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DS-1, a Modified Quillaja Saponin, Enhances Ocular and Nasal Absorption of Insulin
DS-1, a Modified Quillaja Saponin, Enhances Ocular and Nasal Absorption of Insulin DENNISJ. PILLION^^,
JOANNE
RECCHIA~, PINGWANG~, DANTEJ.
MARCiANit5, AND
CHARLOTTE R. KENSILS
Received May 30, 1995, from the +Departmentof Pharmacology, University of Alabama at Birmingham, UAB Station, Accepted for Birmingham, AL 35294-0019, and 'Cambridge Biotech Corporation, 365 Plantation Street, Worcester, MA 01605, §Present address: Phytomed, 2013 Country Ridge Circle, Birmingham, AL 35243-4306. publication August 14, 1995@. Abstract 0 The purpose of this study was to test DS-1, a modified Quillaja saponin, for its efficacy as an absorption enhancer. Anesthetized rats receiving eyedrops or nosedrops formulated with regular pork insulin in saline showed no hypoglycemic response, indicating no systemic absorption of insulin. However, rats receiving eyedrops or nosedrops formulated with insulin plus 0.025-0.1 0% DS-1 showed rapid absorption of insulin and a concomitant decrease in serum D-glucose levels. No response was observed following sublingual or buccal delivery of insulin. In conclusion, the modified saponin DS-1 was efticacious at enhancing nasal or ocular insulin delivery at extremely low concentrations. The mechanism of DS-1 action is not yet known.
Oulllalcadd
oy Glucuronlc xy/oss acid
Fucose
Hq OH
Galaclose
apiose
DS-1 Figure 1-Structure of DS-1.
Introduction Peptide drugs, such as insulin, are administered by injection because oral and topical administration are ineffective. However, several recent studies have shown that insulin applied topically to the eye or nose in the presence of an absorptionenhancing agent could be absorbed into the circulation in a biologically active f ~ r m . l - Among ~ the reagents shown to stimulate systemic insulin absorption from the eye or nose were ionic and nonionic detergents and bile salts, such as glycocholate and deoxycholate.l f 2 However, these reagents caused local irritation when used a t concentrations >0.50%, precluding their widespread use. Subsequently, it was found that a crude mixture of saponins extracted from the Gypsophilla plant was effective a t promoting the absorption of insulin and glucagon applied topically to the rabbit or rat Each of these studies confirmed that little or no systemic absorption of the peptide drugs could take place in the absence of the saponin extract in the formulation. The Gypsophilla saponin extract was also able to stimulate the systemic absorption of insulin when the formulation was infused directly down the nasolacrimal duct of the rat4 and when it was delivered to the rat nose.7 When the efficacy of the Gypsophilla saponin extract was compared to the efficacy of other absorption-enhancingagents, this saponin extract was among the most p ~ t e n t . ~However, ,~,~ the identity and the concentration of the active reagent(s) in this crude mixture is not known, because the Gypsophilla saponin extract was a heterogeneous mixture. Recently, HPLC techniques have been used to purify individual saponin compounds from a different source, the Quillaju saponaria tree.l0J1 One of the saponins found in the Quillaja extract, QS-21, has been shown to be biologically active as an immunological adjuvant. This purified saponin has been further treated to selectively cleave certain portions of the molecule in an attempt to discern the biologically important domains within its molecular structure.l0 One of the hydrolysis fragments of QS-21 was considerably more hydrophilic than the parent compound and was shown to be @Abstractpublished in Advance ACS Abstracts, September 15, 1995.
1276 / Journal of Pharmaceutical Sciences Vol. 84, No. 11, November 1995
DS-112 (Figure l), a product that had been characterized structurally by Higuchi.13 None of these Quillaju saponins had been tested previously for their ability to promote the systemic absorption of insulin, nor was it known if these compounds were chemically related to the as yet uncharacterized active agent(s) present in the extract from the Gypsophilla plant. Neither DS-1 nor QS-21 were found in the Gypsophilla extract (data not shown). Unlike the situation with the complex and uncharacterized mixture of reagents present in the Gypsophilla extract used in previous drug delivery experiment^,^-^ the DS-1 derivative prepared from the highly purified QS-21 saponin isolated from the Quillaja tree was tested individually in this investigation for its potential to serve as an absorption-enhancing agent.14 In addition to this study of DS-1 action on insulin absorption, a parallel study of DS-1 action on antibiotic absorption has revealed that DS-1 enhanced the delivery of aminoglycoside antibiotics applied nasally t o mice.12 Furthermore, DS-1 was shown to behave differently from its parent compound QS-21 in uiuo;whereas QS-21 was a potent immunological adjuvant, the deacylated derivative DS-1 did not stimulate an antibodylo or cytotoxic T-lymphocyte15response when administered to mice; similar experiments have yet t o be conducted in rats. The results described in this article document the effects of DS-1 on the systemic absorption of insulin applied topically to the rat eye or nose. Insulin serves as an excellent model drug in animal studies of drug delivery because it causes a rapid and profound change in blood D-glucose values when it is absorbed systemically. This biological effect is only produced when intact insulin is absorbed; furthermore, blood D-glucose levels and insulin levels can both be measured accurately and promptly during experiments involving insulin delivery to animal^.^-^
Materials and Methods Preparation of DS-1-The naturally occurring saponin QS-21 was purified from a n aqueous extract of Quillaju suponaria Molina bark to > 95% homogeneity by adsorption chromatography and reversedphase HPLC as described previous1y.l' QS-21 was solubilized in water to 10-20 mg/mL. The hydrolysis product DS-1 was prepared
0022-3549/95/3184-1276$09.00/0
0 1995, American Chemical Society and American Pharmaceutical Association
