quantitation of [Nle8,DPhe7]alpha-MSH. The assay was performed as prescribed by the kit with the aliquot size maintained at 50 ul for both standards and sali e samples and assayed in duplicate. The analogue was dissolved in H 0 at 10-9M and diluted in BSA-borate buffer supplied in the kit. The usefu 1 range of the standard curve for [Nle4,g-Phe'lalpha-MSH from 10 to 95% binding ranged from 300 to 1 pg analogue per tube, respectively. Application of the alpha-MSH RIA kit to quFy;;zii,o,' at;; ;T#ogue, although a heterologous system using anti-alpha-labelled] alpha-MSH, yields standard curves that are repeatable and linear (17).Only 13 of the 23 samples were tested by RIA, since earlier samples had been collected and used by FSB before development of the RIA. Microscopy. After 24 hrs at room temperature (20’C) on the permeation cells, all samples were examined grossly for loss of anatomical integrity. In addition, each skin sample was sectioned, stained with henatoxylin and eosin, and examined by light microscopy.
Results By both frog skin bioassay (FSB) and RIA it was determined that [Nle4 DPhe'lalpha-MSH had been delivered transdermally. The results between the & very different assays were in consistent agreement (Fig. 1). For example, it was determined that 15/23 (65%)and 11/13 (85%),respectively, of the samples were positive for the presence of the analogue by FSB and in the RIA. There was a one to one correlation between the two methods with respect to positivity and magnitude of response. What is evident is that low values for the FSB correlated with low values for the RIA, and high FSB readings correlated with high RIA values. These results suggest, therefore, that both assays measure a common entity. Our calculations indicate that approximately 0.05% of the analogue was transdermally delivered through mouse skin exposed to the melanotropin in the PM; cream base vehicle. Using propylene glycol as an alternate vehicle, it appeared that the peptide was delivered to an even greater extent (0.08%) (Fig.2). DMSO, used in a few samples also showed good permeation (Fig. 2).
To our surprise the analogue failed to penetrate to any great extent through the skin of the rat (Fig. 1). Again, there was a close agreement between the two assays. Only one of the 15 FSB's (6.6%) and two of the 17 RIA's (11.8%)were positive for the analogue. These samples were derived from experiments using DMSO as a vehicle. Histologic examination of the stratum corneum, epidermis, and dermis, of all samples (mouse and rat) showed
1114
TransdermalDelivery of a Malanotropin
l Frog Skin Bioassay q Radioimmunoassay
Delivery l
70
Fig. 1. Relative deliv ry of a f ]a lphamelanotropin, [Nle4,D_Phe MSH, through full-thickness skin ofthemouse (C57BL/6JAy) and rat (Sprague-Dawley), using an in vitro permeation cell apparatus, -the relative refractoriness of rat skin compared to mouse skin. Positive samples are those skin samples through which the melanotropincould be delivered as determined by frog skin bioassay (> 10% darkening response)and RIA (greater than 20 pg/50 ul). Note the very good correlation between the two assays employed. The importance of the frog skin bioassay is that it demonstrates the presence of a biologicallyactive peptide.
Rat
Mouse
Transdermol
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of [NleqQ-Phe7]rMSH
Polyethylene
A
Propylene
o
DMSO
Across Mouse Skin
Glycol / Alcohol Glycol
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60 50
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A t
l
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Sample across full-thickness Fig. 2. Transdermal delivery of [Nle4,g-Phe7]alpha-MSH mouse skin _in vitro as determined by the frog skin bioassay. The melanotropin was applied to the skin for 24 hrs in the vehicles indicated. Each point represents the degree of skin darkening obtained from an individual experiment with melanotropinapplied to different mouse skin samples.
