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Transdermal lontophoretic Peptide Delivery: In Vitro and In Vivo Studies with Luteinizing Hormone Releasing Hormone
Transdermal lontophoretic Peptide Delivery: In Vitro and In Vivo Studies with Luteinizing Hormone Releasing Hormone MARK C. HEIT*, PATRICK L. WILLIAMS*, FRIEDERIKE L. JAYES*,SHAO K. CHANG*, JIME. RIVIERE*~
AND
Received March 30,1992, from the *Cutaneous Pharmacologyand Toxicology Center, and the *Departmentof Animal Science, North Carolina State University, Raleigh, NC 27606. Accepted for publication August 17, 1992. Abstract Protein and peptide drugs are not orally active. Their large molecular size and charged character make them poor candidates for passive transdermal delivery.With an applied electromotive force,these drugs can be forced through the skin to be absorbed by the systemic circulation. The present study investigates the transdermal iontophoretic delivery of a peptide hormone in an in vitro model system, the isolated perfused porcine skin flap, as well as in vivo. It is shown that with knowledgeof the systemicdisposition of the drug, transdermal fluxes can be utilized to accurately predict in vivo serum concentrations. It is also shown that the iontophoretically delivered hormone retains both its immunologic and biologic activity.
serum versus time (serum concentration-time profile) can be accurately predicted. These predictions have been shown to be accurate in the pig3 and, more recently, in the human.4.5 The current study investigates transdermal anodal iontophoretic delivery of the reproductive peptide hormone luteinizing hormone releasing hormone (LHRH)both in the IPPSF and in vivo. With pharmacokinetic data obtained from iv bolus injections, the in vitro flap experiments are extrapolated to predict LHRH delivery in vivo in the pig.
Experimental Section Recent advances in biotechnology have made the production of clinically relevant peptide and protein drugs economically feasible.1 Due to proteolytic enzymes in the gastrointestinal tract and extensive first-pass hepatic metabolism, these drugs are not orally active. Although usually potent at very low concentrations, peptides generally have very short half-lives, necessitating frequent dosing. The achievement of the reliable transdermal delivery of peptides would offer several clinical advantages over other more conventional routes of delivery. Because there are no proteolytic enzymes in the skin, presystemic metabolism should be minimal.2 Drug absorbed by cutaneous circulation does not enter the portal circulation, thereby avoiding hepatic first-pass effects. Continual delivery of drug could be achieved by continuous transdermal application, thereby circumventing the problem of rapid drug elimination. Transdermal delivery of drugs is usually painless, hence patient compliance should be excellent. Because peptides and proteins have large molecular weights and radii and are often charged, their passive transdermal delivery is very difficult. Iontophoresis is the process whereby charged molecules are made to move into and through tissues by a n electromotive force. Positive ions are forced away from a positive electrode, whereas negative ions move from the cathode. Iontophoresis provides a potential method for the transdermal delivery of large, charged molecules that cannot be delivered passively. Transdermal systemic delivery of drugs can be simplistically broken down into three phases: (1)delivery from the drug delivery system into the skin, (2) movement from the skin into the vasculature, and (3) systemic disposition and pharmacodynamic effects. The isolated perfused porcine skin flap (IPPSF), in contrast to most in vitro percutaneous absorbtion systems, allows one to investigate the first two phases, resultingin a qualitative and quantitativeestimation of how a drug enters the systemic circulation. With this flap output profile as systemic input [much as one would use an intravenous (iv) infusion pump], and taking into account systemic pharmacokinetics that quantitates distribution, metabolism, and elimination, the profile of concentration in 240 I
Journal of Pharmaceutical Sciences Vol. 82,No. 3,March 1993
IPPSF Iontophoresis-The ability to place drugs on the surface of viable skin with a functional intact microcirculation has made the IPPSF a suitable model for transdermal iontophoretic delivery studies. The model has been described in detail elsewhere.3fj Briefly, two axial pattern tubed skin grafts with the supplying and draining vasculature were harvested from a pig's abdomen and maintained in temperature and humidity regulated chambers. A bicarbonatebuffered albumin containing Krebs-Ringer perfusate was oxygenated and peristaltically pumped through the IPPSF. Viability was monitored throughout the experiment by measurement of glucose utilization and lactate production. The active and indifferent electrodes were circular (1cm'), porex silver screen sandwiches.7 A 1-mg/mL LHRH solution in 154 mM NaCl and 10 mM 2-(N-morpho1ino)ethanesulfonicacid buffer (pH 6.0) was placed in the active positive electrode. The indifferent electrode contained a 50% saturated NaCl/water solution (15.9 g/lOO ml). Isoelectric focusing and capillary zone electrophoresis studies performed in our laboratory and that of Bedon Dickinson Research Center suggest LHRH is highly positively charged and mobile a t physiological pH. A portable power supply (WPI stimulus isolator model A360) was used to deliver a constant current of 0.2 mA. After a 1-h acclimation period, each of the harvested skin flaps received 3 h of