Testing Topicals: Analytical Strategies for the In-Vitro Demonstration of Bioequivalence

Editors' note: An abbreviated version of this article appears in the Pharmaceutical Technology Europe APIs, Excipients, & Manufacturing 2018 Supplement. 

Topical pharmaceutical products usually take the form of lotions, creams, gels, ointments, or pastes and are used to deliver locally-acting drugs to their sites of action. Local anesthetics, dermatological treatments for eczema and dermatitis, and antiviral preparations for cold sores are just some of the medications delivered using formulations applied directly to the skin. As many locally acting drugs are deemed to have complex delivery routes, they fall within the growing pharmaceutical category of complex drug products. Complex drug products include drugs with complex active ingredients or formulations and those such as inhalers and nasal sprays, which rely on specific drug-device combinations. The generic versions of complex drug products are in high demand (1), and pressure is mounting to develop these alternatives more quickly and more efficiently.

One challenge in developing a generic version of a locally-acting drug lies in demonstrating its bioequivalence (BE) to its reference-listed drug (RLD) counterpart. For solid oral-dose formulations intended for systemic delivery, this is an established process involving human pharmacokinetic (PK) studies supported by in-vitro dissolution testing. PK studies based on the systemic in vivo attainment of a therapeutically active drug concentration, however, have limited relevance for locally-acting substances. Measuring directly at the site of action also presents challenges, given the potential variability of factors such as exposure time and size of application area. Alternative approaches are therefore being explored and are exemplified in FDA guidance on establishing bioequivalence for acyclovir (2), the API in the topical cold sore treatment Zovirax (GlaxoSmithKline). In-vitro bioequivalence (IVBE) testing is one of the options proposed. Taking acyclovir as an illustration, this article examines IVBE testing requirements for topical creams and explores some of the analytical techniques necessary.

Complex generics and the regulatory landscape

A whitepaper (3) arising from a 2016 conference at the New York Academy of Sciences (4) divides complex drugs into two categories, based on the challenges involved in proving their pharmaceutical equivalence (PE) and BE. The first category includes those products with complex active ingredients and/or complex formulations, for which the demonstration of both PE and BE is difficult. The second category includes products for which delivery routes, dosage forms, or drug-device combinations are complex, and the establishment of either PE or BE is challenging. Because of their delivery route and the difficulties of assessing BE, locally acting topical drugs fall into this second group.

As discussed, for topical, locally acting drugs, systemic PK testing is not always relevant or easily conducted. Therefore, most require clinical endpoint studies to determine BE. However, these studies are costly, time-intensive, and require high patient numbers, creating a barrier to entry for development of generic formulations. Innovators needing to demonstrate batch-to-batch equivalence for post-approval changes or scale-up face a similar challenge (5). In recognition of this, the FDA has issued various product-specific guidance documents for complex drug products, including some locally acting drugs such as cyclosporine ophthalmic emulsions (6) and acyclovir ointments (7). These guidance documents include simplified product-specific BE recommendations and are published on a case-by-case basis. Importantly, they include the use of in-vitro methods to assess bioequivalence, thereby linking the physical and chemical properties of a drug with its efficacy. For locally acting topical drugs, IVBE is restricted to formulations that have qualitative (Q1) and quantitative (Q2) equivalence (see Table I) to the RLD. The availability of suitable analytical techniques, a thorough evaluation and understanding of the reference drug, and identification of the critical process parameters are all necessary precursors to successful implementation of this approach for the development of generics and the reduction of the need for clinical endpoint studies.

Deformulation and bioequivalence

Applying physicochemical characterization methods to the development of pharmaceutical generics is not a new concept, and the now-familiar deformulation workflow for solid-dosage forms shown in Figure 1 was first published in 2005 (8). Fundamental to this is the concept that having quantitative and qualitative information about key RLD ingredients simplifies the optimization of prototype formulations, reducing both the number of experiments needed and the risks along the developmental pathway.

