Dermal and Underlying Tissue Pharmacokinetics of Lidocaine after Topical Application

PARMINDER SINGH' AND MICHAEL S. ROBERTS^^ Received September 30, 1992, from the "Department of Pharmacy, The University of Queensland, Queensland, Australia 4072. and the $Department of Medicine, The University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia 4 102. Accepted for publication August 16, 1993@.

Abstract 0 The deep-tissue penetration of lidocaine below a dermally applied site was quantified in a rat model. The concentrations of lidocaine in tissues below the applied site were measured and compared with plasma concentrations and concentrations in similar tissues on the contralateral side. The direct penetration of lidocaine was predominant for the first 2 h up to a depth of about 1 cm below the applied site. A physiologically based pharmacokinetic model based on apparent tissue-tissue clearances and local blood flow to tissues is presented which adequately describes the concentration-time profiles of lidocaine in underlying tissues after dermal application. The apparent tissue-tissue clearances were estimated by nonlinear regression assuming first-order diffusional mass transfer of lidocaine between the various tissue compartments below the applied site in anesthetized rats. Tissue levels of lidocaine were estimated using simulations from the model with and without direct penetration and tissue blood supply. Dermal microcirculation is not a perfect sink for lidocaine.

The stratum corneum is the main barrier to drug absorption through the skin.' Attempts have been made to circumvent this barrier both by chemical and physical means.2 We are partic- ularly interested in the fate of drugs when they reach the underlying dermis after topical application to a skin site.3 It is generally believed that cutaneous microcirculation acts as a sink for most topically applied compound^.'^^ However, reports have appeared in the literature giving evidence for the deep-tissue penetration of a number of c0mpounds.~7 This hypothesis can be tested by studying the local tissue penetration of compounds from a dermal site. Applying a solute directly on the dermis avoids the stratum corneum barrier and also places the solute directly on the so-called "sink". The tissues below the application site are then sampled and concentrations compared with plasma concentrations and concentrations in similar tissues from a contralateral site.889

The specific aims of this study were to quantify the depth and extent of the local tissue penetration of lidocaine after topical application and to design a physiologically based pharmaco- kinetic model which can describe the processes of absorption and local tissue distribution of dermally applied lidocaine.

Model Development We have earlier suggested a physiological pharmacokinetic

model to describe local tissue distribution of solutes.8.9 A similar approach can also describe the local tissue penetration of lidocaine after its dermal application. According to Figure 1, each tissue can be described as compartments-in-series, each joined in parallel to a central plasma compartment. After dermal application, solute penetrates to the ith tissue compartment from the tissue compartment overlying it (i + I) and into the tissue compartment underlying it (i - 1). The differential mass-balance equations can then be written for each compartment to describe the inflow, outflow, and disappearance of the drug assuming (1) a pseudoequilibrium exists between a given tissue and the blood

Abstract published in Advance ACS Abstracts, January 15, 1994.

Solution in

cs. vs

C l s w d

C l s c w f a

Fascia I Fascia 1 Qfa Cfa, Vfa - - - t - - - 9 - - - -

S. muscle Csm, Vsm

Cm, Vm

Cb, Vb

kel

Elimination

I I

C l m w f p If I I I

I

I I

I

I I

Cl fpwdm IT 5.

Flgwe 1-A pharmacokinetic model for local tissue penetration of dermally applied lidocaine. Refer to text for meaning of various terms. perfusing that tissue, (2) the lateral spread of a solute from below the applied site is negligible, (3) each tissue is a noneliminating organ and acts as a well-stirred compartment. The rate of change of amount of solute in the cell compartment (Figure 1) can be given by eq 1

dt where PS is the permeability-surface area product from move- ment of the solute from the solution into the dermis, C, and cd are concentrations of solute in solution and the dermis, respectively, and kd is the dermis-water partition coefficient. Noting that Ps = ks-dva, where ks+ is the transfer rate constant

774 / Journal of Pharmaceutical Sciences 0022-3549/94/ 1200- 774$04.50/0 @ 1994, American Chemical Society and American Pharmaceutical Association Vol. 83, No. 6, June 1994

between the cell solution and dermis and V, is the volume of the solution, eq 1 can be reexpressed as

An apparent monoexponential decline in lidocaine concentration was observed after application to the exposed dermis of anesthetized rats, therefore any backward flux from dermis to cell was neglected. The integrated form of eq 2 can then be written as

A, = A, exp(-k,+t) (3) where A0 is the amount in cell solution at zero time. The rate of change in the amount of solute in the dermal compartment is given by eq 4

dAd -= dt k,,dAo exp(-ka-dt) + k&dAb - Izd-bAd +

kac-dAsc - kd-scAd (4)

where k w and kd-b are transfer rate constants between the blood and dermis and vice versa, respectively, k-,j and kd-, are transfer rate constants between the subcutaneous tissue and dermis and vice versa, respectively, and Ab and A,, are the amounts of solute in the blood and subcutaneous tissue, respectively.