current followed by a 2-h passive period during which there was no current. Venous perfusate samples were collected half-hourly and immediately placed at -20°C until assayed. Voltage, media flow, and perfusion pressure were also recorded. In Vivo Iontophoresis-In vivo LHRH iontophoresis was performed on 6-8-week-old female Yorkshire pigs. The pigs were induced with ketaminelxylazine and maintained under anesthesia for the entire experiment with ketaminelqlazine and halothane. The active and indifferent electrodes were rectangular (10 cm'), porex silver screen sandwiches and were placed on the ventral abdomen in close proximity to the site of IPPSF removal. Drug solutions used were identical to those used in the flap experiments. Strong adhesive bandages were applied to insure good skin contact throughout the experiment. A constant current of 2.0 mA (current density = 0.2 mA/cm') was applied with the same power supply used in the IPPSF experiments. After a 1-h predosing period, the electrode patches were applied with no current to assess in vivo passive delivery. After the passive period, the current was turned on for 3 h followed by a 2-h passive period to assess drug elimination. Venous serum and plasma samples were taken every half hour and frozen once separated from the red blood cells. In addition, a 15-min sample was taken after the current was turned on or off. 0022-3549/93/0300-0240$02.50/0 0 1993,American Pharmaceutical Association
Intravenous Pharmacokinetics-Six-to-eight-week-oldfemale Yorkshire pigs were injected with an BO-pg/rnL solution of LHRH at a dose of 2.0 p&g. The pigs were induced with ketamineIxylazine and maintained under anesthesia for the entire experiment with ketaminehylazine and halothane. Injections were made through a different catheter than blood sampling as LHRH may bind to the catheter. Serum and plasma venous samples were taken every 10 min for at least 30 rnin prior to injection and 1, 2,4, 7, 10, 15, 20,30,40,50, 60, 75, 90, 105, and 120 rnin after injection. LHRH Assay-LHRH was quantified in IPPSF perfusate and in serum samples by a single antibody radioimmunoassay. Synthetic LHRH (acetate salt; Sigma) was used as standard and iodinated tracer. Radioiodination* was performed with iodogen,g and monoiodinated hormone was separated from diiodinated, unlabeled hormone and free iodine by anion exchange chromatography.10 Polyclonal antibody (Esp 297)" at a final dilution of 1:370000 per tube was incubated with diluted sample and tracer (-20 000 cpm) for 24 h at 4 "C. Bound hormone was precipitated with 2 mL of ice-cold 100% ethanol (IPPSF perfusate samples) or 1 mL of 15% polyethylene glycol (serum samples), followed by 30 rnin of centrifugation at 1800 x g. For the assay of blood samples, 200 pL of porcine serum were added to the standards to reduce nonspecific interference. This serum did not contain detectable levels of LHRH nor did its addition displace the standard curve. Recovery of LHRH added to serum (0.6-160 pg/tube) was 98 f 5%,and binding was parallel to the standard curve. All samples were run in triplicate. Assay sensitivity ranged between 0.6 and 1 pg/tube at 90%binding as defined as sample binding divided by maximum binding (BIB,). Intra- and interassay coefficients of variation were, respectively, 8.2 and 16.5%at 48 pg/tube and 10.9 and 8.3%at 10.6 pg/tube for perfusate samples and 17.5 and 19.4%at 44 pg/tube and 14.8 and 14.2%at 8.9 pg/tube for serum samples. Porcine Serum Follicle Stimulating Hormone (pFSH) and
Luteinizing Hormone (pLH) Assay-Concentrations of pFSH11J2 and pLH13 were determined by a double antibody, 6%polyethylene glycol separation radioimmunoassay. Anti-pFSH (USDA-398-04P) and [12SIlpFSH(USDA-pFSH-1-1)were used in the FSH assay. FSH concentrations >20 ng/mL and LH concentrations >0.2 ng/mL were detectable. Intraassay variation ranged between 9.1 and 11.1%and between 8.1 and 16.0%for the FSH and LH assays, respectively. All samples were run in the same assay. FSH and LH are both target hormones for LHRH in vivo. Data Analysis-Intravenous pharmacokinetic data were fitted by the method of residuals with an internal SAS program (Statistical Analysis System, Cary, NC). Predictions of in vivo serum concentrations due to iontophoresis were made with the IPPSF output flux as input to a two-compartment mammilary model as described previously.5
Results M e a n LHRH transdermal iontophoretic flux in 21 skin flaps is illustrated in F i g u r e 1. Previous studies indicated that LHRH cannot be delivered passively. The t o t a l amount of LHRH delivered during t h e 5 h was 959 444 ng. LHRH
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Figure 2-LHRH concentrations in serum after iv injection into five anesthetized pigs. The raw data for each time point is illustrated individually (dots) and as a mean f one standard error. The dashed line represents the serum concentration-time profile as predicted by the modeled pharmacokinetic data.
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Figure 1-Mean LHRH fluxes f one standard error in the venous drainage from 21 IPPSFs. The flux has been normalized to electrode area. A 0.2-mNcm2 current density was applied for 0 3 h. Table I-In Vivo Pharmacokinetic Parameters after intravenous Administration of LHRH" to Five Weaniing Pigs Parameter k12 k21
a
Dose, 2.0 pg/kg. Expressed as mean
Resultb
0.095 f 0.039 min-' 0.052 f 0.003 min-' 0.181 f 0.068 min-' 60.7 f 29.9 mUkg 9.1 f 1.6 mUkg/min 4.55 f 2.0 min 2.69 f 1.20 min 22.2 f 2.15 rnin 39.1 2 20.0 ng/mL 0.297 f 0.101 min-' 3.15 2 0.50 ng/mL 0.031 f 0.003 min-l
* standard deviation.
