Protein Pharmacokinetics

• Pharmacokinetics is the time course of drug concentration in body fluids resulting from a specific dosage regimen
• It comprises drug absorption, distribution, metabolism, and excretion processes
• It refers to how the body interacts with the drug
• Protein pharmacokinetics may differ from conventional drugs

Factors that cause the difference in pharmacokinetics

• Structural similarity to endogenous compounds
• Large molecular weights
• Regulatory feedback mechanisms
• Difficulties in analysis (interferences)

Absorption of protein therapeutics

• Poorly absorbed orally
• Mainly administered parenterally (I.V, I.M, and S.C.) depending on protein type
• Potential limitations of SC and IM administrations are pre-systemic degradation processes, local blood flow, injection trauma, and capillaries' sizes
• Ka = F. Kapp.

Absorption Rate Constants

• Ka is the true absorption rate constant
• F is bioavailability compared to IV
• Kapp is the apparent absorption rate constant for IM and SC administrations

Distribution of Protein Therapeutics

• The rate and extent are determined by size, molecular weight, physicochemical properties, protein binding, and dependency on active transport.
• The lymphatic system plays an important role in the distribution of proteins depending on size
• Protein charge is important for electrostatic attraction of positively charged proteins with negatively charged cell membranes containing glycoaminoglycans
• IV administered proteins may follow a one- or two-compartmental model or may be non-compartmental in distribution

Distribution Characterization

• Biopsy or necropsy is used for determination of actual protein concentrations in the tissue
• Bio-distribution studies use radiolabeled compounds and/or imaging techniques
• Binding to endogenous protein structures (specific) can affect the distribution, pharmacodynamics (PD), and disposition properties of proteins
• Binding may be non-specific to plasma proteins (albumin and lipoproteins)
• Site-specific receptor-mediated uptake can also substantially influence and contribute to the distribution, elimination, and PD of proteins

Elimination of Therapeutic Proteins

• The exogenous proteins are subjected to the same catabolic pathways as endogenous ones
• End products of protein metabolism are amino acids that are reutilized in the endogenous amino acids pool for synthesis of endogenous proteins

Elimination pathways

• Proteolysis
• GIT protein metabolism
• Renal protein metabolism and excretion
• Hepatic protein metabolism
• Receptor-mediated protein metabolism

Proteolysis

• Metabolic rate of protein degradation typically increases as the molecular weight decreases from larger proteins to smaller peptides
• The rate is also influenced by factors like size, charge, lipophilicity, functional groups, glycosylation and the presence of secondary or tertiary structures.
• Proteolytic degradation can occur unspecifically nearly everywhere in the body (within blood) or can be limited to a specific organ or tissue
• Proteases and peptidases are found extra and intracellularly

Orally Administered Proteins

• Primarily broken down in the stomach and small intestine by proteolytic enzymes like pepsin (stomach) and trypsin, chymotrypsin, and peptidases (small intestine)
• Absorbed amino acids are transported into the bloodstream for systemic use

Parenterally Administered Proteins

• Most parenterally administered proteins bypass the GIT, some proteins may still undergo metabolism in the intestinal mucosa
• This is relevant for endogenous albumin, which is secreted into the intestine and then reabsorbed

Renal Protein Metabolism and Excretion

• The kidneys are a major site of protein metabolism for smaller sized proteins that undergo glomerular filtration (GF)
• The size-selective cut-off for GF is approximately 60 kD, with optimal efficiency for proteins smaller than 30 kD
• Rate of filtration decreases significantly for proteins larger than 30 kD
• Charge selectivity is crucial, as anionic compounds (TNF-alpha) pass through less readily than neutral compounds, which in turn pass through less readily than cationic compounds

Negative charge of glomerular filter

• Due to the negative charge of the glomerular filter, which has an abundance of glycosaminoglycans
• Renal metabolism of peptides and small proteins is mediated through three highly effective processes, so only trace amounts of intact protein are detectable in urine

First mechanism

• GF of larger, complex peptides and proteins followed by reabsorption into endocytic vesicles in the proximal tubule and subsequent hydrolysis into small fragments and amino acids as for IL-2, GH and insulin

Second mechanism

• GF followed by intraluminal metabolism, predominantly by exo-peptidases in the luminal brush border membrane of the proximal tubule
• The resulting peptide fragments and amino acids are reabsorbed into the systemic circulation as for glucagon and LH-RH (small linear peptides)

Third mechanism

• Peri-tubular extraction of peptides and proteins from post-glomerular capillaries with subsequent intracellular metabolism, as for insulin and IL-2

Hepatic Protein Metabolism

• Rate of hepatic metabolism is largely dependent on the specific amino acid sequence of the protein (endopeptidases or exopeptidases action)
• Mechanisms of hepatic uptake for proteins depend on the size and hydrophobicity
• There are different hepatic cells for uptake mechanisms like hepatocytes, Kupffer cells (specialized macrophages), endothelial cells, and fat-storing cells

Uptake mechanism

• Simple passive diffusion for small peptides with sufficient hydrophobicity in hepatocytes, as for cyclosporine (cyclic peptides), is metabolized by microsomal enzymes in cytosol
• A carrier-mediated transport for cyclic and linear peptides of small size and hydrophobic nature (containing aromatic a.a.) in hepatocytes like cholecystokinin-8 (CCK-8), which is metabolized by cytosolic peptidases and then excreted into bile via active transporters
• Receptor-mediated endocytosis (RME), in which the circulating proteins are recognized by specific hepatic receptor proteins (glycoproteins), as for insulin and epidermal growth factor (large peptides)

Receptor-mediated endocytosis

• Recycling of receptors occurred, so depending on the type of protein receptor, degradation may occur leading to a decrease in receptor concentration on cell surfaces (receptor down-regulation) as for interferon and insulin
• For glycoproteins, if a critical number of exposed sugar groups (such as mannose and galactose) is exceeded, receptor-mediated endocytosis (RME) serves as an efficient hepatic uptake mechanism in hepatocytes, Kupffer cells, and liver endothelial cells
• Low density lipoprotein receptor-related protein (LRP) is a member of the LDL receptor family responsible for endocytosis of several lipoproteins, proteases and protease complex inhibitor in the liver

Transcytotic pathway

• The endocytotic vesicle formed at the cell surface traverses the cell, degradation, and exocytosis into bile, as for polymeric immunoglobulin A

Receptor-mediated protein metabolism

• Occurs for proteins that bind with high affinity to membrane-associated receptors on the cell surface
• It Includes endocytosis and subsequent intracellular lysosomal metabolism
• It is not constant (dose-dependent), decreases with increasing the dose
• It is not limited for a specific organ or tissue type, but depends on the number of protein drug receptors
• An example is the metabolism of some proteins by linking to a receptor-mediated uptake into macrophages