Equations 3 and 4 can also be expressed in terms of physiological pharmacokinetic notation of clearance', and equivalent unbound concentrations:

where CO and c b are concentrations of solute in the cell solution (at zero time) and blood respectively, Vud and v d are the apparent volume of distribution of unbound and total solute (=Vudud) in the dermis, respectively, Cl8-d is the clearance between the cell solution and dermis, C1d-c is the clearance between the dermis and subcutaneous tissue (C1d-e = C L d ) , fudr fW, and fub are the fraction unbound of solute in the dermis, subcutaneous tissue, and blood, and Q d is the blood flow to dermis.

Equation 6 reduces to eq 7when fud = fuse and fudfud = RMd,

where RMd is the dermis-plasma partition coefficient and noting that the concentration in the dermis is expressed per gram of tissue and is estimated by dividing the observed concentration c d by the weight of the tissue assuming a tissue density of unity. By analogy, the rate of change of concentration in all deeper underlying tissue compartments with time for the model in Figure 1 can be described by eq 8

is the clearance between the ith tissue and the i - 1 tissue, CT,i, CT,i+l, and CT.i-1 are the concentrations in the ith tissue, the i + 1 tissue, and the i - 1 tissue, QT,~ is the blood flow to the ith tissue, and RMT,~ is the ith tissue-plasma partition coefficient.

Equation 8 reduces to eq 9 when fuT,i = fuT,i+l = f,,~,i-~:

- (Ch,i+l-i(CT,i+l - cT,i) + QT,i(C&MT,i - CT,~) + dcTi ' T , i X -

clT,i-i-l(cT,i-l - cT,i))fuT (9)

wherefuT represents a common term denoting the equal unbound fraction of solute in the tissues.

A slightly modifed approach was also developed based solely on the direct penetration of the solute. The solute reaches any tissue on the contralateral side predominantly via systemic circulation and possibly by diffusion between the tissues. In the modified model, the concentrations of lidocaine obtained in contralateral tissues were subtracted from the concentrations in similar tissues below the dermal application site to give con- centrations in underlying tissues which can be attributed exclusively to direct penetration. Another feature of this approach is that it takes into account any systemic distribution of metabolites which may occur for drugs which are extensively metabolized. Equation 9 can thus be written as

dC* vT,i+ - - CIT,i+l-i(fuT,i+lC*T,i+l - fuT,ic*T.i) +

QT.i(CbRMT,i - fuT,iC*T,i) + Cb,i-i-l(fuT,i-lC*T,i-l - fuT.ic*T,i) (10)

where C*T,~ represents the concentration of solute in a given underlying tissue obtained exclusively by direct penetration and is defined by c*TJ = CT,~ - c ~ c i (where CT,~ and cTc,i are the concentrations of solute in the underlying treated and con- tralateral tissues, respectively).

Equations 9 and 10 were used to estimate the apparent tissue- tissue clearances by nonlinear regression and to estimate the lidocaine concentrations in tissues. The relative contributions of direct penetration and tissue perfusion were estimated by simulations in the model using eqs 7 and 9, either in the absence of direct penetration (assuming the clearance terms to be zero) or solely in the presence of direct penetration (assuming the blood input function to be zero). Dermal absorption studies combined with penetration fluxes through isolated human epidermis have been used to examine the concentrations in dermis after topical application.ll Simulations were also per- formed to estimate underlying tissue concentrations following a constant input source applied to the epidermis.

Experimental Section Chemicals and Instruments-[ 14C]Lidocaine hydrochloride (spe-

cific activity of 48 mCi/mmol) and tritiated water (1 mCi/g) were purchased from New England Nuclear, and lidocaine hydrochloride waa a gift from Astra Pharmaceuticals Pty. Ltd. Zimmer's electrodermatome (Model 901) was used for removing rat epidermis. Tissue solubilizer (NCS) andliquidscintillation cocktails (OCS, organic countingscintillant, and BCS, biodegradable counting scintillant) for tissue and aqueous samples (respectively) were purchased from Amersham International. All other reagents used were of analytical grade. A liquid scintillation counter (Model MINIAX, Tri-carb 4000 Series, United Technologies Packard) was used to determine the radioactivity in the samples.

Animals-Male Wistar rats (300-350 g) were used in the studies. The animals were housed under standard laboratory conditions at 20.0 f 0.5 "C and 55-75 % relative humidity and supplied with normal pellet diet and water adlibitium. All experiments had previously been approved by the Animal Experimentation Committees of the University of Queensland and the Princess Alexandra Hospital.

Isolated Human Epidermis Penetration Studies-Human skin was obtained from the midabdominal region of cadavers and the

Journal of Pharmceutical Sclences / 775 Vol. 83, No. 6, June 1994

epidermis separated from the dermis by the heat method.12 Upon receipt, the subcutaneous fat was carefully trimmed and the full thickness of skin was immersed in deionized water a t 60 "C for 90 s. The epidermis was then gently peeled off with blunt forceps. The isolated epidermis was dried between the folds of filter paper and refrigerated until further use. It was rehydrated by immersing in deionized water for 1 hour before use. Isolated human epidermis was mounted in side-by-side glass diffusion cells and submergedin a water bath at 37 "C. After equilibration with the buffer of interest, normal saline was added to the receptor compartment and the solution of drug introduced into the donor compartment. The penetration of lidocaine across human epidermis was studied at a donor pH of 3.67, using acetate buffer, and a donor pH of 7.4, using phosphate buffer. The surface area of the epidermis exposed to the drug solution was 4.785 cm2. Samples were removed from the receptor side at predetermined times and analyzed for lidocaine.