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Figure 34oncentrations of LHRH (pg/mL, solid line), LH (ng1100 mL, dashed line), and FSH (ng/mL, dotted line) in serum following in vivo transdermal iontophoresis of LHRH in four pigs. A current of 0.2 mNcm2 was applied for 2-5 h. Mean LHRH concentration f one standard error are representedby the solid line and broken lines represent either LH or FSH concentrations. To insure biological activity, LH was determined in two pigs and FSH was determined in the remaining two pigs. Journal of Pharmaceutical Sciences / 241 Vol. 82, No. 3, March 7993
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Figure &Observed (-) versus IPPSF-predicted (- - - -) LHRH serum concentration-time profiles in vivo in pigs. In all plots, the flux data from 21 IPPSFs are used as input into a two-compartment mammillary model. The pharmacokinetic parameters were determined from five iv experiments. The observed in vivo flux was determined in four anesthetized pigs. Figure 4A depicts the predicted flux if the mean IPPSF flux and the mean iv pharmacokinetic parameters are used; that is, no variability is introduced into the prediction. Figures 48, 4C,and 4D include IPPSF variation,
pharmacokineticvariation, and both, respectively (solid lines represent observed f standard error and dashed lines represent predicted 2 standard error).
concentrations increase throughout the active period and quickly decrease as soon as the current is removed. Table I reports pharmacokinetic parameters obtained after iv LHRH administration. The data were best fit by a twocompartment open model based on sequential F tests. Figure 2 illustrates the serum concentration-time profile for LHRH after iv injection into five pigs. Serum LHRH, LH, and FSH concentrations following in vivo iontophoresis are shown in Figure 3. Two hours of passive delivery caused no LHRH delivery or increases in LH or FSH. LHRH concentrations rise within 0.5 h of current application and remain elevated for the entire active period. LH and FSH concentrations increase after LHRH concentrations and begin to decline when LHRH concentrations decrease. The predicted and observed serum concentrations of LHRH following anodal iontophoresis in the pig are shown in Figure 4. Figure 4A depicts the mean observed in vivo LHRH delivery and the mean predicted in vivo delivery based on the mean flux of 21 IPPSFEand the mean iv parameters from five pigs. Figure 4B demonstrates the effect of flap variability on the predictive envelope. The observed in vivo envelope is pictured with a prediction of the in vivo delivery using all 21 skin flaps with the means of the iv pharmacokinetic data. Figure 4C illustrates the effect of pharmacokinetic variation on predicting in vivo response. The mean skin flap flux is compiled with iv data variability. The wide predictive envelope demonstrated in Figure 4D includes variability of the iv pharmacokinetic parameters as well as variability associated 242 1 Journal of Pharmaceutical Sciences Vol. 82, No. 3, March 1993
with the input flux profile. In all figures, the predicted concentrations approximate the observed data, In Figures 4C and 4D, the predicted envelope fully encloses the observed data. In one instance, the same pig (no. 4814)was used for IPPSF, in vivo, and iv determinations all within 2 weeks. Predictions made from the two flaps removed from that pig, with its own iv pharmacokinetic data, are shown together with the observed in vivo iontophoresis in pig 4814 (Figure 5). For skin flap 1, the prediction is excellent; however, the second flap, which does not show a typical flux profile because the flux peaks prior to 3 h (see Figure 11, is a poor predictor of the in vivo iontophoretic flux.
