**4. Transdermal drug delivery**

## **4.1 Topical application of drugs: advantages and requirements for evaluation**

Topical application of drugs is known as an interesting alternative route to oral and intravenous administration, both aiming to systemic effects and local action, offering advantages such as ease of administration and lack of first-pass effect (Aqil et al*.*, 2007). However, in studies of transdermal drug delivery (penetration of substances into skin), one must employ non-invasive techniques, in order to avoid second order effects that would at least bring difficulties to the interpretation of the results. *In vivo* measurements are particularly desirable in such studies, because the response of excised skin can be affected by dehydration, and the response of artificial skin differs significantly from that of *in vivo* skin at least in some cases – as when we talk about in-depth processes, in which even blood circulation may play a role.

As mentioned earlier in the present chapter, the PA technique can be applied without previous preparation of the samples and even for *in vivo* measurements; as such, transdermal drug delivery studies have been performed employing the PA technique in order to obtain the penetration rate of a wide range of different products topically applied to skin (Bernengo et al*.*, 1998; Hahn et al*.*, 2001; Savateeva et al*.*, 2001; Pedrochi et al*.*, 2005; Truite et al*.*, 2007).

### **4.2 Substrates for transdermal drug delivery studies**

Besides measurements in human skin, penetration rates of topically applied products are frequently evaluated through measurements performed in animal skin tissue. In this case,

amplitude obtained for all measurements (inner face of the forearm) was 1.5mV, this was the cutoff value adopted for separating the volunteers into "P" (for PA signal amplitude above 1.5 mV) and "NP" (under 1.5 mV). Table 2 shows the division of each (clinically evaluated) phototype group into the (experimentally evaluated) P and NP groups. In this way, the PA technique allowed the comparison between two different skin classification forms.

**Skin phototype NP group (%) P group (%)**  II 93 7 III 46 54 IV 31 69 V 20 80 Table 2. Distribution of the volunteers of each phototype in the NP and P groups, according

Table 2 shows that phototype II is highly related to the NP group, while phototypes IV and V concentrate in the P group. Phototype III appears in both groups, showing the variability

The simple methodology presented here and the corresponding results obtained open perspectives for an objective, experimental classification of skin types, based upon PA measurements. Additional work in this field is currently being performed at

Topical application of drugs is known as an interesting alternative route to oral and intravenous administration, both aiming to systemic effects and local action, offering advantages such as ease of administration and lack of first-pass effect (Aqil et al*.*, 2007). However, in studies of transdermal drug delivery (penetration of substances into skin), one must employ non-invasive techniques, in order to avoid second order effects that would at least bring difficulties to the interpretation of the results. *In vivo* measurements are particularly desirable in such studies, because the response of excised skin can be affected by dehydration, and the response of artificial skin differs significantly from that of *in vivo* skin at least in some cases – as when we talk about in-depth processes, in which even blood

As mentioned earlier in the present chapter, the PA technique can be applied without previous preparation of the samples and even for *in vivo* measurements; as such, transdermal drug delivery studies have been performed employing the PA technique in order to obtain the penetration rate of a wide range of different products topically applied to skin (Bernengo et al*.*, 1998; Hahn et al*.*, 2001; Savateeva et al*.*, 2001; Pedrochi et al*.*, 2005;

Besides measurements in human skin, penetration rates of topically applied products are frequently evaluated through measurements performed in animal skin tissue. In this case,

**4.1 Topical application of drugs: advantages and requirements for evaluation** 

to the PA signal level

FASBio/UNIVAP (Brazil).

circulation may play a role.

**4.2 Substrates for transdermal drug delivery studies** 

Truite et al*.*, 2007).

of elements inside this classification.

**4. Transdermal drug delivery** 

rabbit and pig skin are, by far, the most employed alternatives, because of the similarity to human skin.

As pointed by Simon & Malbach (2000), physiological and anatomical similarities between man and pig make this animal a good model for man in biomedical research. The correlation of quantitative data between pig skin and human skin can be frequently classified as very good (Benech-Kieffer et al., 2000); therefore, pharmacological (and even toxicological) skin research is often based on the knowledge of pig skin absorption and percutaneous permeation.

