Figures 4A and 4B compare the concentra-tion profiles of TBF across the SC of five volunteers following delivery of the drug from the control formulation and from the vehicle containing OA, respectively. The TBF concentrations are absolute having been de-termined by HPLC analysis of the extracted tape strips removed after a 2-hour application of the drug. It is first noted that the inter-subject variability in these in vivo data is quite respectable. Second, on gross examination, it
Figure 3: The concentration profile (Cx) of TBF across SC at non-steady state, assuming a constant drug concentration in the vehicle (Cv), and sink conditions within the viable epi-dermis (CL=0). Co is the concentration of TBF in the surface layer of the SC, and K is the SC/vehicle partition coefficient of the drug.
Environment Stratum corneum Viable epidermis
Cv
is clear that the OA-containing formulation results in higher levels of TBF at all depths into the SC; i.e., that the enhancer has in-creased the extent of drug delivery. Third, given that the vehicles were applied for the same amount of time, the deeper penetration induced by OA also implies that the rate of TBF transport across the skin's barrier has also been increased. One may reasonably conclude, therefore, that the formulation with OA has increased the local bioavailability of the drug. The details characterizing this aug-mentation of TBF delivery are revealed by the mathematical fitting of Eq. 1 to the
concentra-tion profiles generated from all the experi-ments performed. It is recalled that the data-fitting procedure permits values of K (the SC-vehicle partition coefficient of TBF) and D/L2 (the drug's characteristic, or kinetic, transport parameter across the SC) to be determined.
These results are summarized in Figures 5 and 6, based on both the semi-quantitative drug analysis in the SC in vivo using IR spectros-copy, and the quantitative determination ex vivo by HPLC.
Figure 5 reveals that the levels of enhan-cers added to the control formulation do not significantly (p>0.05) change the SC-vehicle partitioning behaviour of the drug.
This conclusion is supported by Figure 4:
note that while the control concentration pro-files fall off rapidly with SC depth compared to the OA formulation, the y-axis intercepts of the profiles are not obviously different
be-Figure 4: SC concentration profiles of TBF, determined by HPLC analysis of the drug within the SC tape strips. The indi-vidual data points and the best fits of Eq. (1) to the results are shown for the 5 subjects studied following application, (A) in the control formulation (EtOH:IPM 50:50), and (B) in the
Figure 5: SC/vehicle partition coefficients (mean±SD;
n=5) of TBF, determined either from HPLC (left scale) or IR (right scale) analysis, following drug delivery from four different formulations: (i) the control (CTR), and that same vehicle containing either (ii) 5% oleic acid (OA), (iii) 10% 2-pyrrolidone (2P), or (iv) 1% urea (UR). NS indicates no significant difference (p>0.05; Student's t-test) between the value for the CTR vehicle and that for an "enhancer-containing" formulation. Note that the K values found for the HPLC analysis are quantified abso-lutely. In contrast, the IR results yield only relative values which may be compared directly but have no absolute meaning.
K [HPLC] 102 Apparent K [FTIR]HPLC FTIR
NS
tween the two vehicles.
Direct comparison between the numerical values of K generated by IR and HPLC meth-ods is not possible because the former does not yield an absolute result. In contrast, the HPLC data are quantitative and indicate that TBF partitions almost equally between IPM:EtOH (50:50) and human SC in vivo independent of the enhancer present.
The characteristic transport parameter (D/L2), on the other hand, is formulation-sensitive (Figure 6). Both IR and HPLC ana-lytical methods lead to the conclusion that OA enhances TBF diffusion across the SC. From the TEWL measurements as a function of stripping, SC thickness (L) was found for the skin at each treated site and showed no sys-tematic or significant variation (data not shown).
Any changes in D/L2, therefore, can be at-tributed to changes in D. The more precise HPLC analytical approach implies that OA
increases the diffusivity of TBF by slightly more than 3-fold (D/L2 changing from 3.5 (±0.9) x 10-6 s-1 to 11.2 (±1.8) x 10-6 s-1) whereas the IR results from direct measure-ment of TBF in the SC by reflectance spec-troscopy identify a 2.5-fold increase (D/L2 going from 1.5 (±0.2) x 10-6 s-1 to 3.8 (±1.3) x 10-6 s-1). Of course, the skin permeation-promoting effects of OA, and its mechanism of action, have been studied in-depth before [12,15] and the increases observed in this work, therefore, were to be expected.
