What is Melanin Binding and How to Study It In Vitro?

Melanin is widely distributed in various tissues of the body, including the eyes and skin. Drugs binding to melanin may result in drug retention in these tissues, thereby influencing their distribution and elimination of ophthalmic drugs ¹. This can lead to changes in ocular pharmacokinetics (PK) and pharmacodynamics (PD), potentially affecting normal retinal function. Furthermore, prolonged accumulation of drugs that strongly bind to melanin within tissues can result in adverse effects, such as increased ocular phototoxicity and inner ear toxicity. The 2015 ICH S10 Photosafety Evaluation of Pharmaceuticals: Guidance for Industry recommends assessing the in vitro binding of drugs to melanin. This article discusses in vitro research methods for drug binding to melanin and highlights the significance and impact of drug–melanin binding, aiming to provide valuable insights to facilitate the development of novel drugs.

1. Definition and function of melanin

Melanin, an anionic polymer derived from tyrosine through enzymatic and spontaneous reactions, is synthesized within melanosomes which are organelles covered by a lipid membrane (Figure 1) ². Melanin is widely distributed in the eyes, skin, hair, brain, and inner ears. It is classified into two main types (eumelanin and pheomelanin), which differ in color and molecular structure. Notably, melanin exhibits exceptional antioxidant activity and reduces the generation of reactive oxygen species through the chelation of metal ions. These characteristics play a crucial role in safeguarding pigment cells against the adverse impacts of natural toxins³.

Figure 1. Route of melanin synthesis ²

2. Effects of drug binding to melanin

It is reported that the majority of drugs binding to melanin are small molecules. Although some reports show that proteins and oligonucleotides bind to melanin, large molecules encounter challenges in crossing the lipid membrane and melanosomal membrane due to their poor permeability, making it difficult for them to reach the melanin in vivo. In comparison to in vivo methods for studying melanin binding, in vitro melanin binding methods offer two distinct advantages:

1. In vitro studies have the advantages of low cost and simple operation.
2. In vitro melanin binding serves as a crucial reference for in vivo preclinical studies

2.1 Pharmacological effects of drug binding to melanin

Binding to melanin substantially impacts ocular PK and pharmacodynamics. Drug binding to melanin could increase the drug’s retention in the eyes, leading to a prolonged half-life. Additionally, melanin-bound drugs can be slowly released, allowing the free drug to reach the target site and further enhance drug efficacy (Figure 2). Therefore, melanin binding must be assessed during drug development to evaluate its role in ocular drug retention.

Figure 2. Principles of pharmacological effects resulting from the binding of drugs to ocular melanin

2.1.1 Prolonged drug half-life of drug binding to melanin

The differences in the pharmacological effects of certain ophthalmic drugs in pigmented and albino animals have been investigated since the 1970s. Drug–melanin binding in pigmented animals in vivo could result in a small reservoir that gradually releases the drug into tissues, influencing the PK, efficacy, and safety of the drug. For instance, the retention and action time of atropine ² in pigmented animals were notably longer than that in albino animals (with half-lives of 96h and 43 h, respectively). The accumulation of drugs in melanin tissues extends the drug efficacy and may also lead to potential toxicity. Therefore, research on drug binding to melanin needs to be paid attention.

2.1.2 Enhanced drug targeting of drug binding to melanin

Using the characteristics of drug–melanin binding in drug delivery systems for the treatment of retinal diseases to modify compounds to enhance their basicity and lipophilicity, which can increase the binding of compounds to melanin and targeting of drugs for the treatment of retinal diseases. For example, the antivascular endothelial growth factor (VEGF) drug pazopanib ⁴ forms a drug reservoir in the choroid uvea after a single oral dosing, allowing for a slow release that effectively hindered angiogenesis in a choroidal neovascularization model. These indicate that the drug binding to melanin influences the extent and process of the drug’s pharmacological effects, offering a promising approach for treating choroidal neovascularization (CNV) lesions.

2.2 Potential adverse effects of drug–melanin binding

Due to the binding of drugs to melanin, melanosomes in pigmented ocular cells can serve as reservoirs for drug accumulation compared to non-pigmented tissues. Many drugs, including non-ocular drugs administered systemically (such as chloroquine, and chlorpromazine) may accumulate in melanin-rich tissues and cause adverse reactions. Such as interference with retinal function, unexpected visual consequences, and phototoxicity and ototoxicity. Therefore, studying the binding of drugs to melanin should be prioritized.

2.2.1 Ocular phototoxicity

Certain drugs (e.g., sparfloxacin) can act as photosensitizers, inducing phototoxic effects in the eye ⁵. When these drugs absorb sunlight, they trigger a phototoxic reaction, they generate reactive oxygen species and oxidative stress within the cells, leading to phototoxic reactions and cellular damage ^5,6. Among the melanin-binding drugs, quinolone antibiotics ^7,8 and chlorpromazine ⁹ exhibit phototoxicity. For instance, substantial cellular damage has been observed in the retinal pigment epithelium of mice and rabbits after the administration of sparfloxacin and ofloxacin, respectively ^7,10. Shimoda and Kato ¹¹ also observed a phototoxic response in the retina of albino animals after quinolone treatment followed by exposure to ultraviolet light.