from QS-21 by adding sodium hydroxide to a final concentration of 0.2 M and stirring for 15-60 min. The reaction was terminated by the addition of acetic acid to yield a final pH of 4-5. DS-1 was purified to > 90% homogeneity by reversed-phase HPLC consisting of a Waters 600E system controller, Waters Lambda-Max Model 481 spectrophotometric detector, and a Waters Model 745 data module. A linear water/acetonitrile gradient containing 0.15% trifluoroacetic acid on a Vydac Protein C4 (1.0 x 25 cm) column with detection at 220 nm was used. Identification of the deacyl saponin as DS-1 was confirmed by fast atom bombardment mass spectrometry (data not shown), carbohydrate analysis (data not shown), and comparison to a published structure for DS-l.13 Insulin Delivery Experiments-Male Sprague-Dawley rats were obtained from Charles River Laboratories (Charlotte, NC). Adult (250-350 g) rats were anesthetized with a n intramuscular injection of xylazine (7 mg/kg)ketamine (70 mgkg). This anesthetic mixture caused a hyperglycemic response that was sustained throughout the 120 min sampling interval by redosing as necessary, as reported p r e v i o ~ s l y . Eyedrops ~ ~ ~ , ~ ~ were prepared using the purified saponin derivative formulated in phosphate-buffered saline, pH 7.4. Regular pork insulin (100 unitsiml) was from Novo/Nordisk Pharmaceuticals (Princeton, NJ). In most experiments, insulin was mixed with various concentrations of saponins to a final insulin concentration of 50 units/ mL. Eyedrops were administered to anesthetized rats with a pipetter. An aliquot of 20 pL was administered to each eye. This volume could be accommodated without significant spillage in rats anesthetized with xylazineketamine, in part because this anesthetic combination caused the rats to lose their blink reflex; hence, these rats accommodated more eyedrop volume than rats anesthetized with sodium pentobarbital.I6 In one subchronic study, rats were anesthetized and one eye was treated with eyedrops containing insulin plus 0.05% DS-1, once a day for 15 days. Saline eyedrops were administered to the other eye each day as a n internal control. On days 1, 8 and 15, the efficacy of the eyedrops containing insulin plus DS-1 was evaluated by measuring the change in blood D-glucose levels, as described below. On days 2-7 and 9-14, rats received the eyedrops under anesthesia with halothane and no blood samples were drawn. The two eyes were evaluated for any differential signs of ocular irritation that could be detected visually. In some experiments, nosedrops containing 50 unitdmL insulin with or without DS-1 were applied to anesthetized rats. An aliquot of 20 pL was applied to the right nostril with a pipetter a t time 0 and ~ drops (20 pL again 5 min later, as described p r e v i ~ u s l y .Sublingual a t time 0 and again 5 min later) were administered by a pipetter as well, while buccal administration was achieved by soaking a cotton swab with a n aliquot (150 pL) of the saponidinsulin formulation and placing it on the buccal surface of an anesthetized rat for 10 min. Blood was collected from the tail a t 15 min intervals following eyedrop or nosedrop delivery and applied to Chemstrips for D-glucose determinations using an AccuChek I1 or 111 glucometer (Boehringer Mannheim Corp., Indianapolis, IN). No significant differences were found when paired samples were measured using the AccuChek I1 or the AccuChek 111 glucometers. In some experiments, serum D-glucose values were measured by a spectrophotometric assay (Sigma Chemical Co., St. Louis, MO). The D-glucose values obtained with the glucometer were accurate to within 5% of the values obtained in the spectrophotometric assay. Blood D-glucose values were subsequently converted to represent the percent change that took place following insulin administration compared to the values at time 0. In some experiments, the data were further used to determine the total area under the D-glUCOSe time curve for the 120 rnin period following eyedrop delivery using the trapezoidal method. Measurement of Insulin Levels-Rat serum insulin levels were measured by radioimmunoassay using a human- specific kit that crossreacted completely with porcine insulin (Linco Research Inc., St. Louis, MO). Animal Care-This work was conducted according to the principles outlined in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, National Research Council.
Results Efficacy of DS-1-Blood D-glucose values in the 250-400 mg/dL range were measured in anesthetized rats prior to
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Time (min) Figure 2-Changes in rat blood D-glucoseconcentrationfollowing the administration of eyedrops (A) or nosedrops (B) containing 2 units of regular pork insulin with or without DS-1. Blood D-glucose levels measured at time 0 were used as a baseline, and the percent change from baseline values at each time point is presented. Data represent the mean k SEM; n = 3 or 4. 0, 0.10% DS-1, no insulin; 0, 0.01% CIS-1 + insulin; A, 0.025% DS-1 + insulin; A,0.05% DS-I + insulin; 0 , 0.10% DS-1 + insulin; B, 0.0% DS-1 + insulin.
insulin delivery, presumably as a result of catecholamine and glucocorticoid release in response to the xylazineketamine used for anesthesia.16 This hyperglycemic condition provided a large therapeutic window and this phenomenon was used to our advantage, since insulin administered in eyedrops or nosedrops produced a readily determined reduction in blood D-glucose levels in these animals, as described p r e v i o ~ s l y . ~ ~ ~ When rats received eyedrops containing 0.1% DS-1 without insulin (Figure 2A) or eyedrops containing 2.0 units of regular pork insulin without DS-1 (not shown), there was no hypoglycemic response. Hence, the saponin derivative DS-1 alone did not cause a reduction in blood D-glucose levels; moreover, little or no systemic absorption of insulin was observed when no saponin was added to the eyedrop f o r m u l a t i ~ n .However, ~~~ when rats received eyedrops containing 2.0 units of regular pork insulin plus various concentrations of DS-1(0.01-0.10% or 0.066-0.66 mM), a rapid and substantial decrease in blood D-glUCOSe values was observed (Figure 2A). The hypoglycemic response was greatest in rats receiving eyedrops with a concentration of 0.10% DS-1; blood D-glucose values decreased more than 75%, from 236 f 33 to 58 f 14 mgIdL, following eyedrop delivery. Lower concentrations of DS-1 were proportionately less effective at promoting the hypoglycemic response. At a concentration of 0.01% DS-1, no changes in blood D-glucose values were observed, suggesting that no enhancement of systemic insulin absorption had occurred. The lowest concentration of DS-1 that caused a hypoglycemic response (0.025%= 0.165 mM) was considerably lower than the critical Journal of Pharmaceutical Sciences / 1277 Vol. 84, No. 11, November 1995
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Figure 3-Efficacy of eyedrops containing insulin plus DS-1 in a subchronic study. Rats received eyedrops for 15 consecutive days. Data represent blood o-glucose values on day I , 8, or 15 (mean i: SEM; n = 3 or 4). 0 , day 1; 0, day 8; A, day 15.
