ABSTRACT:During the development of any PEGylated protein or peptide, toxicology in relevant species will be conducted prior to human exposure. Normally, comprehensive metabolism data accompany the toxicity studies for a small molecule. We have examined whether such studies would be relevant in the safety assessment of PEGylated material. Literature data indicate that the polyethylene glycol (PEG) associated with a biological molecule should provide no extra concern because the exposure-toxicity relationship of PEG in animals and humans has been thoroughly investigated and metabolism/excretion of PEG is well understood. Based on the comparisons of PEG exposure from PEGylated biological products and the exposure of PEG associated with toxicity in humans, the therapeutic index is large (approximately 600-fold or greater). Therefore, assuming that toxicological evaluation of a biological molecule of interest is complete and satisfactory therapeutic windows are achieved, the data contained in this review indicate that the PEG associated with a protein or other biological molecule does not represent an additional unquantified risk to humans.The conjugation of small proteins, peptides, and oligonucleotides with polyethylene glycol (PEG), or PEGylation, has become an increasingly common method of improving the half-life of biological products, mainly through reducing the urinary excretion of the molecule (Yang et al., 2004), but also by reducing the enzymic degradation due to the increased steric bulk (Veronese and Pasut, 2005). In addition, PEGylated biological products often exhibit a reduced affinity for the target receptor compared with the native precursor. This reduced affinity can lead to a lower clearance by target-mediated clearance mechanisms. Finally, the addition of the PEG moiety can have beneficial effects on the immunological profile of a molecule by reducing the ability of the compound to raise antibodies in humans (Mehvar, 2000).PEG is a polymer made up of identical ethylene glycol subunits. PEGs have a descriptor associated with them that represents the mean molecular weight of the molecule (i.e., PEG200 has a molecular weight of 200) (Smyth et al., 1955). The PEG molecules conjugated to proteins can also have the terminal hydroxyl group capped with a methyl group (Molineux, 2003). The structures of these PEGs are detailed in Fig. 1. Higher molecular weight PEGs can have some degree of branching. The PEGs used to conjugate biologicals are polydispersed in nature (i.e., have a range of molecular weights), and this can to lead to a range of drug molecules with potentially subtly different biological properties. The impact of polydispersity must be considered when dealing with these conjugated biological agents (Veronese and Pasut, 2005).To couple the PEG to the protein, peptide, or oligonucleotide, the PEG (generally monomethoxy PEG) is first activated. Several methods can be used to achieve this activation and coupling, including cyanuric chloride, 1,1Ј-carbonyldiimidazole, phenylchloroformate, or succi...

Both in vitro and in vivo evidence indicate that cAMP-dependent protein kinase (PKA) mediates some of the acute and chronic cellular responses to alcohol. However, it is unclear whether PKA regulates voluntary alcohol consumption. We therefore studied alcohol consumption by mice that completely lack the regulatory IIbeta (RIIbeta) subunit of PKA as a result of targeted gene disruption. Here we report that RIIbeta knockout mice (RIIbeta-/-) showed incr eased consumption of solutions containing 6, 10, and 20% (v/v) ethanol when compared with wild-type mice (RIIbeta+/+). On the other hand, RIIbeta-/- mice showed normal consumption of solutions containing either sucrose or quinine. When compared with wild-type mice, the RIIbeta-/- mice were found to be less sensitive to the sedative effects of ethanol as measured by more rapid recovery from ethanol-induced sleep, even though plasma ethanol concentrations did not differ significantly from those of controls. Finally, both RIbeta- and catylatic subunit beta1-deficient mice showed normal voluntary consumption of ethanol, indicating that increased ethanol consumption is not a general characteristic associated with deletion of PKA subunits. These data demonstrate a role for the RIIbeta subunit of PKA in regulating voluntary consumption of alcohol and sensitivity to the intoxication effects that are produced by this drug.

Sildenafil citrate, marketed as Viagra s , for the treatment of erectile dysfunction, has a proven record of safety in humans as predicted by the results of extensive pharmacological and toxicological testing in animals and in vitro, and confirmed by pharmacokinetic exposure data. The aim of this paper is to review succinctly the main findings resulting from these experiments. Daily doses of sildenafil, within and far beyond the human therapeutic range, were given to dogs and rodents for up to 1 and 2 y, respectively. Plasma analyses were conducted to determine the exposure to sildenafil. We found species-specific effects in dogs (Beagle pain syndrome), mice (marked intestinal dilatation) and rats (adaptive reversible hepatocellular hypertrophy associated with secondary thyroid hypertrophy). All these effects in rodents and dogs have no relevance to humans. Morphometric thickness measurements of the retinal layers carried out in response to clinical observations of visual disturbances in humans indicated no difference between treated and control rats and dogs after up to 24 months of treatment. There was no evidence of histopathologic damage to any structures of the visual pathway. Sildenafil had no effects on fertility, no teratogenic potential, was not genotoxic and has no carcinogenic potential. In rats and dogs, safety ratios were 40:1 and 28:1, respectively, in terms of exposure over 24 h (AUC 24 h ) and 19:1 and 8:1, respectively, in terms of peak plasma concentration (C max ). These safety ratios illustrate the separation between exposure to sildenafil of animals at large nontoxic doses and the much smaller human therapeutic exposure. This profile highlights the very low risk of human toxicity for sildenafil. The favourable results of the nonclinical safety evaluation of sildenafil in established animal models have been confirmed by many years of clinical experience during the development and marketing of sildenafil.

A pediatric assessment is now a required component of every New Drug Application in North America or Marketing Authorization Application in Europe, unless a waiver has been granted previously. Nonclinical juvenile toxicity studies are usually required as part of this assessment. The protocols for juvenile toxicity studies are devised in consultation with the FDA or EMEA. It is important to approach the regulatory authority well in advance in order not to delay the marketing authorization of the drug and to confirm the need or not to perform a preclinical evaluation in juvenile animals. The choice of species and the design of juvenile studies are based on a series of complex considerations, including: the therapeutic use of the drug, the age at which children will be treated, the duration of treatment, and potential age- or species-specific differences in pharmacokinetics or toxicity.

Since more than 10 years, risk assessment of bisphenol A (BPA) is debated at the international level. In 2008, the U.S. National Toxicology Program (NTP) expressed some concern for adverse effects, at current level of exposure to BPA, on developmental toxicity. In this context, the French Food Safety Agency (AFSSA) decided to review the toxicity data on BPA with a special focus on this endpoint at doses below 5mg/kg bw/day (the no observed adverse effect level set by different regulatory bodies). This paper summarizes the conclusions of a collective assessment conducted by an expert Working Group from AFSSA. Studies were classified into 3 groups: (i) finding no toxicity, (ii) reporting results not considered to be of concern and (iii) indicating warning signals. The term "warning signal" means that no formal conclusion can be drawn regarding the establishment of a health based guidance value but the study raises some questions about the toxicity of BPA at low doses. It was concluded that studies are needed to ascertain the significance for human health of these warning signals and to be able to propose new methodologies for assessing the risks associated with low doses of BPA and more generally of endocrine disruptors.