Recently, Nicoli et al*.* (2008) employed qualitative and quantitative analysis of stratum corneum lipids and permeation experiments to analyze the utilization of rabbit ear skin in transdermal permeation studies, using pig ear skin as a reference. Their results showed that the stratum corneum of both rabbit ear skin and pig ear skin present similar thickness. Probably due to its higher lipophilicity, rabbit ear skin was less permeable to hydrophilic compounds; however, the permeability to progesterone was comparable between isolated pig epidermis and rabbit ear skin. Nicoli and co-workers conclude that the rabbit ear skin can be sucessfully employed in skin permeatin studies.

## **4.3 Photoacoustic evaluation of topically applied products**

Different pharmaceutical formulations for a topically applied drug may present very different transdermal delivery ratios, depending on the product composition (excipients usually play a major role in the penetration kinetics of topically applied products). These penetration rates may be evaluated through the analysis of the time-dependence of the PA signal after topical application of a given product in skin. This methodology can also be applied to the evaluation of sunscreens, that may be characterized in terms of their (photo)stability after topical application (in this case, the lower the rate, the better the product).

Gutierrez-Juarez et al. (2002) employed PA measurements in the analysis of substances topically applied to the human skin. To fulfill this purpose, these authors utilized a doublechamber PA cell; the absorption determination was obtained through the measurement of the thermal effusivity of the binary system substance–skin. The model employed by Gutierrez-Juarez and co-workers (that assumes that the effective thermal effusivity of the binary system corresponds to that of a two-phase system) was experimentally applied to study different topically applied substances, in different parts of the body. The corresponding relative concentrations of substances as a function of time were determined by fitting a sigmoidal function (for ketoconazol and sunscreen) or an exponential function (for nitrofurazona, vaseline and vaporub) to the experimental data.

Pedrochi and co-workers (2005) employed PAS measurements to evaluate the penetration rate of different sunscreens into human skin *in vivo*. Their results showed that the diminution rate of the sunscreen amount in the skin surface depends on the form of the product: sunscreens in cream form tend to present faster reduction after application in skin. This leads to the conclusion that sunscreens in gel form are more adequate (presenting longer protection against UV radiation).

Another transdermal drug delivery study is the work of Truite et al*.* (2007), which employed PAS measurements in the *ex vivo* determination of the penetration rate of different phytotherapic formulations (with and without salicylic acid) for treatment of vitiligo. Measurements were performed as a function of time in rabbits. PA depth monitoring

Photoacoustic Technique Applied to Skin Research:

experimental curves were fitted by the Boltzmann equation:

the penetration process occurs between t0-dt e t0+dt.

(Rosencwaig, 1980).

GraphPad Instat® 3.0.

Boltzmann curve.

**4.3.2 Results and discussion** 

6,0

6,5

7,0

PA amplitude (mV)

7,5

8,0

acquisition.

Characterization of Tissue, Topically Applied Products and Transdermal Drug Delivery 297

frequency employed was 17Hz, so that the foil can be considered as thermally thin

Each measurement series consisted of 40 readings (one each two seconds, for a total time of 80 seconds), repeated 10 times for each application form, with rest intervals of 100 seconds between successive series (total time of 30 minutes for each volunteer and application method). The software "SISCOMF" (developed at UNIVAP) was employed for data

In order to analyze the typical time constant for the penetration of the applied drug, the

A A PA(t) <sup>A</sup> 1 e <sup>−</sup> <sup>−</sup> = +

The Boltzmann curve is a S-shaped curve in which A1 is the initial signal amplitude, A2 the final signal amplitude, t0 is the half-absorption time and dt, the time interval such as 67% of

Analysis of the PA data was performed with the aid of the software Microcal Origin® 7.5 (employed for the generation of the fitting curves); statistical analysis was performed with

Figure 3 shows an example of PA data measurements as a function of time fitted by a

0 5 10 15 20 25

Time (minutes)

Fig. 3. Example of a Boltzmann curve (dashed line) fitting PA data obtained for one of the volunteers after phonophoresis application of the pharmaco *Cordia verbenacea* DC (Acheflan)

1 2 (t t )/dt 2 0

<sup>+</sup> (2)

showed that both formulations propagated through the skin up to the melanocytes region, leading the authors to suggest that the delivery of the active agents may occur even without the use of queratinolitic substances (that are not really recommended, since they are known to induce side effects in animals).