Data from the HPLC ex vivo analysis of SC tape strips also indicated that 2-pyrrolidone (2P) was able to enhance TBF transport across the membrane, causing an increase in D/L2 (from 3.5 (±0.9) x 10-6 s-1 to 12.0 (±4.6) x 10-6 s-1), not significantly differ-ent from that induced by OA. Results from the IR data for the 2P formulation, however, were too noisy (perhaps due to a subtle spec-troscopic interference) to show a similar dif-ference. The mechanism of action of 2P as a permeation enhancer has been less well stud-ied than that of OA, and an effect on the transport of a relatively lipophilic drug like TBF is not obviously anticipated by the pub-lished literature (e.g. [16]). However, sweep-ing generalizations about the alleged speci-ficities of certain enhancers on drugs of par-ticular physicochemical properties must be carefully evaluated (to ensure that the right comparisons are being made [17]) and, at the very least, it should be verified that other
Figure 6: Characteristic diffusion parameter (mean D/L2±SD; n=5) of TBF, determined either from HPLC (left scale) or IR (right scale) analysis, following drug delivery from four different formulations: (i) the control (CTR), and that same vehicle containing either (ii) 5%
oleic acid (OA), (iii) 10% 2-pyrrolidone (2P), or (iv) 1%
urea (UR). Significant differences (p<0.05; Student's t-test) between the value for the CTR vehicle and that for an "enhancer-containing" formulation are denoted by an asterisk. NS indicates a lack of significant difference.
CTR OA 2P UR
components in the vehicles compared are not themselves either capable of promoting trans-port directly, or of acting synergistically with the enhancer under investigation.
In the end, it must be said that, while the methodologies presented here offer a practical and obviously relevant way in which to assess formulation effects on topical drug delivery in man, they do not permit detailed understand-ing into the mechanism of action of specific penetration enhancers.
A final point to be made from Figure 6 is that urea did not significantly enhance TBF diffusion across the SC (whether assessed by IR or HPLC). Although there have been re-ports that urea can enhance the delivery of lipophilic drugs (such as hexyl nicotinate [18]) in vivo in a
concentra-tion-dependent fashion, no effect was observed here, perhaps because the level of urea used was too low or because the application time was too short to elicit a measurable effect relative to the control.
In Figure 7, the semi-quantitative measurements of TBF by reflectance IR spec-troscopy are compared with the quantitative evaluations of the drug using HPLC, for all the vehicles tested. The
graph reveals two features of importance.
First, while linear regression of the data re-sults in reasonable r2 values (0.74 to 0.84), there is obvious noise present as may be an-ticipated for human skin permeation experi-ments in vivo. Second, the data near the origin reveals why the IR results are sometimes more equivocal than those from HPLC; that is, there is a sensitivity problem with the spec-troscopic technique at low TBF concentra-tions, which are measurable by HPLC but which are invisible to reflectance IR. Correla-tion between the data sets is clearly skewed, therefore, by this limitation. Thus, while the IR approach is attractive for its simplicity and rapidity, compared to HPLC and other ap-proaches which have been used previously
0.00 0.01 0.02 0.03 0.04 0.05
0.0 0.5 1.0 1.5
[TBF] m easured by HPLC (M)
Relative [TBF] from IR (AU)
Figure 7: Correlation between the relative TBF concentrations determined in hu-man SC in vivo by FTIR and the absolute values found ex vivo in the tape strips by HPLC. The lines through the data are from simple linear regression (r2 values were in the range 0.74 to 0.84).
[6,19], its usefulness will clearly be greatest for compounds that permeate the skin well and which show an intense IR absorbance band easily distinguished from the spectrum of the SC.
Conclusions
The results of this study demonstrate that a drug's concentration profile across the SC and, by extrapolation, a measure of its local topical bioavailability, can be noninvasively determined in vivo, in man. Furthermore, the comparison between drug uptake levels from different vehicles permits the effect of puta-tive penetration enhancers to be deduced, and their impact on drug diffusion and partition-ing to be assessed.
Although only a single time-point has been considered here, the method can obviously be extended to provide a more complete "der-matopharmacokinetic" profile, if necessary [2]. In addition, the stage is now set to be able to correlate the kinetic and the bioavailability information accessible from this type of study with clinical, pharmacodynamic information (e.g., TBF's minimum inhibitory concentra-tion against different dermatophytes). In this way, and at long last, the efficacy of derma-tological drugs will be quantifiable, and held to the same standards of performance (and bioequivalence in the case of generic prod-ucts), as those given, for example, by the oral
route of administration.
Acknowledgement
This work was supported by Novartis Pharma (Basel, Switzerland), which also pro-vided the HPLC method.
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