Discussion Iontophoresis allows the transdermal delivery of compounds that are too large, charged, or hydrophilic for passive transdermal delivery. LHRH, a decapeptide of molecular weight 1182 and -+2 charge at pH 6.0, was delivered through pig skin by anodal iontophoresis. These results are in agreement with those of previous studies performed in vitro in hairless mouse skin.14 Iontophoretic delivery of LHRH in humans has been achieved with less than maximized electrode ~onditions.15~16 These studies, however, indirectly demonstrate LHRH delivery as increasing LH concentrations. In the present study, increases in FSH and LH concentrations occur concurrently with the iontophoresis and increasing LHRH concentrations. It is concluded that the iontophoreti-
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Figure Wbserved (-) versus IPPSF-predicted (. .* and - - -) LHRH serum concentration-time profiles in vivo using pig 4814 for
determining IPPSF flux profiles, iv pharmacokinetics, and in vivo iontophoresis. The same experimental conditions are utilized as for the experiments demonstrated in Figure 4. cally delivered LHRH is both immunologically and biologically active. In addition, the present study demonstrates for the first time the ability of the IPPSF to predict in vivo serum concentrations of a n iontophoretically delivered peptide. By separating out each contribution to variability, one can better assess the importance that both a n individual animal’s cutaneous absorption profile and systemic pharmacokinetics will have on iontophoretic delivery. The wide predictive envelope demonstrated in Figure 4D includes variability of the systemic pharmacokinetic parameters as well as variability associated with the transdermal input flux profile. This is a worst case scenario, however, because the lower bound predicts the in vivo concentrations if the lowest flux flap occurred in the pig with the highest volume of distribution and most rapid clearance. There is no current evidence to suggest any correlation of these two factors. By noting the relatively small variability seen in the observed in vivo transdermal concentrations compared with the predicted envelope, one might speculate there may be an inverse relationship between the iontophoretic flux and the volume of distribution, The problem of in vivo flux predictions based on input of one individual and iv data of another is eliminated in the experiment utilizing pig 4814;in this experiment, there is excellent agreement between the IPPSF-predicted and the observed LHRH flux. However, it is significant to note that without knowledge of the typical flux profile for these iontophoretic conditions (Figure 11, a relatively poor prediction could have been made. It should also be noted that the IPPSF predictions seem to be more accurate when the iv variability is utilized (Figure 4C)compared with the IPPSF variability (Figure 4B), illustrating the overriding importance of systemic disposition. The physicochemical basis of this needs further study. Passive percutaneous absorbtion depends on the interaction of physicochemical properties of the drug and vehicle, as well as physicochemical properties of the skin. Lipid composition, intercellular spacing, appendageal density, and thick-
ness of individual skin layers will affect the ability of a drug to enter and cross the skin (phase 1).These same parameters often vary greatly between species, thus making extrapolation between species difficult. With iontophoretic delivery, molecules that otherwise could not penetrate the skin barrier are fascilitated through with a n electromotive force. Due to the electrophysics of the electrode design and the use of constant-current delivery devices, this impenetrability to charged compounds being forced into the skin may be more uniform than the selective permeability of the skin to passively delivered compounds. Thus, phase 1 should be less variable for iontophoretic delivery than for passive delivery. If future studies show either that skin composition has little effect on drug transport within the skin (phase 2) or that certain skin factors have predictable effects on delivery, then the IPPSF with its intact macro- and microcirculation could be used to represent the “generalized skin barrier” to iontophoretic delivery. The IPPSF output profile of a drug together with pharmacokinetic parameters obtained for the species in question would be all that was needed to allow fine tuning of serum concentration-time profiles to maximize efficacy while minimizing the side effects of the drug.
References and Notes 1. Chien, Y. W.; Siddiqui, Y. S.; Shi, W. M.; Liu, J. C. Ann. N.Y. &ad. Sci. 1987,507,32-50. 2. Pannatier, P. J.;Testa, B.; Etter, J. C. DrugMetab. Rev. 1978,8, 31 9 ---.
3. Riviere, J. E.; Sage, B.; Williams, P. L. J. Pharm. Sci. 1991,80, 61 - - 5-620. - - -..
4. Riviere, J. E.; Monteiro-Riviere, N. A. CRC Crit. Rev. Toxicol. 1991,21(5), 329-344. 5. Riviere, J. E.; Williams, P. L.; Hillman, R. S.; Mishky, L. M. J. Pharm. Sci., in press. 6. Riviere, J. E.; Bowman, K. F.; Monteiro-Riviere, N. A.; DiU, L. P.; Carver, M. P. Fund. Appl. Toxicol. 1986,7,444-453. 7. Monteiro-Riviere, N. A. Fund. Appl. Toxicol. 1990,15,174-185. 8. Laws, S.C. Ph.D Thesis, North Carolina State University, Raleigh, NC, 1988;p 30ff. 9. Fraker, P. J.; Speck, J. C. Biochem. Biophys. Res. Comm. 1978, 80(4), 849-857. 10. Nett, T. M.; Adams, T. E. Endocrinology 1977,101,1135-1144. 11. Espenshade, K. L.;Britt, J. H. Biol. Reprod. 1985,33,569-577. 12. Guthrie. H. D.: Bolt. D. J. J. Anim. Sci. 1983.57f4). 993-1000. 13. Armstrong, J. D.; I&-aeling, R. R.; Britt, J. H. J. Reprod. Fert. 1988.83.301-308. 14. Miller, L. L.; Kolaskie, C. J.; Smith, G. A.; Rivier, J. J. Pharm. Sci. 1990,79(6), 490493. 15. Meyer, R. B.; Kreis, W.; Eschbach, J.; O’Mara, V.; Rosen, S.; Sibalis, D. Clin. Pharmacol. Ther. 1988,44(6), 607412. 16. Meyer, R. B.; Kreis, W.; Eschbach, J.; O’Mara, V.; Rosen, S.; Sibalis, D. Clin. Pharmacol. Ther. 1990,48,340-345.
Acknowledgments Financial support was provided by the Becton Dickinson Research Center, Research Triangle Park, NC. We thank Dr. Randy Bock and Burt Sage of Becton Dickinson Research Center. We also thank Dr. Jack Britt for his e ertise and use of facilities for the hormone assays. We acknowlexe Dr. G. D. Niswender for supplying antisera to luteinizing hormone, Dr. L. E. Reichert, Jr. for roviding the urified porcine LH (LER 786-3),and Dr. D. J. Bolt, UiDA Hormone Frogram for donating porcine FSH antisera and purified orcine FSH. Lastly, we thank the technical staff at the Cutaneous PIarmacolo and Toxicology Center at North Carolina State University. This wo% was completed in partial fulfillment of a Ph.D. at North Carolina State University (MCH).
