**1. Introduction**

320 Toxicity and Drug Testing

Tirapelli, C. R.; Ambrosio, S. R.; Da Costa, F. B.; Coutinho, S. T.; De Oliveira, D. C. R. & De

*Pharmacology*, Vol.492, No.2-3, (May, 2004), pp. 233-241, ISSN 0014-2999 Valencia, A.; Wens, A.; Ponce-Monter, H.; Pedrón, N.; Gallegos, A. J.; Quijano, L.; Calderón,

56-62, ISSN 0367-326X

102, ISSN 0305-1978

ISSN 1099-1565

ISSN 0378-8741

pp. 434-438, ISSN 0367-326X

pp. 266-270, ISSN 1099-1565

(March/April, 2005), pp. 97-103, ISSN 1071-2690

Vol.68, No.5, (May, 2002), pp. 452-454, ISSN 0032-0943

induced Rat Carotid Contraction. *Fitoterapia*, Vol.73, No.1, (November, 2001), pp.

Oliveira, A. M. (2004). Analysis of the Mechanisms Underlying the Vasorelaxant Action of Kaurenoic Acid in the Isolated Rat Aorta. *European Journal of* 

J.; Gómez, F. & Ríos, T. (1986). Zoapatle XII. *In vitro* effect of kaurenoic acid isolated From Montanoa frutescens and two derivatives upon human spermatozoa *Journal of Ethnopharmacology*, Vol.18, No.1, (October, 1986), pp. 89-94, ISSN 0378-8741 Veneziani, R. C. S.; Camilo, D. & Oliveira, R. (1999). Constituents of *Mikania glomerata*

Sprengel. *Biochemical Systematics and Ecology,* Vol.27, No.1, (January, 1999), pp. 99-

Kaurenoic Acid in *Mikania glomerata* ('guaco') Leaves by Capillary Gas Chromatography. *Phytochemical Analysis*, Vol.8, No.2, (March, 1997), pp. 74-77,

(with Emphasis on Coumarin and Kaurenoic Acid) from *Mikania glomerata* ("guaco") Leaves. *Phytochemical Analisys*, Vol.8, No.5, (September/October, 1997),

Sulfation of 7-hydroxycoumarin in Liver Matrices from Human, Dog, Monkey, Rat, and Mouse. *In Vitro Cellular & Developmental Biology-Animal*, Vol.41, No.3-4,

Bactericidal Activity of the Natural Diterpene Kaurenoic Acid. *Planta Medica*,

(2005). Effects of *Mikania* genus Plants on Growth and Cell Adherence of *Mutans streptococci*. *Journal of Ethnopharmacology*, Vol.97, No.2, (January, 2005), pp. 183-189,

from the Stem Bark of *Mitrephora celebica*. *Fitoterapia*, Vol.73, No.5, (August, 2002),

Vilegas, J. H. Y.; Demarchi, E. & Lancas, F. M. (1997a). Determination of Coumarin and

Vilegas, J. H. Y.; Marchi, E. & Lanças, F. M. (1997b). Extraction of Low-polarity Compounds

Wang, Q.; Jia, R.; Ye, C.; Garcia, M.; Li, J. B. & Hidalgo, I. J. (2005). Glucuronidation and

Wilkens, M.; Alarcon, C.; Urzua, A. & Mendoza, L. (2002) Characterization of the

Yatsuda, R.; Rosalen, P. L.; Cury, J. A.; Murata, R. M.; Rehder, V. L.; Melo, L. V. & Koo, H.

Zgoda-Pols, J. R.; Freyer, A. J.; Killmer, L. B. & Porter, J. R. (2002). Antimicrobial Diterpenes

Progress in the field of biotechnology has accelerated the development of a broad range of novel vaccines, and the composition of vaccine products has evolved from attenuated or inactivated whole-cell organisms, to protein polysaccharide conjugates, peptides, recombinant proteins, DNA vaccines and viral vectors. More recently, there has been a generation of a wide range of complex vaccine products and vaccine technologies (Buckland, 2005) that are often combined with novel adjuvants (Kovarik & Siegrist, 2001; Litvinov, 2009), administered in new delivery systems, and by new routes of inoculation.

In this context, DNA immunization has arisen as a promising strategy for the development of successful vaccines against infectious agents. In fact, some DNA vaccines have been already registered for application in animals (horses, fishes and dogs) against infection with West Nile virus, Infectious haematopoietic necrosis virus or treating melanoma (Liu, 2011). Moreover, thousands of people have already received DNA vaccine candidates in clinical trials without major adverse events (Alvarez-Lajonchere & Dueñas-Carrera, 2009).

DNA vaccination involves the administration of DNA, generally but not always a plasmid, to a host in order to induce a desired immune response. Once into the host, the DNA is taken up by cells, including antigen presenting cells, and the protein(s) expected to be the target of the immune system is/are expressed, processed and presented to specialized cells for induction of immune response. For this purpose, the DNA vaccine must comprise an eukaryotic expression unit, encompassing an enhancer/promoter region, intron, signal sequence, vaccine gene and a transcriptional terminator (poly A), for driving protein synthesis in the host (Glenting & Wessels, 2005). Frequently, DNA vaccines also include immune stimulatory sequences (ISS) for adding adjuvanticity (Glenting & Wessels, 2005). In addition, a unit for the previous propagation of the DNA in the microbial host, in order to obtain the required amounts for vaccination, is normally present, although some compact variants of DNA vaccines are designed for lacking this unit in the final product (Liu, 2011).

