Elimination and Metabolism

Molecular Weight. The molecular size of a protein is a critical factor in determining rate of elimination and duration of action. Small-protein molecules can be filtered in the kidneys and directly excreted, without reabsorption, into urine. The larger the molecular size of a protein, the lower its renal clearance. The limit of glomerular filtration is estimated to be 50 to 70kDa. Because the molecular weights of peptides and proteins range from a few thousand daltons to greater than 150kDa, many bio-pharmaceuticals are eliminated in part by renal excretion. The importance of renal excretion in protein elimination means that changes in renal function (e.g., due to medical disorders) may have an impact on protein therapy.

When proteins exceed a molecular weight of 200 kDa, phagocytosis plays an increasing role in elimination. Phagocytosis is also important when a partially degraded small-protein aggregate exhibits the characteristics of very large proteins. Internal-ization of large proteins or protein aggregates by phagocytes leads to intra-cellular proteolytic inactivation.

N: Native

I : Intermediate

In: Aggregated intermediate

U: Unfolded

Pt: Precipitated

Figure 5.2. Inactivation of protein from native to various denaturation states leading to formation of protein aggregates, detectable as precipitates.

Physical and Chemical Integrity of Proteins. The primary sequence of proteins and peptides is comprised of l-amino acids linked together by covalent amide bonds. Substituent group polarity and/or charge is a critical determinant of secondary and tertiary structure and stability. Secondary structures (a-helices and b-sheets) arise from hydrophobic, ionic, and Van der Waals interactions that fold the primary amino acid chain upon itself. Most therapeutic proteins exhibit tertiary structure vital to functionality and are held together by covalent and noncovalent bonding of secondary structures (Figure 5.2).

All proteins and peptides display chemical and physical instability that affects the way they are distributed and cleared in the body and their delivery to the site of action. Physical and chemical instability is affected by primary sequences and secondary and tertiary structures and the degree of glyco-sylation of protein. Chemical degradation of proteins and peptides involves deamida-tion, racemization, hydrolysis, oxidation, beta elimination, and disulfide exchange. Physical degradation of proteins involves denaturation and aggregation.

Proteases found in blood are catalysts that accelerate hydrolysis and other chemical degradation processes. Furthermore, chemical degradation almost always leads to physical degradation.

Mechanisms of Chemical and Physical Degradation of Proteins. A folded native protein exists in a local minimum-energy conformation. Typically 5 to 15kcal/mol of energy is needed to rapidly (in milli seconds) convert a folded protein to an unfolded conformation. In general, proteins and peptides fold in a manner that minimizes the exposure of hydrophobic regions of the molecule to water or an aqueous environment in a biological milieu. The unique structure that the molecule takes will depend on both the surrounding environment (buffered solution, biologic membrane, blood, plasma, etc.), and the primary amino acid sequence. The unfolding of a native protein leads to denaturation (Figure 5.2). The denatura-tion process may involve a number of intermediate conformations, leading to protein aggregates and precipitates that are readily cleared from the body. In most cases, partially denatured proteins exhibit greatly reduced or no biological activity. In some cases, partially denatured proteins are immunogenic and may play a role in eliciting an antibody response against native therapeutic proteins.

Any chemical or enzymatic process that modifies a therapeutic protein in the body is known as protein metabolism. Unfolded proteins are, in general, more susceptible to metabolism via proteolysis because of increased access to critical peptide sequences. Chemical modification of a protein can "mark" it for further degradation. This is thought to occur by altering the folding equilibrium in favor of the denatured conformation [3].

Glycosylation and Protein Stability. Many endogenous proteins and related biotechnology products exist as glycopro-teins—protein chains linked to carbohydrates (glycosyl groups). Typically the carbohydrate portion of the molecule increases the stability and residence time of the protein in the blood circulation. Partially digested carbohydrates can decrease the residence time of the glycoprotein in blood.