Journal of Pharmaceutical Sciences I 243 Vol. 82, No. 3, March 1993
AND
Received March 30,1992, from the *Cutaneous Pharmacologyand Toxicology Center, and the *Departmentof Animal Science, North Carolina State University, Raleigh, NC 27606. Accepted for publication August 17, 1992. Abstract Protein and peptide drugs are not orally active. Their large molecular size and charged character make them poor candidates for passive transdermal delivery.With an applied electromotive force,these drugs can be forced through the skin to be absorbed by the systemic circulation. The present study investigates the transdermal iontophoretic delivery of a peptide hormone in an in vitro model system, the isolated perfused porcine skin flap, as well as in vivo. It is shown that with knowledgeof the systemicdisposition of the drug, transdermal fluxes can be utilized to accurately predict in vivo serum concentrations. It is also shown that the iontophoretically delivered hormone retains both its immunologic and biologic activity.
serum versus time (serum concentration-time profile) can be accurately predicted. These predictions have been shown to be accurate in the pig3 and, more recently, in the human.4.5 The current study investigates transdermal anodal iontophoretic delivery of the reproductive peptide hormone luteinizing hormone releasing hormone (LHRH)both in the IPPSF and in vivo. With pharmacokinetic data obtained from iv bolus injections, the in vitro flap experiments are extrapolated to predict LHRH delivery in vivo in the pig.
Experimental Section Recent advances in biotechnology have made the production of clinically relevant peptide and protein drugs economically feasible.1 Due to proteolytic enzymes in the gastrointestinal tract and extensive first-pass hepatic metabolism, these drugs are not orally active. Although usually potent at very low concentrations, peptides generally have very short half-lives, necessitating frequent dosing. The achievement of the reliable transdermal delivery of peptides would offer several clinical advantages over other more conventional routes of delivery. Because there are no proteolytic enzymes in the skin, presystemic metabolism should be minimal.2 Drug absorbed by cutaneous circulation does not enter the portal circulation, thereby avoiding hepatic first-pass effects. Continual delivery of drug could be achieved by continuous transdermal application, thereby circumventing the problem of rapid drug elimination. Transdermal delivery of drugs is usually painless, hence patient compliance should be excellent. Because peptides and proteins have large molecular weights and radii and are often charged, their passive transdermal delivery is very difficult. Iontophoresis is the process whereby charged molecules are made to move into and through tissues by a n electromotive force. Positive ions are forced away from a positive electrode, whereas negative ions move from the cathode. Iontophoresis provides a potential method for the transdermal delivery of large, charged molecules that cannot be delivered passively. Transdermal systemic delivery of drugs can be simplistically broken down into three phases: (1)delivery from the drug delivery system into the skin, (2) movement from the skin into the vasculature, and (3) systemic disposition and pharmacodynamic effects. The isolated perfused porcine skin flap (IPPSF), in contrast to most in vitro percutaneous absorbtion systems, allows one to investigate the first two phases, resultingin a qualitative and quantitativeestimation of how a drug enters the systemic circulation. With this flap output profile as systemic input [much as one would use an intravenous (iv) infusion pump], and taking into account systemic pharmacokinetics that quantitates distribution, metabolism, and elimination, the profile of concentration in 240 I
Journal of Pharmaceutical Sciences Vol. 82,No. 3,March 1993
IPPSF Iontophoresis-The ability to place drugs on the surface of viable skin with a functional intact microcirculation has made the IPPSF a suitable model for transdermal iontophoretic delivery studies. The model has been described in detail elsewhere.3fj Briefly, two axial pattern tubed skin grafts with the supplying and draining vasculature were harvested from a pig's abdomen and maintained in temperature and humidity regulated chambers. A bicarbonatebuffered albumin containing Krebs-Ringer perfusate was oxygenated and peristaltically pumped through the IPPSF. Viability was monitored throughout the experiment by measurement of glucose utilization and lactate production. The active and indifferent electrodes were circular (1cm'), porex silver screen sandwiches.7 A 1-mg/mL LHRH solution in 154 mM NaCl and 10 mM 2-(N-morpho1ino)ethanesulfonicacid buffer (pH 6.0) was placed in the active positive electrode. The indifferent electrode contained a 50% saturated NaCl/water solution (15.9 g/lOO ml). Isoelectric focusing and capillary zone electrophoresis studies performed in our laboratory and that of Bedon Dickinson Research Center suggest LHRH is highly positively charged and mobile a t physiological pH. A portable power supply (WPI stimulus isolator model A360) was used to deliver a constant current of 0.2 mA. After a 1-h acclimation period, each of the harvested skin flaps received 3 h of current followed by a 2-h passive period during which there was no current. Venous perfusate samples were collected half-hourly and immediately placed at -20°C until assayed. Voltage, media flow, and perfusion pressure were also recorded. In Vivo Iontophoresis-In vivo LHRH iontophoresis was performed on 6-8-week-old female Yorkshire pigs. The pigs were induced with ketaminelxylazine and maintained under anesthesia for the entire experiment with ketaminelqlazine and halothane. The active and indifferent electrodes were rectangular (10 cm'), porex silver screen sandwiches and were placed on the ventral abdomen in close proximity to the site of IPPSF removal. Drug solutions used were identical to those used in the flap experiments. Strong adhesive bandages were applied to insure good skin contact throughout the experiment. A constant current of 2.0 mA (current density = 0.2 mA/cm') was applied with the same power supply used in the IPPSF experiments. After a 1-h predosing period, the electrode patches were applied with no current to assess in vivo passive delivery. After the passive period, the current was turned on for 3 h followed by a 2-h passive period to assess drug elimination. Venous serum and plasma samples were taken every half hour and frozen once separated from the red blood cells. In addition, a 15-min sample was taken after the current was turned on or off. 0022-3549/93/0300-0240$02.50/0 0 1993,American Pharmaceutical Association
Intravenous Pharmacokinetics-Six-to-eight-week-oldfemale Yorkshire pigs were injected with an BO-pg/rnL solution of LHRH at a dose of 2.0 p&g. The pigs were induced with ketamineIxylazine and maintained under anesthesia for the entire experiment with ketaminehylazine and halothane. Injections were made through a different catheter than blood sampling as LHRH may bind to the catheter. Serum and plasma venous samples were taken every 10 min for at least 30 rnin prior to injection and 1, 2,4, 7, 10, 15, 20,30,40,50, 60, 75, 90, 105, and 120 rnin after injection. LHRH Assay-LHRH was quantified in IPPSF perfusate and in serum samples by a single antibody radioimmunoassay. Synthetic LHRH (acetate salt; Sigma) was used as standard and iodinated tracer. Radioiodination* was performed with iodogen,g and monoiodinated hormone was separated from diiodinated, unlabeled hormone and free iodine by anion exchange chromatography.10 Polyclonal antibody (Esp 297)" at a final dilution of 1:370000 per tube was incubated with diluted sample and tracer (-20 000 cpm) for 24 h at 4 "C. Bound hormone was precipitated with 2 mL of ice-cold 100% ethanol (IPPSF perfusate samples) or 1 mL of 15% polyethylene glycol (serum samples), followed by 30 rnin of centrifugation at 1800 x g. For the assay of blood samples, 200 pL of porcine serum were added to the standards to reduce nonspecific interference. This serum did not contain detectable levels of LHRH nor did its addition displace the standard curve. Recovery of LHRH added to serum (0.6-160 pg/tube) was 98 f 5%,and binding was parallel to the standard curve. All samples were run in triplicate. Assay sensitivity ranged between 0.6 and 1 pg/tube at 90%binding as defined as sample binding divided by maximum binding (BIB,). Intra- and interassay coefficients of variation were, respectively, 8.2 and 16.5%at 48 pg/tube and 10.9 and 8.3%at 10.6 pg/tube for perfusate samples and 17.5 and 19.4%at 44 pg/tube and 14.8 and 14.2%at 8.9 pg/tube for serum samples. Porcine Serum Follicle Stimulating Hormone (pFSH) and
Luteinizing Hormone (pLH) Assay-Concentrations of pFSH11J2 and pLH13 were determined by a double antibody, 6%polyethylene glycol separation radioimmunoassay. Anti-pFSH (USDA-398-04P) and [12SIlpFSH(USDA-pFSH-1-1)were used in the FSH assay. FSH concentrations >20 ng/mL and LH concentrations >0.2 ng/mL were detectable. Intraassay variation ranged between 9.1 and 11.1%and between 8.1 and 16.0%for the FSH and LH assays, respectively. All samples were run in the same assay. FSH and LH are both target hormones for LHRH in vivo. Data Analysis-Intravenous pharmacokinetic data were fitted by the method of residuals with an internal SAS program (Statistical Analysis System, Cary, NC). Predictions of in vivo serum concentrations due to iontophoresis were made with the IPPSF output flux as input to a two-compartment mammilary model as described previously.5
Results M e a n LHRH transdermal iontophoretic flux in 21 skin flaps is illustrated in F i g u r e 1. Previous studies indicated that LHRH cannot be delivered passively. The t o t a l amount of LHRH delivered during t h e 5 h was 959 444 ng. LHRH
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Figure 2-LHRH concentrations in serum after iv injection into five anesthetized pigs. The raw data for each time point is illustrated individually (dots) and as a mean f one standard error. The dashed line represents the serum concentration-time profile as predicted by the modeled pharmacokinetic data.
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Figure 1-Mean LHRH fluxes f one standard error in the venous drainage from 21 IPPSFs. The flux has been normalized to electrode area. A 0.2-mNcm2 current density was applied for 0 3 h. Table I-In Vivo Pharmacokinetic Parameters after intravenous Administration of LHRH" to Five Weaniing Pigs Parameter k12 k21
a
Dose, 2.0 pg/kg. Expressed as mean
Resultb
0.095 f 0.039 min-' 0.052 f 0.003 min-' 0.181 f 0.068 min-' 60.7 f 29.9 mUkg 9.1 f 1.6 mUkg/min 4.55 f 2.0 min 2.69 f 1.20 min 22.2 f 2.15 rnin 39.1 2 20.0 ng/mL 0.297 f 0.101 min-' 3.15 2 0.50 ng/mL 0.031 f 0.003 min-l
* standard deviation.