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micellar concentration (1.09 mM) reported for DS-1 by Recchia et a1.12 DS-1 had a lower effective concentration than any of the other absorption-enhancing agents that had been tested previously in eyedrop formulations with i n ~ u l i n .The ~~~~~ pharmacokinetics of insulin absorption from eyedrops containing DS-1 was similar to that described previously for eyedrops containing other absorption-enhancing agents,3p7i.e. the hypoglycemic response was more rapid in onset and more transient than that observed when insulin was injected subcutaneously. When rats received nosedrops containing 2.0 units of regular pork insulin formulated with or without 0 20 40 60 8 0 100 120 0 various concentrations of DS-1, the hypoglycemic response was even more profound (Figure 2B). Nasal drops containing Time (min) 0.05% DS-1 plus insulin were more efficacious than eyedrops Figure 4-Residual effects of DS-1 on insulin absorption. Data represent the containing the same formulation. In contrast, formulations percent change in D-glucose levels compared to values obtained at time 0 when containing 0.05% DS-1 plus insulin applied sublingually or insulin was administered 5, 30, or 60 min after 0.05% DS-1 (A). Blood samples buccally were not effective at reducing blood D-glucose levels were also collected for determination of serum insulin levels by radioimmunoassay (data not shown). (B). Data represent the mean k SEM; n = 3. 0, DS-1 administered 5 rnin prior to insulin; A,DS-1 administered 30 rnin prior to insulin; A,DS-1 administered60 The studies described in Figure 2A involved the adminisrnin prior to insulin; 0 , DS-1 and insulin co-administered; ., insulin and saline tration of insulin eyedrops to both eyes of the rats, equivalent coadministered. to a total application volume of 40 pL, containing 2 units of regular insulin. When these experiments were repeated using Insulin Levels in Rats following Nasal Deliveryeyedrops administered to only one eye, equivalent to a total Immunoreactive insulin levels were measured in rat blood insulin application of 20 pL, containing 1unit of insulin, the samples following the administration of nosedrops containing hypoglycemic response was once again observed when eyeinsulin formulated either in saline or 0.05% DS-1, in order to drops containing 0.1% or 0.05% DS-1 were used, but no provide direct evidence that the changes observed in blood significant hypoglycemic response to eyedrops containing D-glucose levels were caused by systemic absorption of insulin. 0.025% DS-1 plus insulin was apparent (data not shown). No Administration of nosedrops containing 2 units of regular pork selective ocular irritation was visible in the eyes that received insulin plus 0.05% DS-1 caused an increase in serum immuthe eyedrops containing DS-1. noreactive insulin levels from 9 f 5 microunits/mL at time 0, Acute and Subchronic Efficacy of DS-1-In an effort to 1350 k 31 microunits/mL 20 min after insulin administrato discern if DS-1 eyedrops were safe and efficacious in a tion in nosedrops. Elevated insulin levels were sustained for subchronic study, rats were given eyedrops containing insulin 20-80 min following nasal administration of insulin plus DS-1 plus 0.05% DS-1 in one eye and saline eyedrops in the other and were reduced to 363 f 51 microunits/mL 120 min after eye, once a day for 15 consecutive days. On days 1, 8, and nasal administration of insulin plus DS-1. 15, the efficacy of the eyedrops containing insulin plus DS-1 Residual Effects of DS-1-In addition to the studies was evaluated by measuring the change in blood D-glUCOSe described above, in which insulin and DS-1 were first mixed levels following eyedrop administration under xylazineketin vitro and applied to the rat eye simultaneously, another amine anesthesia; on days 2-7 and 9-14, rats received the experimental protocol involved the administration of DS-1 and eyedrops under anesthesia with halothane, since no blood insulin separately. In this study, rats received eyedrops sampling was required on those days. The efficacy of DS-1 containing 0.05% DS-1 alone and then received eyedrops to enhance systemic insulin absorption, as reflected by a containing insulin a t various time intervals thereafter. The decrease in the blood D-glucose levels, was observed to be effect of DS-1 to promote insulin absorption was observed virtually the same on days 1, 8, and 15 (Figure 3). At this when the insulin was applied 5, 30, or 60 min after the low concentration of DS-1, there was no visible drug-related application of the absorption-enhancing drug (Figure 4A). ocular irritation during the 15-day trial, over and above the When insulin was applied within 30 min of the absorptionsymptoms of keratoconjunctivitis sicca observed in both eyes enhancing agent DS-1, the effect on rat serum D-glucose levels as a result of the anesthesia-induced loss of blink response.I6 appeared to be equivalent to the effect observed when insulin 1278 / Journal of Pharmaceutical Sciences Vol. 84, No. 11, November 1995
and DS-1 were co-administered (Figure 4A vs Figure 2A),but when the insulin was administered 60 min after DS-1, a reduction in the response was observed (Figure 4A). In order to confirm this interpretation of the data, insulin levels were measured by radioimmunoassay in one cohort of rats that received both insulin and DS-1 at time 0 and in another cohort of rats that received insulin 60 min after DS-1 was administered. A third cohort of rats received eyedrops containing insulin plus saline at time 0. Rats that received both insulin and DS-1 a t time 0 showed a greater response than rats that received insulin 60 min after DS-1 administration (Figure 4B), a result that is consistent with the hypoglycemic responses reported in Figure 4A. The area under-the-curve (AUC)in Figure 4B is 27% smaller for the animals that received the insulin 60 min after the DS-1 compared to the A U C for animals who received insulin and DS-1 simultaneously.