In Vivo Dermal Penetration and Local Tissue Uptake Studies (Anesthetized Animals)-The rats were lightly anesthetized by pentobarbitone (35 mg/kg), and their body temperature was maintained at 37 "C by placing them on a beating pad. The epidermis from the dorsum region (4 cmz) of rats was removed by means of an electroder- matome set at a thickness of 80 1m.13 A glass cell (internal diameter, 1.8 cm) was then adhered to the exposed rat dermis and warmed to 37 "C by means of an external heating device.lgJ4

A solution of lidocaine (pH 7.4) previously warmed to 37 "C was introduced into the dermal glass cell and samples removed from the dermal cell at various times. The glass cell containing drug solution was removed from the rat dermis at predetermined times and a blood sample taken from the tail vein. The animals were then sacrificed with an overdose of anesthetic ether and the tissues below the treated site, i.e., dermis, subcutaneous tissue, fascia, muscle lining, or superficial muscle (s muscle), muscle, fat pad, and deep muscle (d muscle) were then dissected and placed in preweighed scintillation vials.13 Similar tissues from the contralateral side were also removed. Tissue and plasma samples were stored at -20 "C prior to analysis.

In Vivo Dermal Penetration and Local Tissue Uptake Studies (Sacrificed Animals)-The rates were initially anesthetized by intraperitoneal injection of pentobarbitone (35 mg/kg) and, after removal of the epidermis as described above, were sacrificed by overdose of ether. Dermal perfusion studies were then conducted in postmortem rats.

Blood-Flow Measurements-The blood flows in skin and underlying tissues estimated in our earlier stud? were used in the present work.

Sample Treatment-Aqueous samples removed from the glass cells in in vivo dermal studies and in vitro epidermal studies were directly mixed with 5 mL of BCS and counted on a liquid scintillation counter. The tissue samples were solubilized with 50 pL of water and 1 mL of NCS at 50 OC for 6-8 h. After cooling of the digested samples to room temperature, 0.03% glacial acetic acid was added to each tissue sample followed by 10 mL of OCS. The plasma samples were solubilized with tissue solubilizer (5 parts for 1 part of plasma) at room temperature and treated with glacial acetic acid before adding OCS. Each sample was then counted on the liquid scintillation counter for 10 min.13

Analysis of Data-Zero-time samples from the cell were used to represent the initial solution concentration, and the 14c activity in the tissues and plasma was converted to the fraction of the initial solution concentration (concentration fraction).8J3 The amount absorbed was calculated as the difference between the initial concentration and concentration remaining in the cell at the end of each dermal perfusion study. Clearance into the dermis was estimated from the plot of percent lidocaine remaining in the dermal absorption cell with time.*3J4 In isolated epidermal penetration studies, the cumulative amount of lidocaine appearing in the receptor compartment was plotted against time. The slope of the linear portion of the plot gave the steady state flux, which when divided by the initial concentration gave the perme- ability coefficient.ls

The tissue to plasma partition coefficient (RMT,~) were calculated using pseudo-steady-state plasma and contralateral tissue concentrations of lidocaine at 10 h post dermal perfusion and also from the ratio of area under the curves for contralateral tissue concentration-time profiles and plasma concentration-time profile according to eq 11

RMT,i = AUCT,i/AUC, (11)

where AUCT,, is the area under the tissue concentration-time profile, AUC, is the area under the plasma concentration-time profile, and RMT, is the tissue-plasma partition coefficient.16

The MINIM computer programI7 was used to numerically integrate eqs 5,7, and 9 (using the concentration-time data in underlying tissues)

4 r

Time (hr)

Flgure 2-Cumulative amount of lidocaine penetrating human epidermis with time. Key: (0) 0.016%, (0) 0.25%, (0) 2% donor concentrations. Values are reported as mean f SD (n = 4).

or 10 (using the underlying tissue concentration minus contralateral tissue concentration data) and simultaneously fit the lidocaine data obtained as the solution for cell, dermis, subcutaneous tissue, and fascia using weighted (l/Ci) least squares with Hartley modification of the Gauss-Newton algorithm. A sixth order polynomial input function representation of the observed solute concentrations in the plasma- time profiles was used. In order to estimate the parameters for deeper tissues, eq 9 or 10 was numerically integrated for subcutaneous tissue, fascia, superficial muscle, muscle, and fat pad concentration-time profiles, and a sixth order polynomial input function representation of the observed solute concentrations of either the dermis-time or subcutaneous-time profdes was used. The final model fitting was deemed acceptable on the basis of the regression goodness-of-fit criteria which included the Akaike information criteria (AIC),18 a lack of systemic deviations in the residuals, and a high correlation coefficient (r2 > 0.9).