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Figure 34oncentrations of LHRH (pg/mL, solid line), LH (ng1100 mL, dashed line), and FSH (ng/mL, dotted line) in serum following in vivo transdermal iontophoresis of LHRH in four pigs. A current of 0.2 mNcm2 was applied for 2-5 h. Mean LHRH concentration f one standard error are representedby the solid line and broken lines represent either LH or FSH concentrations. To insure biological activity, LH was determined in two pigs and FSH was determined in the remaining two pigs. Journal of Pharmaceutical Sciences / 241 Vol. 82, No. 3, March 7993
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Figure &Observed (-) versus IPPSF-predicted (- - - -) LHRH serum concentration-time profiles in vivo in pigs. In all plots, the flux data from 21 IPPSFs are used as input into a two-compartment mammillary model. The pharmacokinetic parameters were determined from five iv experiments. The observed in vivo flux was determined in four anesthetized pigs. Figure 4A depicts the predicted flux if the mean IPPSF flux and the mean iv pharmacokinetic parameters are used; that is, no variability is introduced into the prediction. Figures 48, 4C,and 4D include IPPSF variation,
pharmacokineticvariation, and both, respectively (solid lines represent observed f standard error and dashed lines represent predicted 2 standard error).
concentrations increase throughout the active period and quickly decrease as soon as the current is removed. Table I reports pharmacokinetic parameters obtained after iv LHRH administration. The data were best fit by a twocompartment open model based on sequential F tests. Figure 2 illustrates the serum concentration-time profile for LHRH after iv injection into five pigs. Serum LHRH, LH, and FSH concentrations following in vivo iontophoresis are shown in Figure 3. Two hours of passive delivery caused no LHRH delivery or increases in LH or FSH. LHRH concentrations rise within 0.5 h of current application and remain elevated for the entire active period. LH and FSH concentrations increase after LHRH concentrations and begin to decline when LHRH concentrations decrease. The predicted and observed serum concentrations of LHRH following anodal iontophoresis in the pig are shown in Figure 4. Figure 4A depicts the mean observed in vivo LHRH delivery and the mean predicted in vivo delivery based on the mean flux of 21 IPPSFEand the mean iv parameters from five pigs. Figure 4B demonstrates the effect of flap variability on the predictive envelope. The observed in vivo envelope is pictured with a prediction of the in vivo delivery using all 21 skin flaps with the means of the iv pharmacokinetic data. Figure 4C illustrates the effect of pharmacokinetic variation on predicting in vivo response. The mean skin flap flux is compiled with iv data variability. The wide predictive envelope demonstrated in Figure 4D includes variability of the iv pharmacokinetic parameters as well as variability associated 242 1 Journal of Pharmaceutical Sciences Vol. 82, No. 3, March 1993
with the input flux profile. In all figures, the predicted concentrations approximate the observed data, In Figures 4C and 4D, the predicted envelope fully encloses the observed data. In one instance, the same pig (no. 4814)was used for IPPSF, in vivo, and iv determinations all within 2 weeks. Predictions made from the two flaps removed from that pig, with its own iv pharmacokinetic data, are shown together with the observed in vivo iontophoresis in pig 4814 (Figure 5). For skin flap 1, the prediction is excellent; however, the second flap, which does not show a typical flux profile because the flux peaks prior to 3 h (see Figure 11, is a poor predictor of the in vivo iontophoretic flux.
Discussion Iontophoresis allows the transdermal delivery of compounds that are too large, charged, or hydrophilic for passive transdermal delivery. LHRH, a decapeptide of molecular weight 1182 and -+2 charge at pH 6.0, was delivered through pig skin by anodal iontophoresis. These results are in agreement with those of previous studies performed in vitro in hairless mouse skin.14 Iontophoretic delivery of LHRH in humans has been achieved with less than maximized electrode ~onditions.15~16 These studies, however, indirectly demonstrate LHRH delivery as increasing LH concentrations. In the present study, increases in FSH and LH concentrations occur concurrently with the iontophoresis and increasing LHRH concentrations. It is concluded that the iontophoreti-
0.701
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Figure Wbserved (-) versus IPPSF-predicted (. .* and - - -) LHRH serum concentration-time profiles in vivo using pig 4814 for