Discussion DS-1 is a semisynthetic derivative of QS-21, a natural saponin that can be isolated from the bark of the Quillaja Saponaria Molina tree. DS-1 is distinct from QS-21 in some of its biological properties, most notably in that DS-1 does not enhance immune response in mice,15 whereas QS-21 does enhance antibody response and a cytotoxic T-lymphocyte response in mice.1°J5 However, DS-1 does promote the systemic absorption of aminoglycoside antibiotics applied nasally to mice and rats12,and insulin applied topically to the rat eye or nose (see Figures 2-4), even when used a t a concentration as low as 0.025% (0.16 mM). No other absorption-enhancing agent has been effective when used a t such low concentration^.'-^ The potential for using DS-1 as an absorption-enhancing agent for the ocular or nasal delivery of insulin or other peptide drugs in humans would depend on several factors, including the safety and efficacy of the saponin derivative during chronic administration. In the present investigation, the acute and subchronic effects of DS-1 have been studied. Topical application of very low concentrations of DS-1 plus regular pork insulin caused no visible inflammation or irritation of the rat eye or nose, but morphological changes induced by the enhancer might still be occurring. A more detailed study of enhancer toxicity would be required to fully characterize the effects of DS-1 on ocular and nasal tissues. The body of evidence that has been established to date suggests that peptide drugs applied topically to the rat eye are flushed down the nasolacrimal duct and ultimately delivered to, and absorbed from, the ipsilateral maxillary sinus,l-* while drugs applied in nasal drops are exposed to the larger surface area of the nasal mucosa; this difference in the sites available for peptide drug absorption may explain the distinction between the amount of insulin absorbed following ocular vs nasal delivery, as shown in Figure 2. The precise mechanism of DS-1 action is not known. However, the administration of DS-1 eyedrops to a rat up to 30 min prior to the addition of insulin eyedrops caused a strong hypoglycemic response (Figure 4A). This result was consistent with the hypothesis that DS-1 caused a change in the nasal epithelium that somehow made it more permeable to insulin. Whatever the nature of this change, the effect was diminished considerably 60 min after the purified saponin derivative was applied to the eye. A similar result was reported by Illum et al.17when nosedrops containing insulin were administered 30 min after a nasal formulation containing the absorption-enhancingcationic polysaccharide chitosan. However, when the insulin nosedrops were applied 60 rnin after the chitosan, little insulin absorption could be detected. In contrast, results with DS-1 and antibiotic mixtures administered at separate times demonstrated that antibiotic levels
in blood were elevated by only 10% if applied 60 min after DS-1, compared with administration together.12 Several mechanisms of action for absorption-enhancing agents other than DS-1 have been s ~ g g e s t e d , l including ~-~~ protection of insulin from endogenous proteases, conversion of hexameric insulin to its dimeric or monomeric forms, and increased rate of delivery of insulin to its ultimate site of absorption. Our results do not rule out these possibilities, but they are consistent with a direct interaction between DS-1 and the nasal epithelium. Future studies describing the interaction between DS-1 and membrane proteases, drug compounds, and other membrane components,especiallywhen considered with respect to their duration of action following the addition of DS-1, should provide new insights as to the mechanism(s) of DS-1 action. Studies with other peptide drugs, such as glucagon, GHRH, IGF-1, and interferon, formulated with and without DS-1, should provide additional information about the practicality of using DS-1 to enhance the systemic absorption of other peptide drugs applied topically to the eyes or nose.
References and Notes 1. Hirai, S.; Ikenaga, T.; Matsuzawa, T. Diabetes 1978, 27, 296299. 2. Moses, A. C.; Gordon, G. S.; Carey, M. C.; Flier, J. S. Diabetes 1983,32, 1040-1047. 3. Chiou, G. C. Y.; Chuang, C.; Chang, M. S. Diabetes Care 1988, 11, 750-751. 4. Pillion, D. J.;Bartlett, J. D.; Meezan, E.; Yang, M.; Crain, R. J.; Grizzle, W. E. Invest. Ophthalmol. Vis. Sci. 1991, 32, 30213027. 5. Chiou, G. C. Y.; Chuang, C. Y. J. Ocul. Pharmacol. 1988,4,179186. 6. Pillion, D. J.; McCracken, D. L.; Yang, M.; Atchison, J. A. J . Ocul. Pharmacol. 1992,8, 349-358. 7. Pillion, D. J.;Atchison, J. A.; Stott, J.;McCracken, D.; Garguilo, C.; Meezan, E. J. Ocul. Pharmacol. 1994,10, 461-470. 8. Pillion, D. J.; Atchison, J. A,; Wang, R.-X.; Meezan, E. J . Pharmacol. Exp. Ther. 1994,271, 1274-1280. 9. Pillion, D. J.; Atchison, J. A.; Garguilo, C.; Wang, R.-X.; Wang, P.; Meezan, E. Endocrinol. 1994,135,2386-2391. 10. Kensil, C. R.; Soltysik, S.; Patel, U.; Marciani, D. J. In Vaccines 92; Brown, F., Chanock, R. M., Ginsberg, H. S., Lerner, R. A., Eds.; Cold Spring Harbor Lab.: Plainview, NY,1992; pp 3540. 11. Kensil, C. R.; Patel, U.; Lennick, M.; Marciani, D. J. Zmmunol. 1991,146, 431-431. 12. Recchia, J.; Lurantos, M. H. A.; Amsden, J.; Storey, J.; Kensil, C. R. Submitted for publication. 13. Higuchi, R.; Tokimittsu, Y.; Fujioka, T.; Komori, T.; Kawasaki, T.; Oakenful, D. G. Phytochem. 1987,26, 229-234. 14. Kensil, C. R.; Soltysik, S.; Marciani, D. J. U.S. Patent Number 5,273,965, 1993. 15. Kensil, C. R.; Soltysik, S.; Wheeler, D.; Wu, J.-Y. Submitted for publication. 16. Pillion, D. J.; Wang, P.; Yorks, J.; McCann, P.; Meezan, E. J . Ocul. Pharmacol. In press. 17. Illum, L.; Farraj, N. F.; Davis, S. S. Pharm. Res. 1994,11,11861189. 18. Gordon, G. S.; Moses, A. C.; Silver, R. D.; Flier, J. S.; Carey, M. C. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 7419-7423. 19. Hayakawa, E.; Yamamoto, A,; Shoji, Y.; Lee, V. H. L. Life Sci. 1989.45, 167-174. 20. Tengamnuay, P.; Mitra, A. K. Pharm. Res. 1990, 7, 370-375. 21. Carstens, S.; Danielsen, G.; Guldhammer, B.; Frederiksen, 0. Diabetes 1993,42, 1032-1040.
Acknowledgments We thank Maria H. A. Lurantos for preparation of DS-1. The University of Alabama at Birmingham is fully accredited by the American Association for Accreditation of Laboratory Animals.
