Protein pharmacokinetics

A prerequisite to pharmacokinetic/pharmacodynamic studies is the availability of a sufficiently selective and sensitive assay. The assay must be capable of detecting and accurately quantifying the therapeutic protein in the presence of a complex soup of 'contaminant' molecules characteristic of tissue extracts/body fluids. As described in Chapter 7, specific proteins are usually detected and quantified either via immunoassay or bioassay. Additional analytical approaches occasionally used include liquid chromatography (e.g. HPLC) or the use of radioactively labelled protein.

The macromolecular structure of drugs and the fact that relatively minor structural alterations can potentially have a major influence upon bioactivity are often complicating factors. For example, an immunoassay may be blind to the oxidation of an amino acid residue, or very limited proteolytic processing, although such events can activate or decrease bioactivity.

As outlined previously, i.v. or s.c. administration is by far the most common delivery approach in the context of biopharmaceuticals. Whole-body distribution studies are undertaken mainly in order to assess tissue targeting and to identify the major elimination routes. The large molecular weight of therapeutic proteins, along with additional properties (e.g. charge), generally impairs their passage through biomembranes; hence, their initial distribution is usually limited to the volume of the extracellular space (mainly the plasma volume). Distribution volume usually subsequently increases, as the protein is taken up into tissue during its metabolism/elimination.

The metabolism/elimination of therapeutic proteins occurs via processes identical to those pertaining to native endogenous proteins. Ultimately this entails proteolytic degradation, with amino acid residues released either being incorporated into newly synthesized protein or being further degraded by standard metabolic pathways. Although the therapeutic protein may be subject to limited proteoly-sis in the blood, extensive and full metabolism occurs intracellularly, subsequent to product cellular uptake. Clearance of protein drugs from systemic circulation commences with passage across the capillary endothelia. The rate of passage depends upon the protein's physicochemical properties (e.g. mass and charge). Final product excretion is, in the main, either renal and/or hepatic mediated.

Many proteins of molecular mass <30 kDa are eliminated by the kidneys via glomerular filtration. In addition to size, filtration is also dependent upon the protein's charge characteristics. Owing to the presence of glycosaminoglycans, the glomerular filter is itself negatively charged, so negatively charged proteins are poorly filtered due to charge repulsion.

After initial filtration many proteins are actively reabsorbed (endocytosed) by the proximal tubules and subjected to lysosomal degradation, with subsequent amino acid reabsorption. Thus, very little intact protein actually enters the urine.

Uptake of protein by hepatocytes can occur via one of two mechanisms: (a) receptor-mediated en-docytosis or (b) non-selective pinocytosis, again with subsequent protein proteolysis. Similarly, a proportion of some proteins are likely degraded within the target tissue, as binding to their functional cell surface receptors triggers endocytotic internalization of the receptor ligand complex (Figure 4.7).

Cellular uptake of some glycosylated therapeutic proteins occurs via specific sugar-binding cell surface receptors. Cell surface mannose receptors, for example, are capable of binding glycoproteins whose sugar side chains terminate in mannose, fucose, N-acetyl glucosamine or N-acetyl galactosamine. Evidence suggests that a liver-specific form of the mannose receptor mediates clearance of luteinizing hormone (LH, Chapter 11). The sugar side chains of many glycoproteins exhibit terminal sialic acid residues (sialic acid caps). The hepatic asialoglycoprotein receptor binds glycoproteins whose sialic acid caps have been removed, likely mediating their removal from general circulation.

Pharmacokinetic and indeed pharmacodynamic characteristics of therapeutic proteins can be rendered (even more) complicated by a number of factors, including:

• The presence of serum-binding proteins. Some biopharmaceuticals (including insulin-like growth factor (IGF), GH and certain cytokines) are notable in that the blood contains proteins that specifically bind them. Such binding proteins can function naturally as transporters or activators, and binding can affect characteristics such as serum elimination rates.

lysosome lysosome


Figure 4.7 The process of receptor-mediated endocytosis. Binding of Ligand, in this case a therapeutic protein, to its cell surface receptor (a) triggers invagination of the immediately surrounding area of plasma membrane, internalizing the receptor and its ligand in an intracellular vesicle (b). This is usually followed by fusion of the internalized vesicle with a lysosome and, therefore, degradation of both ligand and receptor by lysosomal hydrolases (c). In some cases, however, a variation can occur in which the ligand disassociated from the receptor (due to a Lower pH in the vesicLe), with subsequent budding off a smaLL receptor-containing section of the vesicle, which returns the receptor to the cell surface. In this situation, only the ligand is available for degradation upon subsequent vesicular fusion with the lysosome (d)

• Immunogenicity. Many, if not most, therapeutic proteins are potentially immunogenic when administered to humans. The likelihood that non-human proteins (e.g. murine monoclonal antibodies; Chapter 13) are immunogenic in humans is an obvious one. However, human proteins can also be potentially immunogenic, as discussed in Box 4.1. Antibodies raised in this way can bind the therapeutic protein, neutralizing its activity and/or affecting its serum half-life.

• Sugar profile of glycoproteins. Expression of a therapeutic glycoprotein in different eukaryotic expression systems results in a product displaying differences in exact glycosylation detail (Chapter 2). The exact glycosylation pattern can influence protein activity and stability in vivo, and some sugar motifs characteristic of yeast-, insect- and plant-based expression systems are immunogenic in man.

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