Simulations in the model were performed by either numerical integration and nonlinear regression of eqs 5, 7, and 9, assuming experimentally determined blood flows as constants in the absence of direct penetration or apparent tissue clearances constants in the absence of input tissue blood flow. Simulations were also performed assuming no blood supply (both input and output blood functions in eqs 7 and 9 = 0) and predicted underlying concentrations compared with those obtained experimentally in sacrificed animals. The simulations de- scribing underlying tissue levels of lidocaine after epidermal application were performed by combining the isolated human epidermal and dermal absorption studies according to the model presented in Figure 1, when the solution compartment was replaced by a constant input source applied to the epidermis.

Each observation is a mean f SD of three or four determinations.

Results and Discussion

Initial lidocaine penetration studies were conducted with isolated human epidermis to estimate the likely concentrations achievable on the dermal side. These concentrations of lidocaine were then applied to the exposed rat dermis in vivo for the tissue uptake studies.

Lidocaine Penetration across Isolated Human Epi- dermis-Figure 2 shows the penetration profile of lidocaine through human epidermis. The flux was proportional to the applied concentration whereas the permeability coefficient (k , values at 0.016, 0.25, and 2% lidocaine were 0.0035 f 0.0009, 0.0050 f 0.0011, 0.0041 f 0.0009 cm/h, respectively) was independent of initial concentration. A lag time of 25 min was observed, which is the time taken by lidocaine to partition into and then diffuse through the epidermis before appearing in the receptor compartment. A permeability coefficient and lag time of a similar order have been reported across dimethylpolysiloxane membrane.lg Applying lidocaine at a pH of 3.67 resulted in a decrease in flux and permeability coefficient (mean k, = 0.000 32 f 0.000 095 cm/h). Epidermal clearance of lidocaine hydro-

778 / Journal of Pharmaceutical Sciences Vol. 83, No. 6, June 1994

0 5 1 0 1 5 2 0 Time (hr)

Figure 3-Disappearance versus time profile of lidocaine from solution applied to anesthetized rat dermis. Values are reported as mean f SD (n = 3).

chloride applied to human stratum corneum was also found to increase with an increase in Lidocaine is a base (pK, = 7.86) and hence 25% un-ionized at pH 7.4, 50% un-ionized at pH 7.9, and almost fully ionized at a pH 3.67; the un-ionized form of any compound is known to penetrate the skin better than the ionized form.21 The integrity of epidermal membranes during penetration studies was checked by simultaneously quantifying the flux of tritiated water. The data were rejected if the permeability coefficient for water exceeded 0.0025 cm/h.

Application of a 2 % solution resulted in a flux of 0.44 f 0.085 pmol/cm2/h across isolated human epidermis. To test the hypothesis whether topically applied lidocaine can bypass the dermal microcirculation to reach underlying tissues, solutions of lidocaine (0.7 mM or 0.7 pmol/mL) were applied directly on the exposed rat dermis (epidermis removed) in uiuo. This treatment places lidocaine directly on the so called “perfect sink” and simulates perfect delivery through epidermis.

Disappearance Kinetics of Lidocaine Applied to the Exposed Rat Dermis-An apparent monoexponential decline in cell concentration was observed (Figure 3) with an absorption rate constant of 0.10 f 0.02 h-1. Levy and Rowland22 have earlier shown a biexponential disappearance curve for lidocaine applied to rat subcutaneous tissue whereas Ballard23 obtained curves which were difficult to analyze in terms of either mono- or biexponential loss of drug from the subcutaneous absorption cell. The probable reason given was the pH shift in the cell with consequent change in ionization and hence distribution of lidocaine between subcutaneous tissue and cell solution.23 An absorption rate constant of 0.17 f 0.04 h-1 for lidocaine has earlier been reported for 2-h dermal perfusion study.l3 The mean absorption rate constant computed from dermal perfusion studies at various times was found to be 0.15 f 0.04 h-1. The apparent variability in absorption rate with time could be due to factors such as microcirculatory changes with time, degree of anesthesia, and possible drug effects. Similar effects and reasons were previously reported for benzyl alcohol absorption from a subcutaneous sitez4 and salicylic acid absorption from a dermal site.9 Membrane thickness can also vary due to interanimal variations.24 An absorption rate constant of 0.17 h-1 from the dermal site is similar in order to that reported by Levy and RowlandZ2 and Menczel et al.Z5 for subcutaneous absorption rate constants of lidocaine in rats and rabbits, respectively. This may suggest that lidocaine is being cleared to an almost equal extent from solutions applied to either rat dermis or subcutaneous tissue, implying diffusion-limited uptake.

Distribution and Pharmacokinetics of Lidocaine in Underlying Tissues-The rats were sacrificed at the end of each dermal perfusion study and tissues below the treated site,

similar tissues from the contralateral side, and a blood sample were removed and analyzed for lidocaine. Figure 4 shows concentration-time profiles of lidocaine in tissues below the site of application and similar tissues from a contralateral site. The concentration of lidocaine in the underlying tissues was higher than those in plasma and the contralateral tissues over a 16-h period.

