Pharmacokinetics and toxicology of therapeutic proteins: Advances and challenges

Table 1 Examples of proteins and peptides administered subcutaneously[10]

INN/BAN (description)	Trade name	MW (kDa)	Absolute bioavailability1	SC animal models used in drug development
Buserelin acetate (LH-releasing hormone analog)	Suprefact	1.30	Human: 70%	Pharm: rat, hamster, guinea pig, rabbit, dog and monkey Tox: mouse, rat, rabbit and dog
Pramlintide acetate (amylin analog)	Symlin	3.95	Human: 30 to 40%	Pharm: rat and dog PK: mouse, rat, rabbit and dog Tox: mouse, rat, rabbit and dog
Insulin lispro (insulin analog)	Humalog	5.81	Human: 55 to 77%	Pharm: rat, rabbit, dog and pig PK: rat and dog Tox: rat, rabbit and dog
Insulin glulisine (insulin analog)	Apidra	5.82	Human: about 70% Dog: 42% Rat: 96%2	Pharm: rat and dog PK: rat and dog Tox: mouse, rat, rabbit and dog
Insulin glargine (insulin analog)	Lantus	6.06	Precipitates in skin-slow uptake in human, dog and rat	Pharm: mouse, rat, guinea pig, rabbit and dog PK: rat and dog Tox: mouse, rat, guinea pig, rabbit and dog
Mecasermin (IGF-1)	Increlex	7.65	Human: about 100% Rabbit: 47% Rat: 38 to 57%	Pharm: mouse, rat, rabbit and monkey PK: rat, rabbit, dog and monkey Tox: rat, dog, rabbit and monkey
IFNβ-1b (cytokine)	Betaseron	18.5	Human: 50% Monkey: 31 to 44%	Pharm: monkey PK: monkey Tox: rabbit and monkey
Somatropin (GH)	Nutropin	22	Human: 81%	Pharm: rat PK: rat and monkey Tox: mouse, rat, dog and monkey
IFNβ-1a (cytokine)	Rebif	22.5	Human: 6 to 62% Monkey: 12 to 38% Rat: 16%	Pharm: mouse and monkey PK: rat and monkey Tox: monkey
PEG-IFNα-2b (cytokine variant)	PEG-Intron	31	Monkey: 57 to 89% Rat: 43 to 51%	Pharm: rat and monkey PK: rat and monkey Tox: mouse, rat, rabbit and monkey
Pegfilgrastim (PEG-G-CSF)	Neulasta	39	Monkey: 49 to 68% Rat: < 10% to 30%	Pharm: mouse, rat and dog PK: mouse, rat and monkey Tox: rat and monkey
Pegvisomant (PEG-GH)	Somavert	42, 47 and 523	Human: 49 to 65% Monkey: 70 to 81% Mouse: 45 to 73%	Pharm: mouse and monkey PK: mouse, rat, rabbit and monkey Tox: mouse, rat, rabbit and monkey
PEG-IFNα-2a (cytokine variant)	Pegasys	60	Human: 61 to 80%	Pharm: mouse PK: rat and monkey Tox: mouse, rat and monkey
Certolizumab pegol (PEG-anti-TNFα Fab' fragment)	Cimzia	91	Human: 76 to 88% Rat: 24 to 34%	PK: rat and monkey Tox: monkey
Canakinumab (anti-IL-1β mAb)	Ilaris	145	Human: 63 to 67% Monkey: 60%	Pharm: mouse, rat and monkey (marmoset) PK: mouse and monkey Tox: mouse and monkey
Adalimumab (anti-TNF mAb)	Humira	148	Human: 64% Monkey: 96%	PK: monkey Tox: rabbit and monkey
Omalizumab (anti-IgE mAb)	Xolair	149	Human: 53 to 71% Monkey: 64 to 104% Mouse: 90%	Pharm: monkey PK: mouse and monkey Tox: monkey
Golimumab (anti-TNF mAb)	Simponi	150	Human: 53% Monkey: 77%	PK: monkey Tox: mouse and monkey
Ustekinumab (anti-p40 mAb)	Stelara	150	Human: 24 to 95% Monkey: 97%	Pharm: monkey PK: monkey Tox: monkey
Etanercept (TNF receptor-Fc-IgG1 fusion protein)	Enbrel	150	Human: 76% Monkey: 73% Mouse: 58%	Pharm: mouse PK: mouse, rat and monkey Tox: mouse, rat, rabbit and monkey
Rilonacept (IL-1 inhibitor, fusion protein)	Arcalyst	251	Human: 43% Monkey: 70% Rat: 60% Mouse: 78%	Pharm: mouse and monkey PK: mouse, rat and monkey Tox: monkey

¹Systemic dose following subcutaneous (SC) injection relative to systemic dose following intravenous injection;

²Assumes linearity of AUC/dose;

³Product is a mixture of three distinct protein variants. GH: Growth hormone; LH: Luteinizing hormone; MW: Molecular weight; Pharm: Pharmacology; PK: Pharmacokinetics; Tox: Toxicology (including safety pharmacology); INN: International nonproprietary name; BAN: British approved name; SC: Subcutaneous; PEG: Polyethylene glycol; IFN: Interferon; TNF: Tumor necrosis factor; IL: Interleukin.

Table 2 Transport mechanisms for proteins from systemic circulation across capillary endothelia[22]

Type of capillary endothelium	Barrier/transport mechanism	Particle size subject to passage	Typical tissues
Continuous (non-fenestrated)	Basal lamina membrane supported by collagen	50-110 nm	Muscle, central nervous system, bone, skin, cardiac muscle
Fenestrated	Large pores, open fenestrae, intracellular junctions, basal lamina	50-800 nm	Renal glomeruli, intestinal villi, synovial tissue, endocrine glands, choroid plexus (brain)
Discontinuous (sinusoidal)	Large pores (fenestrae), pinocytotic vesicles	1000-10  000 nm	Liver, spleen, bone marrow, postcapillary venules of lymph nodes

Table 3 Bioanalytical methods applied to absorption, distribution, metabolism and excretion studies of therapeutic proteins

Methods	Capability to assay for	Current throughput	Currently use	Current sensitivity
Immunoassay	Total, free, intact	High for serum, low for tissues, requires homogenization	Mostly serum/plasma, physiological fluids (e.g., synovial and bronchoalveolar lavage )	Usually high for serum/plasma
Bioassay	Activity of targets, biomarker, ex vivo efficacy	Medium to low, may require fresh samples for certain assays	Serum/plasma and tissues	Varies depending on individual assay
Radioactivity counting	Total, intact and degradants	High, requires probe preparation and characterization	Serum/plasma, tissues, biological fluids, and excreta	Usually high, depending on specific activity of labeled materials
MS	Total, free, intact and degradants	High for peptides in serum/plasma, requires homogenization for tissues	Serum/plasma, tissues, biological fluids, and excreta	Usually high for peptides
		Medium to low for proteins in plasma/serum, requires purification (e.g., immunocapture) and digestion for large MW biologics		Low for large proteins
Imaging	Total, intact and degradants	Medium to low, requires probe preparation and characterization	Live animals, clinical studies in humans, cells and tissues	Varies, depending on probes used and study settings
Auto-radiography	Total, intact and degradants	Low, requires tissue slicing and film developing	Tissues	Varies, depending on specific activity of labeled materials

“Low” and “High” sensitivity is an assessment of likelihood of obtaining a sufficient data-set for quantitative assessment of absorption, distribution, metabolism and excretion properties. MW: Molecular weight; MS: Mass spectrometry.