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structural integrity, and intercellular bridges could be seen in the rat epidermis. Discussion We have presented evidence that a peptide analogue of alpha+nelanotropin can be delivered transdennally through mouse skin in vitro. To our knowledge, this is the first such demonstration that a paptidehze can be delivered systemically by this method. We hat demon,stratedpreviously that topical application of the analogue, [Nle ,g-Phe Ialpha-MSH to the skin of mice resulted in follicular melanogenesis (1,2). The response occurred within 24 hrs of a single application of the analogue, depending upon the concentration employed (1,2). In the yellow strain of mouse (C57BL/6JAy)the analogue caused a shift within follicular melanocytes from pheomelanogenesis (yellow-brown melanin) toeumelanogenesis (dark brown-blackmelanin) (18). Although the vehicle was applied to the posterior dorsum, eLmElanogenesisoccurred in all hair follicles. Presumably the melanotropin was delivered by percutaneous absorption to all follicular melanooytes throughout the skin. These present results demonstrate that the analogue was transportedacross the skin in vitro, as determined by both bioassay (FSB) and by radioimmunoassay (RIA). Other investigators have found great discrepancies in percutaneous absorption of several compounds in different animal species (19). Sane investigators argue that many substancescan pass throughmouse skin since so many hairs are present. Previous studies have ranked skin permeability of different species as determined in vitro. In all cases rodent skin was most permeable (rabbit, mouse, rat, oFg=a pig, 20-22). Other investigators, however, refute this claim (20),and it is noteworthy that in the current studies the analogue was not transported across hairy rat skin. Penetrationmay vary with dose and exposure time. Further studies are needed to evaluate these factors. These results suggest that one can not readily extrapolate results from one experimental animal model to another. The reason for the difference between penetrability of the analogue across the mouse and rat skin is unclear at this stage. The rat stratum corneum is thicker than that of themouse, but may be of similar thickness to that of some areas of human skin. However, measurements of skin thickness do not always correlate with species differences in percutaneous absorption (23). Furthermore, there is evidence that [Nle4@Phe7]alpha-MSH can be delivered across same human skin samples -in vitro as determinedby both bioassay and RIA (24). Although relatively low molecular weight substancesof a lipophilicnature can cross the stratum cornea as do steroid hormones (25),we know of no report of a peptide hormone being delivered by such a route. For example, the percutaneousabsorption of hydrocortisonefrom the forearm is limited to about 1% of the dose applied (21). Compared to themelanotropin described herein, (M-W. 1,833), hydrocortisone is both smaller (M-W. 362) and much more lipophilic. Thus the percutaneous absorption of 0.05 to 0.08% of the melanotropin dose applied is highly significant. As we have discussed elsewhere, the ability of the melanotropin analogue to traverse the skin may relate to some unique three-dimensionalconformation of the peptide analogue (8). Possibly, the analcgue is more amphipathicallowing for the development of a lipophilic surface. The lipid nature of the stratum corneum might therefore favor the movement of the analogue across the barrier. Our studies with other related but structurally different alpha-MSH analogues may provide us with more information on the proc sses of transport across the skin. For 7 Ialpha-MSH was 1000 times more effective example we determined that [Nle4,g-Phe in stimulating eumelanogenesis in mouse skin than the native hormone, alphaMSH, when topically applied to the mouse (1). The shorter fragment analogues,
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TransdermalDelivery of a Helanotropin