The blood flows and tissue-plasma partition coefficients for various tissues are shown in Table 1, the values for different tissues being comparable and consistent with reported values for fat pad and muscle.26 Tissue concentrations of lidocaine for extradural fat, subcutaneous fat, and sciatic nerve tissue have also been shown to be remarkably similar.27 This may be attributed to the properties of the lidocaine molecule and is also supported by the observation that there is no difference in diffusion of lidocaine into myelinated and unmyelinated nerve fibers.28 Table 2 lists the calculated values of the apparent clearances of lidocaine for different tissues in rat estimated by nonlinear regression, using either the treated tissue concentra- tion-time data or the difference between treated and contralat- era1 tissue concentration-time data. The apparent clearances of lidocaine from both approaches were similar (Table 2), thus confirming the general structure of the model. The estimated permeability coefficients are also consistent with the reported average value of 103 cm/s for various tissues for lipophilic solutes.29 The estimated permeability coefficients for lidocaine are 1 order higher than the earlier reported values for water in similar tissues;12 the apparent differences can be attributed to the differences in the physicochemical properties of the two s0lutes.~9 The estimated tissue-tissue clearances and the blood flows were than fitted to the model shown in Figure 1 and also to the modified model based solely on direct penetration. The predicted concentrations in dermis, subcutaneous tissue, fascia, superficial muscle, and muscle compared well with the exper- imentally determined values (Figure 4). The predictions from both approaches were very similar.

The modified approach in which the modeling was done with the data solely attributed to direct penetration takes into account any systemic distribution of metabolites. Lidocaine is extensively metabolized in l i ~ e r ; 3 ~ , ~ ~ therefore, one would accept the un- derlying tissue concentrations attributed to the systemic blood be both due to the parent compound and the metabolites. In our analysis, we subtracted contralateral tissue concentrations (lidocaine + metabolites) from the concentrations in tissues immediately below the application site to obtain concentrations in underlying tissues which can be solely attributed to intact lidocaine. Lidocaine metabolism in blood and local tissues has been shown to be negligible.32

A single plasma peak was observed at 10 h following dermal application while two peaks were seen for underlying tissues, a smaller one at 10 h and an earlier distinct one at 2 h.

The presence of two peaks in underlying tissues has also been observed with salicylic acidg and p i ro~ icam.~~ The concentration time-profiles in all the contralateral tissues resembled that of the dermal plasma, with levels increasing rapidly in the initial 2 h and then showing a gradual rise with the peak concentrations observed at 10 h. The simulations performed in the model assuming either no blood flow to tissues or no direct penetration are shown in Figure 4. In the absence of direct penetration, levels in all the tissues were found to rapidly build up in the initial 2 h and increase rather slowly after that to peak at 10 h (dotted curve in Figure 4 plotted using tissue concentration- time data in contralateral tissues), which is consistent with the plasma levels observed experimentally. On the other hand, a single peak at 2 h was observed in the absence of the blood input function, which very closely corresponds to the first of the two experimentally observed peaks in underlying tissues.

The peak at 10-12 h in tissues below the applied site can be attributed to systemic distribution and probably due to some

Journal of Pharmaceutical Sciences / 777 Vol. 83, No. 6, June 1994

0.20 r

0.010

0.006

0.006

0.004

A

0) a a 0.15

0

Q O - --

0.10

E $ 5 $ 0.05

Y 5

DERMIS

0.00- ' . I 0 4 8 1 2 1 6

Time (hr)

FASCIA

........ ... ...-........ ............................. I ,...... o.Oo04 . ' 0 4 8 1 2 1 6

Time (hr)

0.006 r

0.0051 1 MUSCLE

0 4 8 1 2 1 6

Time (hr)

DEEP MUSCLE - W

0 4 8 1 2 1 6

Time (hr)

0.06

0.04

0.02

0.00

T SUBCUTANEOUS

............... ........ -...-...T ........,...... 4 8 1 2 1 6

Time (hr)

T S.MUSCLE

....... ................ ...................... .... 4 8 1 2 1 6

Time (hr)

1 FAT PAD

O.OO0 0 4 8 1 2 1 6

Time (hr)

PLASMA

I I . I

0 4 8 1 2 1 6

Time (hr)

Flgure 4-Tissue concentration versus time profiles of lidocaine applied to anesthetized rat dermis. Key: (0) underlying tissues, (0) similar tissues from a site on the contralateral side. The solid curve is the total prediction from the model shown in Figure 1 , according to equations 5, 7, and 9 or 10 (the predictions from two approaches were very similar), the broken line is the prediction due to direct penetration, and the dotted line is the prediction in the absence of any direct penetration. The figures for fat pad and deep muscle represent experimentally determined values as the predictions from the model were only possible till underlying muscle. S.MUSCLE = superficial muscle. Values are reported as mean & SD (n = 3 or 4).

direct penetration. However, the earlier peak at 2 h cannot be attributed to the systemic blood supply, at least in tissues up to underlying muscle, and suggests that lidocaine is being directly transferred to underlying tissues from the dermis without prior

pasage into the systemic blood. Even after 30 min sufficient levels build up in underlying tissues, which further increase over 2 h and then show a gradual decline before peaking again at 10 h. During the initial period, lidocaine uptake into local tissues

778 /Journal of phermaceutical Sciences Vol. 83, No. 6. June 1994

Table 1-Blood Flows and Tissue-Plasma Partition Coefficlents of Lldocalne for Various Tissues In the Rat

