2. Use of cytotoxicity tests

successful medication [3]. The formulated delivery system must support the administration of the API to improve patience compliance [4]. Moreover excipients are able to enhance the API's effectiveness in many cases [5]. Besides their role in solubility or stability issues many excipients are used as penetration enchancers, since their effect on biological membranes [6]. It has been concluded that this advantageous property of excipients must be considered before the formulation but toxicity aspect shows great impact as well [7]. To optimize the procedure cost of this part of preclinical phase, simple but reliable methods must be performed [8]. Traditional drug toxicity tests are great possibilities for the pharmaceutical developers, not only because of the associated loss of human life or health, but also because of immense financial loss of investment [9]. Early application of appropriate cell-based assays in drug development offers the single-most impactful solution to the challenge of human toxicity [10]. In vitro cell line models for evaluating toxicity should predict human specific toxicity. This may be due in part to the success that in vitro screening for certain absorption, distribution, metabolism, and elimination (ADME) endpoints has seen over the past 10 years. In vitro systems designed to evaluate permeability, interaction with membrane transporter systems, and metabolic stability in cell models with human relevance reduced this failure rate to less than 10% [11]. To ensure success in toxicology evaluation methods, comprehensive and tiered screening methods must be employed. To avoid adverse results or conclusions, potential labilities and limitations of the experiments must be investigated [12]. It is unlikely that a single in vitro viability test or cell line model would be sufficient as a final decision point for toxicity [13]. In a well-built toxicity

132 Cytotoxicity

screening procedure, different test types on different cell cultures must be performed.

In tiered approach, in vitro toxicity screening models are based on the cell viability alteration of different human cell lines. These determinations must be well characterized and predictive of in vivo effects with a low incidence of false positive or negative results. The methods must have the capacity to evaluate various molecules in a short period of time with a minimum amount of compound. The results should provide information on potential mechanisms of toxicity, and subcellular targets as well. These results might ensure useful data for the inventors to modify structures of API or alter the amount or types of excipients that are applied in the delivery system development. The modified compositions can be re-screened for toxicity without large cost. The first decisive step is the cell line selection. According to the desired administration route, developers are able to select the most appropriate cell cultures for the in vitro assessments [7]. Nowadays, a wide range of different immortalized or primary cell and tissue models are available from safe sources for in vitro toxicity evaluation. However a well selected cell type ensures valuable information about the developed dosage form, the investigators often faced difficulties; since the main strengths and weakness of the models must be considered wisely. Routine toxicity screening procedures require a robust cell culture model that can be maintained easily in flasks and 96-well culture plates or inserts. The cells must be genetically stable and provide reproducible results in each assessment. The cells should be well characterized in terms of their doubling time, optimal growth conditions, and biochemistry. Various human cell lines are used routinely to evaluate toxicity. For example, Caco-2, HaCaT, CaLu, HeLa, 3 T3, HEK293, and many more. During the cell line selection process, it is important to totally characterize the morphology and understand the relevant biochemistry of the cells. These data are required to understand the background of potential mechanisms of toxicity [14]. In vitro cytotoxicity assays offer special and early identification of potential

### 2.1. Application of cytotoxicity tests

Nowadays the development of a new drug molecule costs around 1 billion dollars and out of 10,000–30,000 possible candidates, only 1 will find its way to the drug market [19]. This means that the number of companies, which have actual financial background for such a research, is decreasing and the whole process of drug development is slowing down. This is the consequence of ICH's GCP protocol, which in one hand, not only created a worldwide secure standard for clinical trials of new drug molecules, but also radically increased the expanses. On the other hand, non-drug related medical researches, such as medical devices (insulin pumps, implants, etc.), also appeared on the market and the requirements of GCP was too complicated and usually unnecessary.

Such circumstances lead to the increased popularity and development of cell culture model systems. Cell lines are a cheap way to investigate the effect of any questioned molecule or device on a given cell type. They can primarily be purchased from cell banks such as European Collection of Authenticated Cell Cultures (ECACC) and the American Type Culture Collection (ATCC). Cell lines can either be primary as they are directly isolated from a tissue or organ. Their structure, protein expression patterns, metabolism, and genetical code are identical with the in vivo cells. Also, they are very sensitive to any effect during cultivation and they have a determined lifespan, meaning that they can only endure a limited number of passages. Secondary cell lines are immortalized by some method, which means that they have a hypothetically infinite lifespan. However, we can say from our own experience that for example Caco-2 cells are best suited for transport experiments (where they have to create a monolayer on an insert) between the 20th and the 30th passages. After 50 passages, the cells are hardly able to reproduce their own number, thus, they are no longer sufficient for cell viability tests.