JS950214S Journal of Pharmaceutical Sciences / 1279 Vol. 84, No. 11, November 1995
JOANNE
RECCHIA~, PINGWANG~, DANTEJ.
MARCiANit5, AND
CHARLOTTE R. KENSILS
Received May 30, 1995, from the +Departmentof Pharmacology, University of Alabama at Birmingham, UAB Station, Accepted for Birmingham, AL 35294-0019, and 'Cambridge Biotech Corporation, 365 Plantation Street, Worcester, MA 01605, §Present address: Phytomed, 2013 Country Ridge Circle, Birmingham, AL 35243-4306. publication August 14, 1995@. Abstract 0 The purpose of this study was to test DS-1, a modified Quillaja saponin, for its efficacy as an absorption enhancer. Anesthetized rats receiving eyedrops or nosedrops formulated with regular pork insulin in saline showed no hypoglycemic response, indicating no systemic absorption of insulin. However, rats receiving eyedrops or nosedrops formulated with insulin plus 0.025-0.1 0% DS-1 showed rapid absorption of insulin and a concomitant decrease in serum D-glucose levels. No response was observed following sublingual or buccal delivery of insulin. In conclusion, the modified saponin DS-1 was efticacious at enhancing nasal or ocular insulin delivery at extremely low concentrations. The mechanism of DS-1 action is not yet known.
Oulllalcadd
oy Glucuronlc xy/oss acid
Fucose
Hq OH
Galaclose
apiose
DS-1 Figure 1-Structure of DS-1.
Introduction Peptide drugs, such as insulin, are administered by injection because oral and topical administration are ineffective. However, several recent studies have shown that insulin applied topically to the eye or nose in the presence of an absorptionenhancing agent could be absorbed into the circulation in a biologically active f ~ r m . l - Among ~ the reagents shown to stimulate systemic insulin absorption from the eye or nose were ionic and nonionic detergents and bile salts, such as glycocholate and deoxycholate.l f 2 However, these reagents caused local irritation when used a t concentrations >0.50%, precluding their widespread use. Subsequently, it was found that a crude mixture of saponins extracted from the Gypsophilla plant was effective a t promoting the absorption of insulin and glucagon applied topically to the rabbit or rat Each of these studies confirmed that little or no systemic absorption of the peptide drugs could take place in the absence of the saponin extract in the formulation. The Gypsophilla saponin extract was also able to stimulate the systemic absorption of insulin when the formulation was infused directly down the nasolacrimal duct of the rat4 and when it was delivered to the rat nose.7 When the efficacy of the Gypsophilla saponin extract was compared to the efficacy of other absorption-enhancingagents, this saponin extract was among the most p ~ t e n t . ~However, ,~,~ the identity and the concentration of the active reagent(s) in this crude mixture is not known, because the Gypsophilla saponin extract was a heterogeneous mixture. Recently, HPLC techniques have been used to purify individual saponin compounds from a different source, the Quillaju saponaria tree.l0J1 One of the saponins found in the Quillaja extract, QS-21, has been shown to be biologically active as an immunological adjuvant. This purified saponin has been further treated to selectively cleave certain portions of the molecule in an attempt to discern the biologically important domains within its molecular structure.l0 One of the hydrolysis fragments of QS-21 was considerably more hydrophilic than the parent compound and was shown to be @Abstractpublished in Advance ACS Abstracts, September 15, 1995.
1276 / Journal of Pharmaceutical Sciences Vol. 84, No. 11, November 1995
DS-112 (Figure l), a product that had been characterized structurally by Higuchi.13 None of these Quillaju saponins had been tested previously for their ability to promote the systemic absorption of insulin, nor was it known if these compounds were chemically related to the as yet uncharacterized active agent(s) present in the extract from the Gypsophilla plant. Neither DS-1 nor QS-21 were found in the Gypsophilla extract (data not shown). Unlike the situation with the complex and uncharacterized mixture of reagents present in the Gypsophilla extract used in previous drug delivery experiment^,^-^ the DS-1 derivative prepared from the highly purified QS-21 saponin isolated from the Quillaja tree was tested individually in this investigation for its potential to serve as an absorption-enhancing agent.14 In addition to this study of DS-1 action on insulin absorption, a parallel study of DS-1 action on antibiotic absorption has revealed that DS-1 enhanced the delivery of aminoglycoside antibiotics applied nasally t o mice.12 Furthermore, DS-1 was shown to behave differently from its parent compound QS-21 in uiuo;whereas QS-21 was a potent immunological adjuvant, the deacylated derivative DS-1 did not stimulate an antibodylo or cytotoxic T-lymphocyte15response when administered to mice; similar experiments have yet t o be conducted in rats. The results described in this article document the effects of DS-1 on the systemic absorption of insulin applied topically to the rat eye or nose. Insulin serves as an excellent model drug in animal studies of drug delivery because it causes a rapid and profound change in blood D-glucose values when it is absorbed systemically. This biological effect is only produced when intact insulin is absorbed; furthermore, blood D-glucose levels and insulin levels can both be measured accurately and promptly during experiments involving insulin delivery to animal^.^-^
Materials and Methods Preparation of DS-1-The naturally occurring saponin QS-21 was purified from a n aqueous extract of Quillaju suponaria Molina bark to > 95% homogeneity by adsorption chromatography and reversedphase HPLC as described previous1y.l' QS-21 was solubilized in water to 10-20 mg/mL. The hydrolysis product DS-1 was prepared
0022-3549/95/3184-1276$09.00/0
0 1995, American Chemical Society and American Pharmaceutical Association