The PA technique can be employed to study the penetration kinetics of topically applied products not only as a function of product composition, but also according to the application method. Phonophoresis is the utilization of ultrasound (US) waves to enhance the delivery of topically applied substances (Byl, 1995). In physiotherapy practice, phonophoresis is one of the various strategies developed to overcome the skin's resistance in transdermal drug delivery, enhancing skin permeability (Duangit et al., 2011).

In the last years, comparative studies between massage and phonophoresis (in its different modes) as application methods for different anti-inflammatories have been in the front line of research at the FASBio/UNIVAP; in the experiments, transdermal drug delivery has been evaluated through PA measurements as a function of time after topical application of different drugs in the forearm region, using manual massage or phonophoresis.

Results indicate that different products present distinct absorption times (depending on the vehicle employed, for example); the application method also affects the typical time constant of drug penetration into skin, though not for all tested formulations.

In the following subsections, we present one experiment performed at FASBio/UNIVAP in which the penetration kinetics of the pharmaco *Cordia verbenacea* DC (Acheflan) in the human skin was evaluated through PA measurements as a function of time for each of the application methods: massage and phonophoresis.

### **4.3.1 Materials and methods**

The pharmaco *Cordia verbenacea* DC (Acheflan) is a topic usage anti-inflammatory medication widely employed in medicine, having alpha-humulen and trans-caryophyllen as active agents. Our experiment aimed to evaluate the penetration kinetics of Acheflan in the human skin (massage *versus* phonophoresis) through *in vivo* PA measurements.

The survey was conducted in 10 volunteers (four men and six women) aged between 18 and 30 years. The following inclusion criteria were adopted: (i) absence of ulcers or any change in dermatology distal forearm and wrist; (ii) not being allergic to any component of the formula topically applied; (iii) absence of metal implants in the wrist or forearm; (iv) absence of stomach pain complaints; and (v) not being pregnant.

The protocol for cleaning prior to drug application consisted of cleaning the skin area to be evaluated (region near the distal forearm and right ulnar artery) with cotton soaked in 70% alcohol. The area of topical application was then demarcated and *Cordia verbenacea* DC (essential oil 5.0mg/g) was applied by rubbing the head of the ultrasound therapy equipment configured for continuous mode and intensity of 1.2W/cm2, but switched off, for five minutes. This procedure was repeated on the opposite forearm with the ultrasound therapy equipment turned on, also for five minutes.

PA measurements employed the same experimental system described in section 3.2.1. During measurements, the volunteers were positioned adjacent to the assembly with an aluminum foil (60μm thick) sealing the PA cell and the distal forearm positioned in direct contact with it, as proposed by Bernengo et al. (1998). For an aluminum foil with this thickness, the cutoff frequency is approximately 7kHz; in the present study, the modulation frequency employed was 17Hz, so that the foil can be considered as thermally thin (Rosencwaig, 1980).

Each measurement series consisted of 40 readings (one each two seconds, for a total time of 80 seconds), repeated 10 times for each application form, with rest intervals of 100 seconds between successive series (total time of 30 minutes for each volunteer and application method). The software "SISCOMF" (developed at UNIVAP) was employed for data acquisition.

In order to analyze the typical time constant for the penetration of the applied drug, the experimental curves were fitted by the Boltzmann equation:

$$\text{PA(t)} = \frac{\mathbf{A}\_1 - \mathbf{A}\_2}{1 + \mathbf{e}^{(t - t\_0)/4t}} + \mathbf{A}\_2 \tag{2}$$

The Boltzmann curve is a S-shaped curve in which A1 is the initial signal amplitude, A2 the final signal amplitude, t0 is the half-absorption time and dt, the time interval such as 67% of the penetration process occurs between t0-dt e t0+dt.

Analysis of the PA data was performed with the aid of the software Microcal Origin® 7.5 (employed for the generation of the fitting curves); statistical analysis was performed with GraphPad Instat® 3.0.