2.2.2 Inner ear toxicity

Aminoglycoside antibiotics are extensively used as bacteriostatic drugs but potentially induce severe nephrotoxicity and ototoxicity. These antibiotics display notably higher inner ear toxicity in pigmented guinea pigs than in albino guinea pigs, suggesting a possible correlation between their ototoxic effects and melanin ¹². This could lead to the accumulation of antibiotics in the pigmented tissues of the inner ear and promote the toxic effects on surrounding cells.

Summary: The long-term accumulation of drugs in ocular tissues can result in various adverse reactions. Therefore, it is necessary to develop research methods for drug binding to melanin in vitro and corroborate them with in vivo assays to elucidate the specific mechanisms of drug-melanin binding. This will help to reduce the toxicity arising from the long-term accumulation of drugs in the eye or other tissues.

3. Research methods for drug binding to melanin in vitro

The presently available in vitro methods for studying drug binding to melanin include equilibrium dialysis, rapid equilibrium dialysis, and centrifugation. WuXi AppTec DMPK has selected equilibrium dialysis and centrifugation for validation and established an in vitro research method for evaluating drug binding to melanin. Melanin is derived from various sources ² (Table 1), with the most prevalent natural melanin being derived from the eyes of cattle, pigs, and squid ink.

Table 1. Melanin from different sources ².

3.1 Equilibrium dialysis method

The equilibrium dialysis method was employed to determine the binding rate of compounds to melanin in vitro and it closely resembles the plasma equilibrium dialysis. The bound and free drugs are separated through equilibrium dialysis after a specific incubation period (Figure 3). The unbound, bound, and recovery rates of the compounds with melanin are subsequently calculated to determine the distribution of the compounds within melanin.

Nine approved commercial drugs, including atenolol, chloroquine, propranolol, diclofenac, nadolol, quinidine, fluconazole, timolol maleate, and chlorpromazine, were selected as test compounds. Their binding to squid melanin was confirmed through equilibrium dialysis (bound%). The study results strongly correlated with the values in the literature, with a goodness of fit R² value exceeding 0.9 (Figure 4).

Figure 3. Equilibrium dialysis procedure

Figure 4. Correlation between drug binding to melanin as determined using the equilibrium dialysis method (In-house %Bound) and the values in the literature (Literature %Bound)

3.2 Centrifugation method

Centrifugation was employed to assess the binding of various compounds to melanin in vitro. Under specific conditions of speed, time, etc., the bound and free drugs are separated through centrifugation (Figure 5). LC-MS/MS analysis is used to detect the F (free sample), T (total sample), and T0 (sample at time zero) of the compounds. These data are then used to calculate the unbound, bound, and recovery rates of the compounds with the melanin, which is followed by assessing the distribution of the compounds within the melanin.

To verify the feasibility of the method, the nine commercially available standard compounds mentioned above were selected to verify the binding of these compounds to melanin using the centrifugation method, obtaining the binding rate (Bound%) in cuttlefish melanin. The study results strongly correlated with the values in the literature, with a goodness of fit R² value exceeding 0.9 (Figure 6).

Figure 5. Centrifugation procedure.

Figure 6. The correlation between drug binding to melanin was determined using the centrifugation method (In-house %Bound) and the values in the literature (Literature %Bound).

4. Integrated application of in vitro melanin–drug binding models in drug research and development

Despite numerous examples demonstrating the biological relevance of melanin binding in drug development, the PK transformation from in vitro to in vivo is not fully elucidated ¹³. Studies have shown that alkaline, lipophilic, and aromatic molecules exhibit the highest degree of binding to melanin when assessed using melanin that was isolated in vitro. Although melanosome binding degree can be quantified using the dissociation constant (K_d), the accurate measurement of K_d at various concentrations and time points is expensive and prone to error. Our in vitro assay generally determined the free fraction (f_u) value for drug–melanin binding and obtained results with low error and cost, providing a reference for in vivo preclinical studies. Therefore, employing this in vitro model and comprehending the mechanistic interactions in drug–melanin binding with in vivo metabolic processes may aid in applying in vitro assay data to predict the in vivo binding of drugs to melanin. This approach offers guidance for the rational design of new drug candidates with desirable physicochemical properties and absorption, distribution, metabolism, and excretion (ADME) characteristics. 

Conclusions

WuXi AppTec DMPK has successfully established a high-throughput in vitro model for drug–melanin binding studies, which is fast, efficient, and cost-effective. This model facilitates a more precise understanding of the accumulation and distribution of topical ophthalmic drugs and systemic drugs in melanin, providing a valuable reference for relevant pharmacokinetics and pharmacological studies.

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Authors: Chunhong Lu, Xue Tang, Jie Wang, Xiangling Wang, and Genfu Chen

Committed to accelerating drug discovery and development, we offer a full range of discovery screening, preclinical development, clinical drug metabolism, and pharmacokinetic (DMPK) platforms and services. With research facilities in the United States (New Jersey) and China (Shanghai, Suzhou, Nanjing, and Nantong), 1,000+ scientists, and over fifteen years of experience in Investigational New Drug (IND) application, our DMPK team at WuXi AppTec are serving 1,500+ global clients, and have successfully supported 1,200+ IND applications.