from QS-21 by adding sodium hydroxide to a final concentration of 0.2 M and stirring for 15-60 min. The reaction was terminated by the addition of acetic acid to yield a final pH of 4-5. DS-1 was purified to > 90% homogeneity by reversed-phase HPLC consisting of a Waters 600E system controller, Waters Lambda-Max Model 481 spectrophotometric detector, and a Waters Model 745 data module. A linear water/acetonitrile gradient containing 0.15% trifluoroacetic acid on a Vydac Protein C4 (1.0 x 25 cm) column with detection at 220 nm was used. Identification of the deacyl saponin as DS-1 was confirmed by fast atom bombardment mass spectrometry (data not shown), carbohydrate analysis (data not shown), and comparison to a published structure for DS-l.13 Insulin Delivery Experiments-Male Sprague-Dawley rats were obtained from Charles River Laboratories (Charlotte, NC). Adult (250-350 g) rats were anesthetized with a n intramuscular injection of xylazine (7 mg/kg)ketamine (70 mgkg). This anesthetic mixture caused a hyperglycemic response that was sustained throughout the 120 min sampling interval by redosing as necessary, as reported p r e v i o ~ s l y . Eyedrops ~ ~ ~ , ~ ~ were prepared using the purified saponin derivative formulated in phosphate-buffered saline, pH 7.4. Regular pork insulin (100 unitsiml) was from Novo/Nordisk Pharmaceuticals (Princeton, NJ). In most experiments, insulin was mixed with various concentrations of saponins to a final insulin concentration of 50 units/ mL. Eyedrops were administered to anesthetized rats with a pipetter. An aliquot of 20 pL was administered to each eye. This volume could be accommodated without significant spillage in rats anesthetized with xylazineketamine, in part because this anesthetic combination caused the rats to lose their blink reflex; hence, these rats accommodated more eyedrop volume than rats anesthetized with sodium pentobarbital.I6 In one subchronic study, rats were anesthetized and one eye was treated with eyedrops containing insulin plus 0.05% DS-1, once a day for 15 days. Saline eyedrops were administered to the other eye each day as a n internal control. On days 1, 8 and 15, the efficacy of the eyedrops containing insulin plus DS-1 was evaluated by measuring the change in blood D-glucose levels, as described below. On days 2-7 and 9-14, rats received the eyedrops under anesthesia with halothane and no blood samples were drawn. The two eyes were evaluated for any differential signs of ocular irritation that could be detected visually. In some experiments, nosedrops containing 50 unitdmL insulin with or without DS-1 were applied to anesthetized rats. An aliquot of 20 pL was applied to the right nostril with a pipetter a t time 0 and ~ drops (20 pL again 5 min later, as described p r e v i ~ u s l y .Sublingual a t time 0 and again 5 min later) were administered by a pipetter as well, while buccal administration was achieved by soaking a cotton swab with a n aliquot (150 pL) of the saponidinsulin formulation and placing it on the buccal surface of an anesthetized rat for 10 min. Blood was collected from the tail a t 15 min intervals following eyedrop or nosedrop delivery and applied to Chemstrips for D-glucose determinations using an AccuChek I1 or 111 glucometer (Boehringer Mannheim Corp., Indianapolis, IN). No significant differences were found when paired samples were measured using the AccuChek I1 or the AccuChek 111 glucometers. In some experiments, serum D-glucose values were measured by a spectrophotometric assay (Sigma Chemical Co., St. Louis, MO). The D-glucose values obtained with the glucometer were accurate to within 5% of the values obtained in the spectrophotometric assay. Blood D-glucose values were subsequently converted to represent the percent change that took place following insulin administration compared to the values at time 0. In some experiments, the data were further used to determine the total area under the D-glUCOSe time curve for the 120 rnin period following eyedrop delivery using the trapezoidal method. Measurement of Insulin Levels-Rat serum insulin levels were measured by radioimmunoassay using a human- specific kit that crossreacted completely with porcine insulin (Linco Research Inc., St. Louis, MO). Animal Care-This work was conducted according to the principles outlined in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, National Research Council.
Results Efficacy of DS-1-Blood D-glucose values in the 250-400 mg/dL range were measured in anesthetized rats prior to
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Time (min) Figure 2-Changes in rat blood D-glucoseconcentrationfollowing the administration of eyedrops (A) or nosedrops (B) containing 2 units of regular pork insulin with or without DS-1. Blood D-glucose levels measured at time 0 were used as a baseline, and the percent change from baseline values at each time point is presented. Data represent the mean k SEM; n = 3 or 4. 0, 0.10% DS-1, no insulin; 0, 0.01% CIS-1 + insulin; A, 0.025% DS-1 + insulin; A,0.05% DS-I + insulin; 0 , 0.10% DS-1 + insulin; B, 0.0% DS-1 + insulin.
insulin delivery, presumably as a result of catecholamine and glucocorticoid release in response to the xylazineketamine used for anesthesia.16 This hyperglycemic condition provided a large therapeutic window and this phenomenon was used to our advantage, since insulin administered in eyedrops or nosedrops produced a readily determined reduction in blood D-glucose levels in these animals, as described p r e v i o ~ s l y . ~ ~ ~ When rats received eyedrops containing 0.1% DS-1 without insulin (Figure 2A) or eyedrops containing 2.0 units of regular pork insulin without DS-1 (not shown), there was no hypoglycemic response. Hence, the saponin derivative DS-1 alone did not cause a reduction in blood D-glucose levels; moreover, little or no systemic absorption of insulin was observed when no saponin was added to the eyedrop f o r m u l a t i ~ n .However, ~~~ when rats received eyedrops containing 2.0 units of regular pork insulin plus various concentrations of DS-1(0.01-0.10% or 0.066-0.66 mM), a rapid and substantial decrease in blood D-glUCOSe values was observed (Figure 2A). The hypoglycemic response was greatest in rats receiving eyedrops with a concentration of 0.10% DS-1; blood D-glucose values decreased more than 75%, from 236 f 33 to 58 f 14 mgIdL, following eyedrop delivery. Lower concentrations of DS-1 were proportionately less effective at promoting the hypoglycemic response. At a concentration of 0.01% DS-1, no changes in blood D-glucose values were observed, suggesting that no enhancement of systemic insulin absorption had occurred. The lowest concentration of DS-1 that caused a hypoglycemic response (0.025%= 0.165 mM) was considerably lower than the critical Journal of Pharmaceutical Sciences / 1277 Vol. 84, No. 11, November 1995
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Figure 3-Efficacy of eyedrops containing insulin plus DS-1 in a subchronic study. Rats received eyedrops for 15 consecutive days. Data represent blood o-glucose values on day I , 8, or 15 (mean i: SEM; n = 3 or 4). 0 , day 1; 0, day 8; A, day 15.