determining IPPSF flux profiles, iv pharmacokinetics, and in vivo iontophoresis. The same experimental conditions are utilized as for the experiments demonstrated in Figure 4. cally delivered LHRH is both immunologically and biologically active. In addition, the present study demonstrates for the first time the ability of the IPPSF to predict in vivo serum concentrations of a n iontophoretically delivered peptide. By separating out each contribution to variability, one can better assess the importance that both a n individual animal’s cutaneous absorption profile and systemic pharmacokinetics will have on iontophoretic delivery. The wide predictive envelope demonstrated in Figure 4D includes variability of the systemic pharmacokinetic parameters as well as variability associated with the transdermal input flux profile. This is a worst case scenario, however, because the lower bound predicts the in vivo concentrations if the lowest flux flap occurred in the pig with the highest volume of distribution and most rapid clearance. There is no current evidence to suggest any correlation of these two factors. By noting the relatively small variability seen in the observed in vivo transdermal concentrations compared with the predicted envelope, one might speculate there may be an inverse relationship between the iontophoretic flux and the volume of distribution, The problem of in vivo flux predictions based on input of one individual and iv data of another is eliminated in the experiment utilizing pig 4814;in this experiment, there is excellent agreement between the IPPSF-predicted and the observed LHRH flux. However, it is significant to note that without knowledge of the typical flux profile for these iontophoretic conditions (Figure 11, a relatively poor prediction could have been made. It should also be noted that the IPPSF predictions seem to be more accurate when the iv variability is utilized (Figure 4C)compared with the IPPSF variability (Figure 4B), illustrating the overriding importance of systemic disposition. The physicochemical basis of this needs further study. Passive percutaneous absorbtion depends on the interaction of physicochemical properties of the drug and vehicle, as well as physicochemical properties of the skin. Lipid composition, intercellular spacing, appendageal density, and thick-
ness of individual skin layers will affect the ability of a drug to enter and cross the skin (phase 1).These same parameters often vary greatly between species, thus making extrapolation between species difficult. With iontophoretic delivery, molecules that otherwise could not penetrate the skin barrier are fascilitated through with a n electromotive force. Due to the electrophysics of the electrode design and the use of constant-current delivery devices, this impenetrability to charged compounds being forced into the skin may be more uniform than the selective permeability of the skin to passively delivered compounds. Thus, phase 1 should be less variable for iontophoretic delivery than for passive delivery. If future studies show either that skin composition has little effect on drug transport within the skin (phase 2) or that certain skin factors have predictable effects on delivery, then the IPPSF with its intact macro- and microcirculation could be used to represent the “generalized skin barrier” to iontophoretic delivery. The IPPSF output profile of a drug together with pharmacokinetic parameters obtained for the species in question would be all that was needed to allow fine tuning of serum concentration-time profiles to maximize efficacy while minimizing the side effects of the drug.
References and Notes 1. Chien, Y. W.; Siddiqui, Y. S.; Shi, W. M.; Liu, J. C. Ann. N.Y. &ad. Sci. 1987,507,32-50. 2. Pannatier, P. J.;Testa, B.; Etter, J. C. DrugMetab. Rev. 1978,8, 31 9 ---.
3. Riviere, J. E.; Sage, B.; Williams, P. L. J. Pharm. Sci. 1991,80, 61 - - 5-620. - - -..
4. Riviere, J. E.; Monteiro-Riviere, N. A. CRC Crit. Rev. Toxicol. 1991,21(5), 329-344. 5. Riviere, J. E.; Williams, P. L.; Hillman, R. S.; Mishky, L. M. J. Pharm. Sci., in press. 6. Riviere, J. E.; Bowman, K. F.; Monteiro-Riviere, N. A.; DiU, L. P.; Carver, M. P. Fund. Appl. Toxicol. 1986,7,444-453. 7. Monteiro-Riviere, N. A. Fund. Appl. Toxicol. 1990,15,174-185. 8. Laws, S.C. Ph.D Thesis, North Carolina State University, Raleigh, NC, 1988;p 30ff. 9. Fraker, P. J.; Speck, J. C. Biochem. Biophys. Res. Comm. 1978, 80(4), 849-857. 10. Nett, T. M.; Adams, T. E. Endocrinology 1977,101,1135-1144. 11. Espenshade, K. L.;Britt, J. H. Biol. Reprod. 1985,33,569-577. 12. Guthrie. H. D.: Bolt. D. J. J. Anim. Sci. 1983.57f4). 993-1000. 13. Armstrong, J. D.; I&-aeling, R. R.; Britt, J. H. J. Reprod. Fert. 1988.83.301-308. 14. Miller, L. L.; Kolaskie, C. J.; Smith, G. A.; Rivier, J. J. Pharm. Sci. 1990,79(6), 490493. 15. Meyer, R. B.; Kreis, W.; Eschbach, J.; O’Mara, V.; Rosen, S.; Sibalis, D. Clin. Pharmacol. Ther. 1988,44(6), 607412. 16. Meyer, R. B.; Kreis, W.; Eschbach, J.; O’Mara, V.; Rosen, S.; Sibalis, D. Clin. Pharmacol. Ther. 1990,48,340-345.
Acknowledgments Financial support was provided by the Becton Dickinson Research Center, Research Triangle Park, NC. We thank Dr. Randy Bock and Burt Sage of Becton Dickinson Research Center. We also thank Dr. Jack Britt for his e ertise and use of facilities for the hormone assays. We acknowlexe Dr. G. D. Niswender for supplying antisera to luteinizing hormone, Dr. L. E. Reichert, Jr. for roviding the urified porcine LH (LER 786-3),and Dr. D. J. Bolt, UiDA Hormone Frogram for donating porcine FSH antisera and purified orcine FSH. Lastly, we thank the technical staff at the Cutaneous PIarmacolo and Toxicology Center at North Carolina State University. This wo% was completed in partial fulfillment of a Ph.D. at North Carolina State University (MCH).
Journal of Pharmaceutical Sciences I 243 Vol. 82, No. 3, March 1993