chemistry, new molecules are synthetized, and even an older compound might be re-evaluated for a new indication. Secondary cell lines are ideal for the screening of the enormous amount of test subjects. If the right cell line is chosen, not just the cytotoxicity, but the biological activity of the chosen material can be measured. In short time, multiple experiments can be carried out, with high reproducibility in the same test system. If we use more than one method, with different signaling mechanisms and different cell lines, we will have a more complex view on the in vitro toxicity data. This means more information when planning the in vivo experiments. Generally, it can be said, that if a compound proves to be non-toxic, then in vivo it will be tolerable. If it is moderately toxic, there is a chance that the 3D structure and the different cell types of an organ or the human body can effectively recover from the cytotoxic damage or the

Role of Cytotoxicity Experiments in Pharmaceutical Development

http://dx.doi.org/10.5772/intechopen.72539

135

The results of the cytotoxicity tests require additional consideration. They—even if the right cell lines were used—are not an automatic green light for in vivo application. If a given chemical proves to be non-toxic, it only means that we do not necessarily have to start our next experiment in an animal with the smallest dosage, but from a medium or a high concentration, to determine the maximum single dose, maximum daily dose and the LD50 value. Also, if we do not use the appropriate cell line—like testing an ointment preservative on enterocytes—then the scientific value of our study will be questioned. The cytotoxicity tests usually end up in a specific IC50 value. These values usually cannot be compared, because these tests are highly dependent on the parameters of the test system. Such parameters can be: cell line, passage number of the cells, number of cells/well, volume of medium/well, growth time of a plate, concentration/volume of reagent, manufacturer of the reagent, length of incubation, reaction time, solubilization solution (if needed) even the performance of the spectrophotometer. We suggest that instead of using static IC50 values, trends, comparisons should be seen. Stating that compound A has an IC50 value of 0.5 mg/ml and B has 0.25 mg/ml should be changed that A is about twice as tolerable, than B. This fact does not decrease the significance of the cytotoxicity tests, but prevents to deny of a scientifically accurate study, because the written IC50 value cannot be reproduced. Another issue is that a specific compound can interact with the reagents or the mechanism of the method, thus a false positive or negative result can be detected. Such interaction can only be found if we use more than one method or in the scientific literature. If we only use one type of cytotoxicity test, the scientific value will be low and the chance of detecting false results will be high. This is not necessarily caused by a specific interaction, but because of the given test system, the whole method will over- or underestimate cytotoxicity. Thus, it is advisable to use different types of tests, so link MTT with LDH or RT-CES, but not with XTT, because they are both tetrazolium

The cellular damage caused by different chemical compounds can be various and thus, the methods to measure this effect are numerous. To select the proper test, we must know: number

damage will only be minimal.

2.3. Disadvantages of cytotoxicity tests

based assays and have the same limitations.

3. Description of different cytotoxicity tests

Also, it is crucial to choose the right cell line for the given experiment. If the question is the biocompatibility of a chemical compound, which is about to be used on humans, then a human cell line must be chosen for the given experiment and even, an appropriate organ should be selected. A good summarizing table was created by Amelian et al. [20] (Table 1).

As it can be seen, there are multiple available cell lines with the same origin. The selection and the test system must be based on the later application of the device/compound, as each cell line has a different medium requirement and cultivation method as they can act differently in cell viability tests.

Also, because anti-proliferative drugs main attribute is their cytotoxicity, the following methods are ideal for testing these substances on cell lines. Because the secondary, immortalized cells can be seen as cancer cells (because their apoptotic or growth stop signals are suppressed by mutations), they are capable to react to certain promising anti-cancer molecules like their in vivo counterparts.

#### 2.2. Advantages of cytotoxicity tests

As scientific and medical studies are getting more expensive over the recent decades, most of the universities, research institutes are underfinanced, the importance of a certain method's price is greater than ever before. Animal experiments are expensive and the administrative burden is overwhelming, so they are only used when no other test is suitable. Also, every year new plants and their respective metabolites are described and through the various methods of


Table 1. Most common cell lines used in cytotoxicity studies [20].

chemistry, new molecules are synthetized, and even an older compound might be re-evaluated for a new indication. Secondary cell lines are ideal for the screening of the enormous amount of test subjects. If the right cell line is chosen, not just the cytotoxicity, but the biological activity of the chosen material can be measured. In short time, multiple experiments can be carried out, with high reproducibility in the same test system. If we use more than one method, with different signaling mechanisms and different cell lines, we will have a more complex view on the in vitro toxicity data. This means more information when planning the in vivo experiments. Generally, it can be said, that if a compound proves to be non-toxic, then in vivo it will be tolerable. If it is moderately toxic, there is a chance that the 3D structure and the different cell types of an organ or the human body can effectively recover from the cytotoxic damage or the damage will only be minimal.