### **4.3.2 Results and discussion**

296 Acoustic Waves – From Microdevices to Helioseismology

showed that both formulations propagated through the skin up to the melanocytes region, leading the authors to suggest that the delivery of the active agents may occur even without the use of queratinolitic substances (that are not really recommended, since they are known

The PA technique can be employed to study the penetration kinetics of topically applied products not only as a function of product composition, but also according to the application method. Phonophoresis is the utilization of ultrasound (US) waves to enhance the delivery of topically applied substances (Byl, 1995). In physiotherapy practice, phonophoresis is one of the various strategies developed to overcome the skin's resistance

In the last years, comparative studies between massage and phonophoresis (in its different modes) as application methods for different anti-inflammatories have been in the front line of research at the FASBio/UNIVAP; in the experiments, transdermal drug delivery has been evaluated through PA measurements as a function of time after topical application of different drugs in the forearm region, using manual massage or

Results indicate that different products present distinct absorption times (depending on the vehicle employed, for example); the application method also affects the typical time constant

In the following subsections, we present one experiment performed at FASBio/UNIVAP in which the penetration kinetics of the pharmaco *Cordia verbenacea* DC (Acheflan) in the human skin was evaluated through PA measurements as a function of time for each of the

The pharmaco *Cordia verbenacea* DC (Acheflan) is a topic usage anti-inflammatory medication widely employed in medicine, having alpha-humulen and trans-caryophyllen as active agents. Our experiment aimed to evaluate the penetration kinetics of Acheflan in the

The survey was conducted in 10 volunteers (four men and six women) aged between 18 and 30 years. The following inclusion criteria were adopted: (i) absence of ulcers or any change in dermatology distal forearm and wrist; (ii) not being allergic to any component of the formula topically applied; (iii) absence of metal implants in the wrist or forearm; (iv)

The protocol for cleaning prior to drug application consisted of cleaning the skin area to be evaluated (region near the distal forearm and right ulnar artery) with cotton soaked in 70% alcohol. The area of topical application was then demarcated and *Cordia verbenacea* DC (essential oil 5.0mg/g) was applied by rubbing the head of the ultrasound therapy equipment configured for continuous mode and intensity of 1.2W/cm2, but switched off, for five minutes. This procedure was repeated on the opposite forearm with the ultrasound

PA measurements employed the same experimental system described in section 3.2.1. During measurements, the volunteers were positioned adjacent to the assembly with an aluminum foil (60μm thick) sealing the PA cell and the distal forearm positioned in direct contact with it, as proposed by Bernengo et al. (1998). For an aluminum foil with this thickness, the cutoff frequency is approximately 7kHz; in the present study, the modulation

human skin (massage *versus* phonophoresis) through *in vivo* PA measurements.

absence of stomach pain complaints; and (v) not being pregnant.

therapy equipment turned on, also for five minutes.

in transdermal drug delivery, enhancing skin permeability (Duangit et al., 2011).

of drug penetration into skin, though not for all tested formulations.

application methods: massage and phonophoresis.

**4.3.1 Materials and methods** 

to induce side effects in animals).

phonophoresis.

Figure 3 shows an example of PA data measurements as a function of time fitted by a Boltzmann curve.

Fig. 3. Example of a Boltzmann curve (dashed line) fitting PA data obtained for one of the volunteers after phonophoresis application of the pharmaco *Cordia verbenacea* DC (Acheflan)

Photoacoustic Technique Applied to Skin Research:

in the kinetics of transdermal drug delivery.

**6. Acknowledgment** 

York, NY.

July 19-23, 2009.

**7. References** 

application form of a wide range of topically applied products.

Characterization of Tissue, Topically Applied Products and Transdermal Drug Delivery 299

measurements have been sucessfully employed in transdermal drug delivery studies, allowing a quantitative analysis of the kinetics and effectivity of drug delivery. Different PA experiments point to the fact that gel formulations tend to be more adequate for topical use. Depending on the topically applied product, the form of application can also be determinant

Measurements already performed indicate various perspectives for future research, such as: i) the determination of the skin oiliness level; ii) analysis of skin lesions; iii) studies on the photostability of sunscreens and even determination of the sun protection factor (SPF) of sunscreens (through PAS measurements); and iv) further studies on formulation and

The authors acknowledge Fapesp and CNPq for financial support of biomedical research being developed at FASBio/UNIVAP, São José dos Campos (SP), involving the characterization of human skin and transdermal drug delivery of topically applied products.