The glycosyl groups on glycoproteins contain monosaccharides that are linked to each other by glycosidic bonds to form straight or branched chain polysaccharides. Three monosaccharide derivatives are key players in the formation and stability of glycoproteins. They are N-acetylgalactosamine, which links to the hydroxyl group of threonine or serine (O-glycosyl links), N-acetylglucosamine, which links to the terminal amine of asparagine residues (N-glycosyl links), and N-acetyl-neuraminic acid (sialic acid), which forms the terminal "cap" of a polysaccharide chain and prevents cell surface receptor's binding to other sugar residues and uptake of the entire glycoprotein by hepatocytes and macrophages.

For example, erythropoietin (epoetin) is a heavily glycosylated 165 amino-acid protein (30.4kDa), which stimulates red blood cell production. The carbohydrate moiety comprises 40% of the total molecular size (Figure 5.3). The carbohydrate moiety is not necessary for the biologic activity of the molecule (in fact it reduces it somewhat), but the carbohydrate chain greatly reduces the clearance of the therapeutic protein. Fully glycosylated epoetin has a half-life in blood of 6 to 12 hours, whereas desialyated epoetin is cleared within minutes. Loss of the terminal sialic acid residue results in the rapid uptake of the molecule by the liver through a galactose receptor on the surface of the hepatocytes.

The carbohydrate moiety of immuno-globulin G can be an important determinant of both function and stability (Figure 5.4). It is attached to the protein via an N-linked glycosyl group in the constant domain (Fc). The polysaccharide chain influences tissue distribution (via cell surface recognition and binding) and the stability of IgG. Fully gly-cosylated IgG with a sialic acid cap has a half-life of 21 to 27 days. Cleavage of the terminal sialic acid residue of the poly-saccharide chain promotes rapid uptake and destruction by the liver (Figure 5.5).

Chemical "Marking" Modifications. Chemical marking accelerates the

Figure 5.3. Glycosylated erythropoietin (epoetin) with an N-linked glycosyl group and terminal sialic acid cap.

Polysaccharides (2 chains per IgG molecule)

Figure 5.4. Schematic representation of glycosylated IgG. Asn, asparagines; Fuc, fucose; GlcNAc, N-acetyl-glucoseaimine; Man, Mannose; Gal, galactose; Sia, sialicacid.

Circulating blood_

(Fully glycosylated)

Renal elimination -4-

Loss of terminal sialic acid on IgG leads to exposure of galactose

Metabolized IgG ± in blood circulation

Galactose receptor-mediated IgG uptake by liver

Metabolized IgG fragments in liver

Figure 5.5. Disposition of IgG.

elimination of a protein through enhanced metabolism. The process involves one of six protein modifications:

1. Deamidation of basic amino acids, glutamine and asparagine, involves spontaneous acid-catalyzed hydrolysis of the terminal amide on these amino acids. Deamidation of these basic amino acids results in formation of acidic amino-acid residues. Soma-totropin, IgG, and insulin are subject to this modification. Conversion of a single basic amino acid to an acidic amino acid can have a profound effect on tertiary structure (unfolding) and the susceptibility of the amino acid backbone to proteolysis. In general, proteins rich in glutamic acid (serine, threonine, and proline) are rapidly degraded in eukaryotic cells.

2. Oxidation of methionine residues involves hydrogen peroxide (H2O2)-catalyzed conversion of the terminal sulfide to a sulfoxide. This marking occurs with a1-antitrypsin and parathyroid hormone and leads to the introduction of a net charge, which can affect tertiary and secondary structure.

3. Nonspecific cytochrome P450-mediated oxidation involves enzyme-catalyzed formation of reactive oxygen species (superoxide anions and hydroxyl radicals), which oxidize susceptible amino acids such as proline, arginine, lysine, and histidine.

4. Substrate-specific cytochrome P450-mediated hydroxylation involves NADPH-dependent oxidation of some cyclic peptides such as cyclosporine. Hydroxylated products of cyclosporine are biologically inactive and readily cleared from the body.