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AC-[Nle4 D-Phe71alpha-MSH even mori;ffective in "i"$\i-NHPand Ac-[Nle4,D-Phe71 alPha+q_l@-q were 00, 00 times) than the larger tridecapeptide -analogue (2). The incorporation of lipophilic amino acids into melanotropin analogues may provide even better transdermal transport molecules. We have also synthesized several melanotropins that were conjugated to relatively large ligands such as biotin (26) and fluorescein, (27). It will be interestingto determine whether melanotropins acting as transport vehicles will be able to deliver these conjugates to the systemic circulation. If so, then it might be possible to provide for the slow continuous.delivery of a melanotropin-drug conjugate to specific target cells (e.g.,melanoma). Acknowledgments This research was supported by grants from the U.S. Public Health Service (AM-17420, V.J.H.;and AM-36021, M.E.H.). References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
M.E. HADLEY, S.H. WOOD, A.M. LEMUS-WILSON, B.V. DAWSON, N. LEVINE, R.T. DORR and V.J.HRUBY, Life Sci.40, 1889-1895 (1987). N. LEVINE, A. LEMUS-WILSON, S.H. WOOD, Z.A. ABDEL MALEK, F. AL-OBEIDI, V.J.HRUBY and M.E.HADLEY, J. Invest.Dermatol.89, 270-273 (1987). T.J. FRANZ, in: Current Problems in Dermatology 7, p. 58-68; G.A. Simon, Paster, M.A. Klingberg & M. Kaye (eds)Karger, Easel (1978). J.E. SHAW, M.E. PREVO and A.A. AMKRAUT, Arch. Dermatol. 123, 1548-1556 (1987). E.R. COOPER, J. Pharm. Sci., 67, 1469 (1979). A.M.L.CASTRUCCI, M.E. HADELY, T.K. SAWYER and V.J. HRUBY, Comp. Biochem. Physiol. E, 519-524 (1984). T.K. SAWYER, P.J. SANFILIPPO, V.J. HRUBY, M.H. ENGEL, C.B. HEWARD, J.B. BURNETT and M.E. HADLEY, Proc. Natl. Acad. Sci. U.S.A.2, 1432-5758 (1980). V.J. HRUBY, B.C. WILKES, W.L. CODY, T.K. SAWYER and M.E. HADLEY, Pept. Protein Rev. 3, l-64, M.T.W.Hearn, ed. Marcel Dekker, Inc. (1984). M.M. MARWAN, Z.A.ABDEL MALEK, K.L.KREUTZFELD, M.E. HADLEY, B.C.WILKES, V.J. HRUBY and A.M.L.CASTRUCXX, Mol. Cell. Endocrinol.4& 171-177. M.E. HADLEY, Z.A. ABDEL MALEK, M.M. MARWAN, K.L. KREUTZFELD and V.J. HRUBY, Endocrinol. Res. Conmun. 2, 157-170 (1986). Z.A. ABDEL MALEK, K.L. KREUTZFELD, M.M. MARWAN, M.E. HADLEY, V.J. HRUBY and B.C.WILKES,Cancer Res.45, 4735-4740 (1985). M.E. HADLEY, B. ANDERSON, C.B. HEWARD, T.K. SAWYER and V.J. HRUBY, Science 213, 1025-1027 (2981). T.K. SAWYER, V.J. HRUBY, M.E. HADLEY and M.H. ENGEL, Amer. Zool.23, 529540 (1983). K. AKIYAMA, H.I. YAMAMURA, B.C. WILKES, W.L. CODY, V.J. HRUBY, A.M.L. CASTRUCCI and M.E. HADLEY, Peptides 2, 1191-1195 (1984). M.E. HADLEY, J.H. MIEYR, B.E. MARTIN and A.M.L.CASTRUCCI, Comp. Biochem. Physiol.e, l-6 (1985). A.M.L.CASTRUCCI, M.E. HADLEY and V.J. HRUBY, Gen. Comp. Endocrinol. 55, 104-111 (1984). K.L. KREUTZFELD, S.T. WILSON and J.T. BAGNARA, J. Invest. Dermatol. 87, 384-448 (1987). J.L. UPTON, R. WOODS, G.L. JESSEN, A.M.L. CASTRUCCI, M.E. HADLEY, B.C. WILKES and V.J. HRUBY, in Pigment Cell 1985, p. 139-143, J.T. Bagnara, S.N.Klaus and M. Schartl (eds.)Univ.Tokyo Press, Tokyo (1984).
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19.
20. 21. 22. 23. 24. 25. 26. 27.
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M.J. BARLETE, J.A. La BUDDE, in: Animal Models in Dermatology, p. 103120, H.I. Maibach (ed)Churchill Livingstone, New York (1975). R.T. TREGEAR, Physical Function of Skin. Academic Press, New York (1966). H.I. MAIBACH, and R.B. STOUGHTON, Med. Clin. N. Amer. -57: 1253-1260 (1973). A.H. McGREESH, Toxicol. Appl. Pharmacol. Suppl. 2, 20-26 (1965). R.L.BRONAUGH, R.F. STEWART, and E.R.COUGDON, Toxicol. Appl. Pharmacol. 62, 481-488 (1982). B.V. DAWSON, M.E. HADLEY, S. DON and V.J. HRUBY, J. Invest. Dennatol. 87, p. 416 (1987). 0-J.WEPIERRE and M.P. MARTY, Trends Pharmacol. Sci.1, 23-26 (1979). D.N. CHATURVEDI, J.J. KNITTEL, V.J. HRUBY, A.M.L. CASTRUCCI and M.E. HADLEY, J. Med.Chemz, 1406-1410 (1984). D.N. CHATURVEDI, V.J. HRUBY, A.M.L.CASTRUCCI, and M.E. HADLEY, J. Pharm. Sci.2, 237-240 (1985).