Figure 1: A well-accepted deformulation pathway for solid dosage forms. (Figure courtesy of authors)

This application of physicochemical methods to deformulation can also be used directly in the development of complex generic drugs. As described in Figure 2, FDA sets out three levels of equivalence requirements. Q1 equivalence asks if there is qualitative similarity between the RLD and test (generic) drug product: do they have the same active ingredient and excipients? Q2 equivalence asks if the active and excipient ingredients are present in the same amounts in both products. Q3 equivalence examines whether both the RLD and the test product have the same physicochemical attributes or microstructure. The resulting proposed decision tree in Figure 2 illustrates that showing similarity at the Q3 level delivers the opportunity to move to in-vitro bioequivalence testing and provides the possibility of approval without clinical endpoint studies (9). Not only does this offer considerable cost savings, but potentially a faster time to market. Different Q3 parameters being considered in product-specific guidance documents include: appearance, pH, globule size distribution, rheological behavior, drug release profile, drug particle size distribution, drug polymorphic form, and specific gravity (10). Choosing the correct analytical techniques to measure these parameters and applying them effectively can significantly reduce the time, cost, and risk involved in developing a complex generic product.

Figure 2: Proposed decision tree for bioequivalence (BE) testing (9). (Figure courtesy of the authors).

Acyclovir and FDA guidance for the development of generic versions

Acyclovir was the world’s first successful antiviral drug and has been available in topical form since the early 1980s, with oral and intravenous preparations also prescribed for systemic use. Although marketed originally and still available as Zovirax since its patent expiration in the 1990s, acyclovir has been available under many brand names worldwide. The drug treats viruses of the herpes family, most notably herpes simplex (HSV) Types 1 and 2 (cold sores and genital herpes) and Varicella-zoster virus (chickenpox and shingles). The topical formulation is marketed primarily as a cold sore treatment.

FDA guidance covering topical acyclovir (2) defines the criteria that must be met for formulations to qualify as suitable for IVBE studies. Key among these is that the “test and RLD products are physically and structurally similar based upon an acceptable comparative physicochemical characterization.” More specifically, testing should include the following:

• Assessment of appearance

• Analysis of the acyclovir polymorphic form in the drug product

• Analysis of particle size distribution and crystal habit with representative microscopic images at multiple magnifications

• Analysis of the rheological behavior, which may be characterized using a rheometer that is appropriate for monitoring the non-Newtonian flow behavior of semi-solid dosage forms

• Analysis of specific gravity, water activity, pH, and any other potentially relevant physical and structural similarity characterizations.

The case studies described below illustrate the use of morphological imaging, including Morphologically-Directed Raman Spectroscopy (MDRS) and rheological measurements in the physicochemical characterization of acyclovir cream, and discuss the insight they offer. Specifically, these case studies look at a comparison of an innovator product and a generic version.

Case study: Particle size and shape of the generic and innovator products

Many drug substances exist in more than one crystal form. Such polymorphism can affect a drug’s chemical and physical properties and influence its processing or manufacture. These variations can also affect drug product stability, dissolution, and bioavailability, with consequences for quality, safety, and efficacy. It is, therefore, vital to characterize and understand polymorphs in terms of their particle size, shape, and chemistry. In topical formulations, the particle size and crystal habit of the API will, for example, influence the speed at which it permeates the skin. In-vitro permeation data indicate that for acyclovir, small size rectangular crystals are more rapidly absorbed than large oval ones (11).

MDRS can be used to determine the polymorphic form of the API in a formulation, its particle size, and its crystal habit, enabling direct comparison between test products and the RLD. This technique combines automated static particle imaging with Raman spectroscopy to measure particle size and shape and provide chemical identification of individual particles (Figure 3), enabling the generation of component-specific size and shape distributions.

Figure 3: Morphologically-Directed Raman Spectroscopy (MDRS) provides in-depth understanding of particulate samples, chemically identifying each component in a blend by its Raman spectrum. (Figure courtesy of the authors)

Figure 4 illustrates the measurement process whereby images of a well-dispersed suspension or dry powder sample are captured using automated imaging (Morphologi G3-ID, Malvern Panalytical). Particles of interest are identified from their size and shape, allowing them to be specifically targeted for Raman spectral identification. The combination of data acquired through automated selection, targeting, and chemical identification of thousands of individual particles makes MDRS an efficient technique for understanding the form of specific components within a formulation.

Figure 4: Schematic showing Morphologically-Directed Raman Spectroscopy (MDRS) measurement process. (Figure is courtesy of the authors).