5. Disulfide exchange is a spontaneous reaction that occurs under neutral or basic conditions and involves cleavage of a disulfide bond and incorrect reformation. As an example, disul-fide bridges in insulin may unfold and be conjugated to glutathione. This process can be catalyzed by transhydrogenase cleavage of the interchain disulfide bridge and reformation with the cysteine residue of glutathione. Disulfide exchange will invariably change the tertiary structure of a protein.

6. Phosphorylation of serine or threo-nine residues involves an ATP-dependent addition of a phosphate group to a primary (serine) or secondary (threonine) alcohol. Phos-phorylated proteins are often subject to rapid degradation.

Proteolysis. Proteolysis is the cleavage of amide bonds that comprise the backbone of proteins and peptides. The reaction can occur spontaneously in aqueous medium under acidic, neutral, or basic conditions. This process is accelerated by proteases, ubiquitous enzymes that catalyze peptide-bond hydrolysis at rates much higher than occur spontaneously. In humans, these enzymes only recognize sequences of l-amino acids but not d-amino acids. They are found in barrier tissues (nasal membranes, stomach and intestinal linings, vaginal and respiratory mucosa, ocular epithelium), blood, all internal solid organs, connective tissue, and fat. The same protease may be present in multiple sites in the body.

Several different proteases can attack a single protein at enzyme-selective amino-acid sequences. Proteases can be divided into two categories. Endopeptidases are enzymes that cleave peptide bonds between specific, nonterminal amino acids.There are endopeptidases specific for just about every amino acid. Exopeptidases are enzymes that cleave terminal peptide bonds at either the C-terminus or N-terminus.

Protein Metabolism in Eliminating Organs. Peptidases in the gastrointestinal mucosa represent the major barrier to orally administered protein or peptide drugs. Pepsins, a family of proteases specific for aspartatic acid, reside in the stomach lining. The intestinal lining contains chy-motrypsin, an endopeptidase specific for hydrophobic amino acids (e.g., phenylala-nine, leucine, methionine, tryptophan, and tyrosine); trypsin, an endopeptidase specific for basic amino acids (e.g., arginine, lysine); elastase, an endopeptidase specific for amino acids with small, unbranched, nonaromatic functional groups (e.g., glycine, alanine, serine, valine, leucine); and carboxypeptidase A, a C-terminus exopep-tidase selective for l-amino acids with aromatic or bulky functional groups (e.g., tyrosine).

The liver eliminates proteins on first pass after oral administration and on each pass of hepatic blood flow. Hepatocytes, Kupffer cells, adipocytes, and endothelial cells can all be involved in proteolysis (Figure 5.6). Proteolysis can occur in lysosomes after endocytosis of a protein and lysosomal fusion. Endocytosis of a protein may be a nonspecific or receptor-mediated process. Proteolytic products are eliminated from the liver through biliary excretion, and subsequently digested further in the intestinal tract.

The kidneys filter and metabolize proteins and peptides (Figure 5.7). The efficiency of filtration depends on the average radius of the molecule; the molecular charge of a protein may influence the apparent radius. Reabsorption, although uncommon for proteins, can take place along the luminal surface of the proximal tubule. Ordinarily proteins can bind to the brush border (luminal surface) of proximal tubules through an electrostatic interaction with lysine residues on the protein. Endo-cytosis and fusion with lysosomes follows binding and leads to proteolytic digestion. Resulting fragments could diffuse across the basolateral surface of tubules and be reabsorbed into the blood. Kidney metabolism is a major route of elimination for

Intracellular Metabolism
Figure 5.6. Metabolism of proteins by proteases found at the cell surface and internal cellular organelles. Intracellular uptake of proteins often involved receptor-mediated endocytosis; initial surface binding can be specific for a hormone (e.g., insulin, glucagon).

interleukins, interferons, tumor necrosis factor, and colony-stimulating factors.

Was this article helpful?

0 0

Post a comment