Acosta-Avalos, D.; Alvarado-Gil, J.J.; Vargas, H.; Frías-Hernández, J.; Olalde-Portugal, V.;

Aqil, M.; Abdul, A.; Yasmin, S.; Asgar, A. (2007) Status of terpenes as skin penetration enhancers. *Drug Discovery Today (Oxford)*, v.12, n.23/24, pp.1061-1067. Balderas-Lopez, J.A.; Moreno-Márquez, M.M.; Martínez, J.L.; Sánchez-Sinencio, F. (1999)

Balderas-Lopez, J.A.; Mandelis, A. (2001) Thermal diffusivity measurements in the

Balogun, O.; Regez, B.; Zhang, H.F.; Krishnaswamy, S. (2009) Real time, full-field imaging of

Baumann, L. (2006b) *The Skin Type Solution*. Bantam Books, ISBN-10: 0-553-80422-7, New

Beard, P. (2009) High resolution spectroscopic photoacoustic imaging for characteristic

Benamar, N.; Laplante, A.F.; Lahjomri, F.; Leblanc, R.M. (2004) Modulated photoacoustic

dihydroxyacetone. *Physiological Measurement*, v.25, n.5, pp.1199-1210. Benech-Kieffer, F.; Wegrich, P.; Schwarzenbach, R.; Klecak, G.; Weber, T.; Leclaire, J.;

v.119, n.1-2, pp.183-190; doi:10.1016/0168-9452(96)04454-8

techniques. *Joumal of Applied Physics*, v.90, n.5. pp.2273-2279.

Baumann, L. (2006a) New Skin Typing System. *Skin & Aging* , v.14, n.2, pp.60-64.

discontinuites. *Superfícies y Vacio*, v.8, pp.42-45.

pp.77, Leuven, Belgium, July 19-23, 2009.

Miranda, L.C.M. (1996) Photoacoustic monitoring of the influence of arbuscular mycorrhizal infection on the photosynthesis of corn (Zea mays L.). *Plant Science*,

Thermal characterization of some dental resins using the photoacoustic phase lag

photoacoustic open-cell configuration using simple signal normalization

photoacoustic generated signals for biomedical applications, *ICPPP15 - Book of Abstracts, 15th International Conference on Photoacoustic and Photothermal Phenomena*,

tissue structure and function, *ICPPP15 - Book of Abstracts, 15th International Conference on Photoacoustic and Photothermal Phenomena*, pp.79, Leuven, Belgium,

spectroscopy study of an artificial tanning on human skin induced by

Schaefer, H. (2000) Percutaneous Absorption of Sunscreens in vitro: Interspecies

The experimental results obtained (average values for each parameter of the fitting curve) are summarized in Table 3; interpretation of such results must consider that the initial amplitude (A1) of the PA signal corresponds to the system formed by the applied drug+skin, while the final amplitude (A2) corresponds to the skin only, with the product having penetrated beyond the layer responsible for the generation of the PA signal (about 30µm, in the present case).


Table 3. Average values (± standard error) for A1 and A2 (in arbitrary units), t0 and dt (in minutes), for each of the application methods (N=10)

In order to understand if penetration was effective, first of all, it is imperative to evaluate if the difference between A1 and A2 is statistically significant. Therefore, initially a paired t-test was carried on to verify if there was significant difference between A1 and A2 for each application method employed (indicating significant penetration of the applied product). This was verified for both application methods; however, this difference is more evident for phonophoresis application, in which the difference between the initial and the final signal presents p=0,011 (p=0,066 was found for massage application).

Statistical tests were also employed in the comparison between the two application methods (massage and phonophoresis); no statistical significance was found for t0 and dt. Considering tS = (t0 + dt) as the total effective penetration time for the epidermal layer under study, the results obtained (average ± error, N=10) are 11(±2) minutes (for massage application) and 7(±1) minutes (for phonophoresis application). The paired t-test for this parameter shows p=0,073.

Experiments performed at FASBio/UNIVAP show that the form of application can influence the kinetics of transdermal drug delivery, depending on the applied product. In the experiment presented here, significant penetration has been reported for both forms of administration (massage and phonophoresis); PA measurements showed that effective penetration is at least more evident after phonophoresis application, when compared to massage application.