Blood Flow Tissue-Plasma Tissue-Plasma Tissue-Plasma Tissue (mL/h/g) Partition Coefficienta Partiiion Coefficientb Partition Coefficientc

Dermis 3.11 f 0.15 1.56 f 0.30 1.71 Subcutaneous 2.16 f 0.15 1.26 f 0.33 1.25 Fascia 1.86 f 0.59 1.60 f 0.25 1.40 S Muscled 4.15 f 0.27 1.17 f 0.29 0.97 Muscle 4.28 f 0.82 1.04 f 0.18 0.87 0.92 Fat Pad 0.36 f 0.12 1.47 f 0.31 1.40 1.54 Deep Muscle 3.25 f 0.62 0.93 f 0.17 0.71

a Estimated from the ratio of pseudo-steady-state concentrations in contralateral tissues and plasma at 10 h after dermal perfuslon. * Estimated from the ratio of area under the curves for contralateral tissues and plasma according to eq 11. From ref 26. Superficial muscle.

Table 2-Apparent Clearance and Permeability Coefficient for Lidocaine in Various Tissues In the Rat

Apparent Apparent Permeability Clearance8 Clearanceb CoefficientC

Tissue (mL/hr) (mL/h) (cm/s X lo5)

Dermis 1.50 f 0.31 1.65 f 0.40 16.40 f 3.39 Subcutaneous 2.06 f 0.31 2.39 f 0.55 22.53 f 3.39 Fascia 4.31 f 0.45 5.29 f 0.67 47.13 f 4.92 S Muscled 4.76 f 0.96 5.62 f 1.19 52.06 f 10.50 Muscle 4.30 f 1.59 4.95 f 1.54 47.03 f 17.39 Fat pad 0.34 f 0.20 0.46 f 0.31 3.72 f 2.19

Estimated from nonlinear regression analysis according to eqs 5, 7, and 9 (underlying tissue concentration-time data). Estimated from nonlinear regression analysis according to eqs 5, 7, and 10 (treated tissue minus contralateral tissue concentration-time data). Estimated by dividing apparent clearance (see footnote a) by area of application. Superficial muscle.

competes and predominates against systemic absorption. Keen- aghan and Boyles studied the intravenous and oral disposition of lidocaine in rats and reported blood levels of 1.1-1.9% for intravenous and 1.5-2.8% for oral administration (expressed as percent of dose recovered) over a 24-h period. The rapid tissue distribution of lidocaine was also evident in their studies by the fact that <2 % of the administered radioactivity was found in the bl00d.~0 After the initial rapid distribution into local tissues, systemic absorption is likely to become directly dependent on tissue/blood partitioning.34

Considerable accumulation of lidocaine in the rat subcutaneous tissue following subcutaneous application has also been ob- served.22 The accumulation in underlying tissues cannot be due to the vasoconstrictor activity of lidocaine because much higher concentrations are required to produce vasoconstriction than were used in the present st~dies.22,~~ It is therefore obvious that dermally applied lidocaine is bypassing the dermal blood supply to attain significant levels in the underlying tissues. Increasing the applied concentration proportionally increased lidocaine concentrations in underlying tissues (Figure 5).

The tissue:plasma ratios for the tissues on the contralateral side were fairly constant with time, indicating a pseudoequi- librium between plasma and tissues. However the tissue:plasma ratios for tissues (up to superficial muscle) below the applied site were much higher than the ratios for the similar tissues on the contralateral side. Ackerman et al. have reported local levels of lidocaine in neural tissue which were 4-5 times the blood levelsover a 2-h period.36 In deeper underlying tissues, the tissue: plasma ratios were again higher, though less pronounced, then contralateral tissue:plasma ratios, suggesting that the efficiency of direct penetration decreases with increasing depth. The treated tissue:plasma concentration ratios decline with time, indicating systemic removal of lidocaine. During the early period, the concentrations in tissues below the application site (up to

10 r

e

m . a al 0 - 5 .01

,001

Figure 5-Effect of applied concentration of lidocalne on underlylng tissue levels after dermalappllcation. Key: (0) 0.016%, (0) 0.25%. SUBCUT = subcutaneous tissue, S.MUSCLE = superficial muscle, D.MUSCLE = deep muscle. Values are reported as mean f SD (n = 3).

the muscle) were also much higher than the concentrations in similar tissues on the contralateral side, again supporting the hypothesis of direct penetration. The treated:contralateral concentration ratios again declined with time, suggesting removal by systemic blood supply. In tissues deeper than muscle, the concentrations were higher, though comparable to contralateral tissue concentrations. These results strongly suggest that the high concentrations of lidocaine in tissues immediately below the applied site (-1 cm) arise from direct penetration.