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micellar concentration (1.09 mM) reported for DS-1 by Recchia et a1.12 DS-1 had a lower effective concentration than any of the other absorption-enhancing agents that had been tested previously in eyedrop formulations with i n ~ u l i n .The ~~~~~ pharmacokinetics of insulin absorption from eyedrops containing DS-1 was similar to that described previously for eyedrops containing other absorption-enhancing agents,3p7i.e. the hypoglycemic response was more rapid in onset and more transient than that observed when insulin was injected subcutaneously. When rats received nosedrops containing 2.0 units of regular pork insulin formulated with or without 0 20 40 60 8 0 100 120 0 various concentrations of DS-1, the hypoglycemic response was even more profound (Figure 2B). Nasal drops containing Time (min) 0.05% DS-1 plus insulin were more efficacious than eyedrops Figure 4-Residual effects of DS-1 on insulin absorption. Data represent the containing the same formulation. In contrast, formulations percent change in D-glucose levels compared to values obtained at time 0 when containing 0.05% DS-1 plus insulin applied sublingually or insulin was administered 5, 30, or 60 min after 0.05% DS-1 (A). Blood samples buccally were not effective at reducing blood D-glucose levels were also collected for determination of serum insulin levels by radioimmunoassay (data not shown). (B). Data represent the mean k SEM; n = 3. 0, DS-1 administered 5 rnin prior to insulin; A,DS-1 administered 30 rnin prior to insulin; A,DS-1 administered60 The studies described in Figure 2A involved the adminisrnin prior to insulin; 0 , DS-1 and insulin co-administered; ., insulin and saline tration of insulin eyedrops to both eyes of the rats, equivalent coadministered. to a total application volume of 40 pL, containing 2 units of regular insulin. When these experiments were repeated using Insulin Levels in Rats following Nasal Deliveryeyedrops administered to only one eye, equivalent to a total Immunoreactive insulin levels were measured in rat blood insulin application of 20 pL, containing 1unit of insulin, the samples following the administration of nosedrops containing hypoglycemic response was once again observed when eyeinsulin formulated either in saline or 0.05% DS-1, in order to drops containing 0.1% or 0.05% DS-1 were used, but no provide direct evidence that the changes observed in blood significant hypoglycemic response to eyedrops containing D-glucose levels were caused by systemic absorption of insulin. 0.025% DS-1 plus insulin was apparent (data not shown). No Administration of nosedrops containing 2 units of regular pork selective ocular irritation was visible in the eyes that received insulin plus 0.05% DS-1 caused an increase in serum immuthe eyedrops containing DS-1. noreactive insulin levels from 9 f 5 microunits/mL at time 0, Acute and Subchronic Efficacy of DS-1-In an effort to 1350 k 31 microunits/mL 20 min after insulin administrato discern if DS-1 eyedrops were safe and efficacious in a tion in nosedrops. Elevated insulin levels were sustained for subchronic study, rats were given eyedrops containing insulin 20-80 min following nasal administration of insulin plus DS-1 plus 0.05% DS-1 in one eye and saline eyedrops in the other and were reduced to 363 f 51 microunits/mL 120 min after eye, once a day for 15 consecutive days. On days 1, 8, and nasal administration of insulin plus DS-1. 15, the efficacy of the eyedrops containing insulin plus DS-1 Residual Effects of DS-1-In addition to the studies was evaluated by measuring the change in blood D-glUCOSe described above, in which insulin and DS-1 were first mixed levels following eyedrop administration under xylazineketin vitro and applied to the rat eye simultaneously, another amine anesthesia; on days 2-7 and 9-14, rats received the experimental protocol involved the administration of DS-1 and eyedrops under anesthesia with halothane, since no blood insulin separately. In this study, rats received eyedrops sampling was required on those days. The efficacy of DS-1 containing 0.05% DS-1 alone and then received eyedrops to enhance systemic insulin absorption, as reflected by a containing insulin a t various time intervals thereafter. The decrease in the blood D-glucose levels, was observed to be effect of DS-1 to promote insulin absorption was observed virtually the same on days 1, 8, and 15 (Figure 3). At this when the insulin was applied 5, 30, or 60 min after the low concentration of DS-1, there was no visible drug-related application of the absorption-enhancing drug (Figure 4A). ocular irritation during the 15-day trial, over and above the When insulin was applied within 30 min of the absorptionsymptoms of keratoconjunctivitis sicca observed in both eyes enhancing agent DS-1, the effect on rat serum D-glucose levels as a result of the anesthesia-induced loss of blink response.I6 appeared to be equivalent to the effect observed when insulin 1278 / Journal of Pharmaceutical Sciences Vol. 84, No. 11, November 1995
and DS-1 were co-administered (Figure 4A vs Figure 2A),but when the insulin was administered 60 min after DS-1, a reduction in the response was observed (Figure 4A). In order to confirm this interpretation of the data, insulin levels were measured by radioimmunoassay in one cohort of rats that received both insulin and DS-1 at time 0 and in another cohort of rats that received insulin 60 min after DS-1 was administered. A third cohort of rats received eyedrops containing insulin plus saline at time 0. Rats that received both insulin and DS-1 a t time 0 showed a greater response than rats that received insulin 60 min after DS-1 administration (Figure 4B), a result that is consistent with the hypoglycemic responses reported in Figure 4A. The area under-the-curve (AUC)in Figure 4B is 27% smaller for the animals that received the insulin 60 min after the DS-1 compared to the A U C for animals who received insulin and DS-1 simultaneously.