Figure 5 shows how the Morphologi G3-ID first conducts automated morphological analysis for all particles in the acyclovir sample, establishing particle size and shape distributions. The instrument then returns to a selected subset of the total particle population to obtain Raman spectra. This makes it possible to identify which particles are API (the acyclovir 3:2 hydrate form). Figure 4 illustrates the measurement process whereby images of a well-dispersed suspension or dry powder sample are captured using automated imaging (Morphologi G3-ID, Malvern Panalytical). Particles of interest are identified from their size and shape, allowing them to be specifically targeted for Raman spectral identification. The combination of data acquired through automated selection, targeting, and chemical identification of thousands of individual particles makes MDRS an efficient technique for understanding the form of specific components within a formulation.

Figure 5: Using Morphologically-Directed Raman Spectroscopy (MDRS) to determine API polymorphic form in acyclovir cream. (Figure is courtesy of the authors)

Once particle classes have been established, MDRS can be used to determine the particle size distribution (PSD) specifically for the API (Figure 6). In this case, comparing PSDs for the API in the innovator (blue) and generic (red) preparations shows that the API particles in the generic formulation are smaller than those in the innovator. This would be expected to have a significant impact on the solubility rate of the API and, therefore, its bioavailability.

Figure 6: Using Morphologically-Directed Raman Spectroscopy (MDRS) to determine API particle size in acyclovir cream (generic formulation shown in red and innovator in blue). (Figure courtesy of the authors)

Figure 7 shows a comparison of particle shape distributions, using both circularity (left) and elongation (right) parameters. The API in the generic (red) is more circular and less elongated than that in the innovator (blue), which contains more-rectangular API particles. This difference may suggest that, although both formulations contain a similar polymorph, the processing of the generic formulation has led to particle attrition (crystal breakage). This may also explain the differences in particle size.

Figure 7: Using Morphologically-directed Raman Spectroscopy (MDRS) to determine API crystal habit in acyclovir cream (generic formulation shown in red and innovator in blue). (Figure courtesy of authors)

Case study: Rheological parameters

As a complex formulation, acyclovir cream typically consists of a base comprising an oil-wax phase (e.g., liquid paraffin, white soft paraffin and silicone oils), stabilizers, and thickening agents, into which go the API and preservatives, together with additives to improve sensory or functional behavior. Formulation, composition, and processing of the cream all impact its underlying microstructure (droplet volume and size and lamellar structure), in turn influencing the final product’s rheological behavior, evident in such characteristics as flow and deformation. Understanding the rheology of a product is essential in controlling and optimizing the physical properties that deliver the appropriate stability, texture, delivery, and appearance. Rheology is therefore a crucial link between formulation, processing, and final product performance, and measuring a range of rheological parameters provides the information needed to engineer a product’s microstructure toward the desired end-product properties.

FDA guidance on Q3 testing of acyclovir topical creams recommends the following in respect of rheological measurements (2):

• A complete flow curve of shear stress (or viscosity) vs. shear rate should consist of multiple data points across the range of attainable shear rates, until low- or high-shear plateaus are identified

• Yield stress values should be reported if the material tested exhibits plastic flow behavior

• The linear viscoelastic response (storage and loss modulus vs. frequency) should be measured and reported.

Examples for both an innovator and a generic product, with measurements made on a rotational rheometer (Kinexus, Malvern Panalytical), are discussed in the following.

Yield stress. Many emulsions behave like solids at rest because emulsion droplets flocculate to form a floc network. The stress that must be applied to break down this structure and cause the material to flow is known as the yield stress. Generally, the higher the yield stress, the more solid-like the structure. A cream with a higher yield stress will need more force to be squeezed from a tube but will better retain its structure when applied to the skin, allowing a greater amount to stay at the desired location. The cream will also appear and feel thicker.

Measuring yield stress using a rotational rheometer involves linear ramping of shear stress over time and identifying when the viscosity of the sample starts to decrease. Figure 8 shows measurements for the innovator (blue) and a generic (green) acyclovir product, showing that the innovator has a much higher yield stress than the generic product in this case. This structure may make the innovator product easier to apply to the area of skin requiring treatment.

