Reproducibility of In Vitro Release Rate Testing in Human Skin Models
Although FDA does not officially require it yet, the human cadaver skin model is a powerful and sensitive tool by which to accurately quantitate a semisold drug’s rate of percutaneous absorption. This has application within multiple areas of the semisolid drug development process. The area of greatest application historically has been in preclinical development, where it is used to screen and select the optimum formulation for further development.
However, as new data continue to emerge to support the validity of the model as a surrogate for in vivo bioavailability and bioequivalence measurement, its application within other phases of the drug development process have become evident. These include:
Reformulation during clinical studies Phases I–III
Development of new semisolid products with enhanced delivery
Scale-up and post-approval changes (SUPAC-SS)
Generic drug product development
Use as a surrogate for bioequivalence
Greater recognition by FDA of its standing as a scientifically sound test method and acceptance of its use as a valid surrogate for more costly and time-consuming human and animal clinical studies would help to simplify the development of both new and generic topical and transdermal drug products and accelerate the process of bringing these products to market.
The product comparisons should be conducted side-by-side on skin sections from the same donors. Normally, three replicate sections from each of 3 donors (n=9) have to be used for each product.
General information can be obtained from in vitro percutaneous absorption studies. The major parameters are rate and time of drug penetration: flux and the total amount absorbed. The flux data can also be converted to cumulative penetration results. Release profiles of the drug from the investigated vehicle can be obtained by plotting the amount of analyte released (mg/cm2) as function of time.
When mass balance studies are conducted, the residual surface dose and the distributed content within the stratum corneum, epidermis and dermis can also be determined. These parameters can be reported as both µg/cm2 and as percent of applied dose. The amount of analyte retained in the skin can be determined at the end of the in vitro permeation experiment, typically 24 or 48 hour.
For drug release experiments, the permeated active calculated by the amount of active in the receptor medium. Mean cumulative analyte amounts are plotted versus the square root of time: Qt = K x t1/2, where, Qt is the cumulative drug amount recovered in the receiving compartment (μg), K is the kinetic constant indicative of the release rate (μg/h1/2) and t1/2 is the square root of time (h1/2). The kinetic constant K (slope of the plot) and the release Lag-time (abscissa intercept, Lag-time) will be calculated by linear regression.
The permeability coefficient P and Lag-time will be calculated for the individual experiments assuming constant skin thickness and diffusion area resulting in an equation only consisting of the slope of the linear portion of the permeation curve divided by the applied donor concentration: P = (V/A x Ci) x (dCa/dt), where, P is the permeability coefficient (cm/s), V is the volume of the receiver chamber (12 cm3 ), A is the area of the skin surface exposed to the receptor medium (1.7671 cm2), Ci is the initial concentration of the applied substance in μg/cm3 and dCa/dt is the increasing concentration of the drug in the receptor medium with increasing time. All data that is presented as mean ± SD will be evaluated by explorative data analysis. Thus, p ≤ 0.05 will indicate a significant difference.
Also, the permeability coefficient can be calculated according to Fick’s first law of diffusion: Kp = Jmax/S, where Kp (cm/h) is the permeability coefficient, Jmax (μg/cm2 per hour) is the flux obtained with the saturated solution, and S is the drug donor concentration (μg/cm3), corresponding to the drug solubility in the vehicle at 32oC.
The reportable data should include analyte absorption that is measured in the skin model from the topical formulations. The primary pharmacokinetic parameters calculates:
Total absorption at 24 hours (Total Abs, μg)
Maximum rate of absorption (Fmax, /cm2/h)
Time when maximum rate of absorption occurred (Tmax, hour)
The steady state flux (J), representing the absorption rate per unit area can be determined from the slope of the linear portion of the plots. The estimated error standard deviation can be used to compute the 90% confidence intervals (CI) for the ratios of the means (Test/Reference) of the generated parameters in 90% CI.
For Lot-to-lot (test/reference) comparison for transdermal formulations, in vitro test results are expressed as cumulative amounts of drug permeated as a function of time for the different formulations. Data illustrates the cumulative amount permeated relative to the reference product using the same human skin donor. The mean flux of drug permeated will be calculated from the slope of the linear portion of the cumulative amount released versus the time plot and expressed as μg/cm2/h. The result is expressed as a ratio of flux of the test formulation and reference formulation. The cumulative amount of drug permeated through the skin per cm2 area would be also compared with that of reference formulation and expressed as a ratio (test/reference). Therefore, each transdermal formulation would be compared to the reference product for its cumulative permeation and flux value and the results are compiled. The confidence interval for all three primary endpoints should be within the interval 0.80-1.25 in order to meet regulatory requirements for bioequivalence. Useful conclusions can be draw from mass balance comparison of test/reference formulations
Distribution within the skin at a time period of 24 hours can be determined by measuring the level of drug within the stratum corneum and the epidermis plus any remaining lower stratum corneum by tape stripping the skin. The skin is wiped prior to tape stripping to remove any drug remaining at the surface. Additionally, any drug in the filter paper epidermal membrane support, donor chamber sealing and the cell donor chamber surfaces (due to evaporation from the skin surface, follow by condensation on the cooler, upper part of the diffusion cell) can be quantified. Levels in the donor chamber wipe and wash should be combined to produce donor chamber values. The individual tape strip group data as well as other distribution and permeation data expressed as % drug released at incubation time (24 hours) should be reported for the compartments.