Discussion DS-1 is a semisynthetic derivative of QS-21, a natural saponin that can be isolated from the bark of the Quillaja Saponaria Molina tree. DS-1 is distinct from QS-21 in some of its biological properties, most notably in that DS-1 does not enhance immune response in mice,15 whereas QS-21 does enhance antibody response and a cytotoxic T-lymphocyte response in mice.1°J5 However, DS-1 does promote the systemic absorption of aminoglycoside antibiotics applied nasally to mice and rats12,and insulin applied topically to the rat eye or nose (see Figures 2-4), even when used a t a concentration as low as 0.025% (0.16 mM). No other absorption-enhancing agent has been effective when used a t such low concentration^.'-^ The potential for using DS-1 as an absorption-enhancing agent for the ocular or nasal delivery of insulin or other peptide drugs in humans would depend on several factors, including the safety and efficacy of the saponin derivative during chronic administration. In the present investigation, the acute and subchronic effects of DS-1 have been studied. Topical application of very low concentrations of DS-1 plus regular pork insulin caused no visible inflammation or irritation of the rat eye or nose, but morphological changes induced by the enhancer might still be occurring. A more detailed study of enhancer toxicity would be required to fully characterize the effects of DS-1 on ocular and nasal tissues. The body of evidence that has been established to date suggests that peptide drugs applied topically to the rat eye are flushed down the nasolacrimal duct and ultimately delivered to, and absorbed from, the ipsilateral maxillary sinus,l-* while drugs applied in nasal drops are exposed to the larger surface area of the nasal mucosa; this difference in the sites available for peptide drug absorption may explain the distinction between the amount of insulin absorbed following ocular vs nasal delivery, as shown in Figure 2. The precise mechanism of DS-1 action is not known. However, the administration of DS-1 eyedrops to a rat up to 30 min prior to the addition of insulin eyedrops caused a strong hypoglycemic response (Figure 4A). This result was consistent with the hypothesis that DS-1 caused a change in the nasal epithelium that somehow made it more permeable to insulin. Whatever the nature of this change, the effect was diminished considerably 60 min after the purified saponin derivative was applied to the eye. A similar result was reported by Illum et al.17when nosedrops containing insulin were administered 30 min after a nasal formulation containing the absorption-enhancingcationic polysaccharide chitosan. However, when the insulin nosedrops were applied 60 rnin after the chitosan, little insulin absorption could be detected. In contrast, results with DS-1 and antibiotic mixtures administered at separate times demonstrated that antibiotic levels
in blood were elevated by only 10% if applied 60 min after DS-1, compared with administration together.12 Several mechanisms of action for absorption-enhancing agents other than DS-1 have been s ~ g g e s t e d , l including ~-~~ protection of insulin from endogenous proteases, conversion of hexameric insulin to its dimeric or monomeric forms, and increased rate of delivery of insulin to its ultimate site of absorption. Our results do not rule out these possibilities, but they are consistent with a direct interaction between DS-1 and the nasal epithelium. Future studies describing the interaction between DS-1 and membrane proteases, drug compounds, and other membrane components,especiallywhen considered with respect to their duration of action following the addition of DS-1, should provide new insights as to the mechanism(s) of DS-1 action. Studies with other peptide drugs, such as glucagon, GHRH, IGF-1, and interferon, formulated with and without DS-1, should provide additional information about the practicality of using DS-1 to enhance the systemic absorption of other peptide drugs applied topically to the eyes or nose.
References and Notes 1. Hirai, S.; Ikenaga, T.; Matsuzawa, T. Diabetes 1978, 27, 296299. 2. Moses, A. C.; Gordon, G. S.; Carey, M. C.; Flier, J. S. Diabetes 1983,32, 1040-1047. 3. Chiou, G. C. Y.; Chuang, C.; Chang, M. S. Diabetes Care 1988, 11, 750-751. 4. Pillion, D. J.;Bartlett, J. D.; Meezan, E.; Yang, M.; Crain, R. J.; Grizzle, W. E. Invest. Ophthalmol. Vis. Sci. 1991, 32, 30213027. 5. Chiou, G. C. Y.; Chuang, C. Y. J. Ocul. Pharmacol. 1988,4,179186. 6. Pillion, D. J.; McCracken, D. L.; Yang, M.; Atchison, J. A. J . Ocul. Pharmacol. 1992,8, 349-358. 7. Pillion, D. J.;Atchison, J. A.; Stott, J.;McCracken, D.; Garguilo, C.; Meezan, E. J. Ocul. Pharmacol. 1994,10, 461-470. 8. Pillion, D. J.; Atchison, J. A,; Wang, R.-X.; Meezan, E. J . Pharmacol. Exp. Ther. 1994,271, 1274-1280. 9. Pillion, D. J.; Atchison, J. A.; Garguilo, C.; Wang, R.-X.; Wang, P.; Meezan, E. Endocrinol. 1994,135,2386-2391. 10. Kensil, C. R.; Soltysik, S.; Patel, U.; Marciani, D. J. In Vaccines 92; Brown, F., Chanock, R. M., Ginsberg, H. S., Lerner, R. A., Eds.; Cold Spring Harbor Lab.: Plainview, NY,1992; pp 3540. 11. Kensil, C. R.; Patel, U.; Lennick, M.; Marciani, D. J. Zmmunol. 1991,146, 431-431. 12. Recchia, J.; Lurantos, M. H. A.; Amsden, J.; Storey, J.; Kensil, C. R. Submitted for publication. 13. Higuchi, R.; Tokimittsu, Y.; Fujioka, T.; Komori, T.; Kawasaki, T.; Oakenful, D. G. Phytochem. 1987,26, 229-234. 14. Kensil, C. R.; Soltysik, S.; Marciani, D. J. U.S. Patent Number 5,273,965, 1993. 15. Kensil, C. R.; Soltysik, S.; Wheeler, D.; Wu, J.-Y. Submitted for publication. 16. Pillion, D. J.; Wang, P.; Yorks, J.; McCann, P.; Meezan, E. J . Ocul. Pharmacol. In press. 17. Illum, L.; Farraj, N. F.; Davis, S. S. Pharm. Res. 1994,11,11861189. 18. Gordon, G. S.; Moses, A. C.; Silver, R. D.; Flier, J. S.; Carey, M. C. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 7419-7423. 19. Hayakawa, E.; Yamamoto, A,; Shoji, Y.; Lee, V. H. L. Life Sci. 1989.45, 167-174. 20. Tengamnuay, P.; Mitra, A. K. Pharm. Res. 1990, 7, 370-375. 21. Carstens, S.; Danielsen, G.; Guldhammer, B.; Frederiksen, 0. Diabetes 1993,42, 1032-1040.
Acknowledgments We thank Maria H. A. Lurantos for preparation of DS-1. The University of Alabama at Birmingham is fully accredited by the American Association for Accreditation of Laboratory Animals.
JS950214S Journal of Pharmaceutical Sciences / 1279 Vol. 84, No. 11, November 1995