Table 3 shows lidocaine appearing in the underlying tissues expressed as percent of the amount absorbed. Almost 100% of the absorbed dose can be accounted for in the underlying tissues at 0.5, 1 and 2 h; thereafter, the levels in underlying tissues decrease, implying removal by systemic blood supply and consequent distribution and elimination. Similar behavior is also reflected in the decreased treated:contralateral and treated: plasma ratios for different tissues after 2 h as explained above. Direct penetration of lidocaine is thus occurring predominantly in the first 2 h. Higher local tissue levels of lidocaine are maintained over a period of time relative to plasma and contralateral tissue concentrations, implying gradual removal of lidocaine accumulated in the tissues immediately below the application site during the first 2 h of application. The greater tissue affinity coupled with lipophilic nature of lidocaine allows it to preferentially distribute in the local tissues in the initial stages of its application before systemic absorption and elim- ination lower the tissue concentrations. Plasma levels of lidocaine (Table 3) at all times were <0.35% (0.2 WglrnL), which are below those normally observed with other modes of regional anesthesia and well below the toxic threshold and intravenous regional anesthesia.31 Low plasma levels coupled with high local tissue concentrations suggest likely efficacy of topical lidocaine for

Journal of Pharmaceutical Sciences / 779 Vol. 83, No. 6. June 1994

Table 3-Lldocalne Recovered as Percent of the Amount Absorbed In Underlylng Tlssues and Plasma

Tissue 0.5 h l h 2 h ~

Dermis Subcutaneous Fascia S Musclee Muscle Fat Pad D Muscleb Plasma

70 f 6.0 25 f 7.10 5 f 0.69 2.6 f 0.40 0.7 f 0.13 0.5 f 0.04 0.41 f 0.11 0.33 f 0.06

60f 15 23 f 7.30 8.6 f 1.60 3.5 f 0.52 1.2 f 0.41 0.68 f 0.12 0.64 f 0.14 0.34 f 0.03

49 f 16 25 f 8.10 19 f 4.20 7.0 f 1.10 2.1 f 0.61 1.3 f 0.40 1.3 f 0.44 0.30 f 0.02

4 h

23 f 8.90 4.0 f 1.30 2.7 f 0.38 1 . 1 f 0.30 0.80 f 0.27 0.72 f 0.25 0.46 f 0.19 0.27 f 0.04

a Superficial muscle. Deep muscle.

Table 4-Relative Contributions of Diffusion and Perfuslon In the Tlssue Clearance of Lldocalne after Dermal Appllcatlon In Rats

Tissue Diffusion (%) Perfusion (%)

Dermis 33 Subcutaneous 49 Fascia 70 S Muscleb 53 Muscle 50 Fat Pad 49

67 51 30 47 50 51

BEstimated from eq 12 using the apparent clearances listed in Table 2. Superficial muscle.

local skin and deep-tissue anaesthesia. In fact higher concen- trations of lidocaine in the underlying muscle or synovium may be expected when there is minimum or no overlying fat. The presence of the biological fat below the area of application can prevent a significant portion of applied dose from reaching its site of action.27 It may be noted that the experiments in this study were conducted on the dorsum of rats and inspite of the presence of fat below the applied site, significant levels of lidocaine were observed as deep as underlying muscle.

Analysis of Blood Supply and Direct Penetration Con- tributions to the Distribution of Lidocaine in the Various Tissues-The relative contributions of tissue diffusion and perfusion in removing a dermally applied solute can be estimated from the estimated clearance due to diffusion, CldiffTj and the blood flows to individual tissues QT,j (eq 1 2 ) ? s 9

CldiffT,i C1diffT.i + QT,~

(12) percent contribution of tissue diffusion =

Table 4 lists the relative contributions of tissue diffusion and perfusion in removing topically penetrating lidocaine. The relative efficiencies of diffusion and perfusion processes in removing lidocaine from underlying tissues (Table 4) are proportional to the clearances estimated due to direct diffusion (Table 2) and blood flow to individual tissues (Table 1).

Table 5 lists the fractionalcontributions of direct penetration (due to the overlying solution/tissue and underlying tissue) and blood supply in determining the lidocaine flux in any tissue below a dermally applied site. The area under the curves determined for underlying tissues using the observed lidocaine concentrations in underlying tissues or concentration difference between treated and contralateral tissues were comparable. The contribution of direct penetration dominates the observed lidocaine flux in any tissue (Table 5). The contribution of blood supply increases with increasing depth suggesting decrease in the efficiency of direct penetration with depth.

The direct penetration of salicylic acid has been shown to be predominant only to a depth of 3-4 mm below the application site and any drug in deeper underlying tissues reaches mainly via systemic blood supply.9 In contrast, direct penetration of lidocaine is evident as deep as underlying deep muscle (- 1-cm

7 h

9.1 f 3.00 2.0 f 0.22 1.4 f 0.30 0.95 f 0.27 0.67 f 0.14 0.51 f 0.10 0.24 f 0.03 0.15 f 0.03

10 h

6.94 f 1.70 3.02 f 1.12 1.75 f 0.34 0.66 f 0.21 0.48 f 0.13 0.32 f 0.10 0.30 f 0.09 0.27 f 0.06

12h

8.42 f 2.63 4.02 f 1.33 1.50 f 0.44 1.18 f 0.37 0.68 f 0.19 0.20 f 0.07 0.06 f 0.02 0.09 f 0.02

l 6 h

9.24 f 2.80 1.26 f 0.39 0.81 f 0.23 0.33 f 0.10 0.15 f 0.04 0.17 f 0.05 0.02 f 0.006 0.04 f 0.007

Table 5-Fractional Contribution to Lldocalne Flux Into a Given Tlssue by Dlffuslon and Perfuslon