Figure 8: Measuring yield stress in topical acyclovir creams. (Figure courtesy of the authors)

Linear viscoelastic response. Viscoelastic properties are representative of the underlying microstructure of a material before it yields and provide information on its stiffness and elasticity. Measurement involves oscillatory testing on a rotational rheometer, whereby the sample is sheared back and forth using small forces and displacement so as not to disturb the structure. An oscillation frequency of 1 Hz, generally representative of textural processes (in this case, touching), was used to generate the data in Figure 9. G*, the complex shear modulus, which is a measure of total stiffness (from elasticity and viscosity combined) and the phase angle (δ) can be considered as a ratio between viscosity and elasticity and provide information about the viscoelastic properties, or springiness/stiffness of the material. Figure 9 shows that the generic formulation (blue) has a more elastic character, compared to the much higher G* value of the innovator (red), which will deform less with a given amount of force and will feel stiffer to the touch. This corresponds with the yield stress results, because a stiffer structure will require more stress to rupture. Again, the stiffer structure of the innovator product may aid patients in applying the formulation.

Figure 9: Texture mapping showing the linear viscoelastic response of acyclovir creams. (Figure courtesy of the authors)

Viscosity versus shear rate.Figure 10 shows viscosity as a function of shear rate for the innovator (red) and generic (blue) products. Both are shear-thinning, so viscosity decreases as shear rate increases, the result of microstructural reorganization. The innovator has a higher viscosity across all shear rates than the generic and is likely to flow less readily and spread less easily in use. It will also feel thicker on rubbing because of its high viscosity at high shear rates and is likely to more readily hold its structure on application and stay in place for longer, potentially providing more effective treatment.

Figure 10: Viscosity vs shear rate for acyclovir creams. (Figure courtesy of the authors)

Conclusion

In-vitro bioequivalence is of significant interest across the generic drug industry and is especially welcomed by those working with complex generic products, where traditional BE testing may not yield the most appropriate results. FDA bioequivalence guidance documents are now available for a range of complex drug products, including various topical formulations. These documents set out in detail the Q3 physicochemical analysis required for a product to be considered for approval on the basis of IVBE testing, in place of clinical endpoint testing. Selecting the most appropriate analytical techniques for physicochemical characterization will ensure a more complete understanding of the product at both the deformulation stage of generic development, through to manufacture, quality control, and post-approval testing.

Taking acyclovir cream as an example, this article presents data generated using two key technologies: MDRS and rotational rheometry. The data illustrate the type of information required to compare the properties of innovator and generic forms and demonstrate the power of these techniques in providing real insight into topical products and their behaviors. Between these two technologies, it is possible to answer three of the five criteria from the FDA guidance for the physicochemical (Q3) IVBE testing of generic acyclovir products (2).

References

1. R. Lionberger, “Developing New Bioequivalence Approaches for Complex Products,” Presentation delivered at GPhA Fall Technical Meeting, Oct. 29, 2014.
2. FDA, Draft Guidance on Acyclovir (FDA, December 2016).
3. L. Hussaarts, et al., Annals of the New York Academy of Sciences, Vol. 1407, 39–49, DOI 10.1111/nyas.13347 (Apr. 26, 2017)
4. The New York Academy of Sciences, Equivalence of Complex Drug Products: Scientific and Regulatory Challenges, Academy eBriefings, 2016.
5. S. Bhoopathy, et al., AAPS News Magazine18 (2) (February 2015).
6. FDA, Draft Guidance on Cyclosporine (FDA, October 2016).
7. FDA, Draft Guidance on Acyclovir (FDA, March 2012),
8. A. Bansal and V. Koradia, Pharmaceutical Technology 9 (8) (August 2005).
9. Martinez and Fahmy, The AAPS Journal, 17 (2) (March 2015).
10. R. Lionberger, “Pharmaceutical Science of Generic Drugs: The Science of Equivalence,” NIPTE Research Conference, April 30, 2015.
11. R. Lionberger, “FDA, GDUFA Regulatory Science Update,” GDUFA Regulatory Science Public Meeting May 20, 2016.

Article Details

Pharmaceutical Technology Europe
Supplement: APIs, Excipients, & Manufacturing2018
September 2018
Pages: s31­–s34, s36

Citation

When referring to this article, please cite it as P. Kippax, J., and C. O’Grady, " Testing Topicals: Analytical Strategies for the In-VitroDemonstration of Bioequivalence,"Pharmaceutical Technology Europe APIs, Excipients, & Manufacturing2018 (September 2018).

About the Authors

Paul Kippax is director of Product Management–Morphology Group; Jennifer Burt is applications specialist; and Carolyn O’Grady is UK applications manager, all at Malvern Panalytical.