DNA immunization has many possible advantages. No dangerous infectious agents are involved, while the expression of the antigen of interest, in its native form, is possible. DNA vaccines can induce innate and adaptive, both humoral and cell mediated, immune

Evaluation of Drug Toxicity for DNA

**2. Safety evaluation** 

from the exposition to the xenobiotics.

applicable to vaccine products.

Vaccine Candidates Against Infectious Diseases: Hepatitis C as Experimental Model 323

vaccine against this pathogen and current therapies are generally effective in only approximately half of patients treated (Ghany et al., 2009). However, some vaccine candidates against HCV are being currently evaluated on clinical trials; two of them being based on DNA immunization (reviewed by Alvarez-Lajonchere & Dueñas-Carrera, 2009).

In addition to immunogenicity demonstration, regulatory agencies require sufficient preclinical data supporting safety to approve initiation of clinical trials of novel vaccines, including DNA vaccine candidates. The regulatory frame has been abundantly settled (Guidelines for assuring the quality of DNA vaccines, 1998; Guidelines on clinical evaluation of vaccines: regulatory expectations, 2004; Guidelines on nonclinical evaluation of vaccines, 2006). Precisely, the principal aim of non-clinical safety examination is to understand the toxicity of the candidate drug well enough to make judgment that the risk/benefits profile is adequate to initiate clinical trials (Contrera, 1993). Toxicity is complex, and impacted by several factors, such as: the xenobiotic, the dosage, the route, the action mechanism and the products of biotransformation. The distribution of many xenobiotics in the body may only affect certain key organs. Others, however, may damage any cell or tissue it enters in contact with. In addition, the toxicity can result in cellular/biochemical or adverse macromolecular changes. Some examples are: cell substitution, as fibrosis; damage to an enzyme system; interruption of protein synthesis; production of undesired chemical reagents in the cells and damages in the DNA. The distribution of toxic substances and toxic metabolites in the whole body determines the organs and tissues where the toxicity is produced. Many toxic substances are stored in the body, and the most common deposits of storage are fatty structures, the bones and highly vacularized organs involved in blood detoxification, such as the liver and the kidneys. The safety evaluation involves the experimental studies directed to determine the toxicity, identifying and quantifying effects and establishing parameters (as dose, toxic and lethal concentrations, etc.) of the substances, using *in vivo* or *in vitro* models. With the information provided by these studies and other data, the Evaluation and the Estimate of the Risk are carried out, as determination of the probability and nature of the effects that can be derived

As for other vaccination strategies, evaluation of safety in the case of DNA immunization requires several considerations and tests. The lots of vaccine candidates to be used in preclinical studies should have been released according to the specifications required for their use in humans. Manufacturers need to establish a reproducible process for producing the

The main challenge in establishing a predictive non-clinical safety assessment comes from the fact that vaccines act through complex multi-stage mechanisms. Thus, the detection of the toxicity of vaccines is likely to be more complex than for conventional chemicallyderived drug products, because safety concerns regarding the immune response to the vaccine add to the general concerns related to exogenous substances administration. Thus, toxicity testing programs recommended for conventional drug products may not always be

The non-clinical safety assessment of vaccines represents a new and evolving field. And clearly, consensus is needed among industry, academia, and regulatory authorities regarding the most appropriate approaches to this area. Depending on the target population

DNA vaccine candidate in a sterile and free of endotoxins condition.

responses. There is a potential for encoding multiple immunogenic epitopes with the purpose of raising protection against several diseases by a single vaccine. Compared with many conventional vaccines, DNA vaccines are relatively stable. Moreover, DNA vaccines are rapid to construct and their manufacture is generic (Liu, 2011).

The above mentioned advantages have resulted attractive for the application of DNA vaccination in the infectious disease field in humans. This immunization strategy has been widely evaluated against a variety of human pathogens; some of them without a current vaccine solution available like hepatitis C virus (HCV) and human immunodeficiency virus (HIV). In fact, DNA immunization has even reached the phase of clinical evaluation in several infectious diseases (Table 1).

The mechanism of action for DNA vaccines and their potential use for therapeutic and preventive purposes imposes relevant challenges for the evaluation of their safety. In addition, knowledge about potential undesirable side effects at long term is still limited. So far, all DNA vaccine candidates entering to clinical evaluation in humans have been previously evaluated for immunogenicity and toxicity in animal models with good results. However, immunogenicity in humans of naked DNA vaccine candidates has not generally fulfilled the expectations. Therefore, several strategies are currently being evaluated for enhancing the immune response, but some of them involve incorporation of components which are potentially able to also increase the toxicity, or might raise the risk for noncontrolled or non-desired immune responses. Consequently, evaluation of toxicity related to DNA-based immunization is a continuously challenged field.


Table 1. Infectious diseases for which DNA vaccines have entered to clinical trials

In this chapter we discuss relevant elements to be considered during the evaluation of toxicity related to DNA vaccines applied to infectious diseases. We will focus on local reactogenicity and systemic toxicity studies, biodistribution, persistence, and integration analysis, as well as immune-related studies for detecting potential adverse events after immunization with DNA-based vaccines candidates against HCV, as a model. We focus on HCV infection since it is a worldwide health problem, causing chronic hepatitis, frequently progressing to cirrhosis and hepatocellular carcinoma. There is no currently available vaccine against this pathogen and current therapies are generally effective in only approximately half of patients treated (Ghany et al., 2009). However, some vaccine candidates against HCV are being currently evaluated on clinical trials; two of them being based on DNA immunization (reviewed by Alvarez-Lajonchere & Dueñas-Carrera, 2009).