Tissue

Dermis Subcutaneous Fascia S Musclee Muscle Fat Pad D Muscle'

AUCTf 0.92 0.27 0.14 0.076 0.042 0.024 0.023

AUCTC, P 0.018 0.013 0.015 0.0096 0.0086 0.014 0.0070

faC - 0.02 0.05 0.11 0.13 0.20 0.58 0.30

fdn.T(*l)+(Alt

0.98 0.95 0.89 0.87 0.80 0.42 0.70

a AUCT,, = Area under the curve of the i th Underlying tissue concentration-time profile. AUCT,,, = Area under the curve of the i th contralateral tissue concentration-time profile. Fractional con- tribution of blood supply (Ma). Fractional contribution of diffusion (1 - c) due to both overlying and underlying tissue. Superficial muscle. ' Deep muscle.

depth). The inconsistency seen with the fat pad arises from the observed higher AUC for contralateral fat pad due to the slow removal of lidocaine both because of the poor blood supply to the fat pad and also due to the inherently greater tissue affinity of fatty tissue for lidocaine. The observed differences between deep tissue penetration of salicylate and lidocaine can be interpreted in terms of the physicochemical differences between the two solutes. Lidocaine is a lipophilic compound (log P = 1.64), being about 25 96 un-ionized at the tissue pH of 7.4 whereas salicylic acid is completely ionized at a pH of 7.4. The un-ionized form of any compound is known to penetrate the biological membranes better than the ionized form. Also, lidocaine is considerably less protein bound relative to salicylate,37 allowing unbound lidocaine to diffuse among the tissues and between tissues and blood. The tissue-plasma partition coefficient is >1 for lidocaine as compared to salicylate (of the order of 0.25), which shows greater tissue affinity for lidocaine than salicylate. Thus the physicochemical properties of lidocaine make it ideal for local direct penetration after topical administration,

P r e d i c t e d C o n c e n t r a t i o n s in U n d e r l y i n g Tis- sues-Sacrificed Animal-Figure 6a shows the simulated curves for underlying tissues in the absence of blood supply (both input and output blood functions in eqs 7 and 9 equal 0). Figure 6b compares the concentrations of lidocaine predicted from the above simulations with the experimentally obtained lidocaine levels in underlying dermis, subcutaneous tissue, fascia, super- ficial muscle, and muscle, after dermal application in sacrificed rats. The predicted and observed values compare well, proving further the validity of the approach.

Epidermis Present-The concentrations in underlying tissues after application to epidermis were predicted according to the model in Figure 1 when the solution compartment was replaced by a constant input source applied to epidermis. For a constant input into the dermis, either no significant depletion of lidocaine should occur from the topical product or the formulation should

780 /Journal of Pharmaceutical Sciences Vol. 83, No. 6, June 1994

C 0

r)

U

- c

z

o.6 r a 1 DERMIS 2 SUBCUT 3 FASCIA 4 S.MUSUE 5 MUSCCE

0

o-8 r -,-

4 8 1 2 1 6

Time ( h r )

b

0.6 - -

0.4 -

DERMIS SUBCUT FASCIA SMUSCLE MUSCLE Tissue

c 0.25 r

0 I I

0.05* 0.00 0 4 8 1 2 1 6

Time ( h r )

Figure 6-Simulations in the model shown in Figure 1 using equations 5, 7, and 9 when the input and output blood functions were assumed to be zero (a), and the concentration fraction of lidocaine in underlying tissues applied to the sacrificed rat dermis (b). Key: (0) predicted from the model, (m) experimentally determined values. (c) The simulated con- centration-time profiles of lidocaine in underlying tissues after topical application according to the model in Figure 1 when the solution compartment was replaced by a constant input source appiii to epidermis. Key: (A) dermis, (B) subcutaneous tissue, (C) fascia, (D) superficial muscle.

be such that the diffusion within the product becomes the rate- limiting step in the absorption process. Figure 6c shows the simulated concentration-time profiles in underlying tissues following topical application of lidocaine with an epidermal permeability coefficient of 0.0042 cm/h and a lag time of 0.42

h (from isolated human epidermal section discussed earlier). The simulated profiles were generated at an initial lidocaine concentration of 2 % . The peak concentrations in the underlying tissues are attained at 2 h and remain fairly constant thereafter over a 16-h period.

Conclusion The direct penetration of lidocaine in tissues below a dermally

applied site in predominantly evident to a depth of about 1 cm for the first -2 h after application. Dermal microcirculation is not a perfect sink for lipophilic solutes such as lidocaine. A physiological pharmacokinetic model based on first-order dif- fusional mass transfer between the tissue compartments below a dermal site of application adequately describes the underlying tissue concentration-time profile of lidocaine. The physico- chemical properties of lidocaine may serve as an index for designing compounds for local deep-tissue targeting after topical application to skin.

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Acknowledgments This work was supported by the National Health & Medical Research

Council of Australia. M.S.R. acknowledges the generous support of Queensland and Northern NSW Lions Kidney and Medical Research

1966,24,389-409. Foundation.

702 / Journal of Pharmaceutical Sciences Vol. 83, No. 6, June 1994