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MINIREVIEW Pharmacokinetic Aspects of Biotechnology Products

NTHER HOCHHAUS,2 BERND MEIBOHM1 LISA TANG,1 ADAM M. PERSKY,2 GU Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 874 Union Avenue, Suite 5p, Memphis, Tennessee 38163
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Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, Florida 32610

Received 10 September 2003; revised 11 January 2004; accepted 20 March 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20125

ABSTRACT: In recent years, biotechnologically derived peptide and protein-based drugs have developed into mainstream therapeutic agents. Peptide and protein drugs now constitute a substantial portion of the compounds under preclinical and clinical development in the global pharmaceutical industry. Pharmacokinetic and exposure/ response evaluations for peptide and protein therapeutics are frequently complicated by their similarity to endogenous peptides and proteins as well as protein nutrients. The rst challenge frequently comes from a lack of sophistication in various analytical techniques for the quantication of peptide and protein drugs in biological matrices. However, advancements in bioassays and immunoassaysalong with a newer generation of mass spectrometry-based techniquescan often provide capabilities for both efcient and reliable detection. Selection of the most appropriate route of administration for biotech drugs requires comprehensive knowledge of their absorption characteristics beyond physicochemical properties, including chemical and metabolic stability at the absorption site, immunoreactivity, passage through biomembranes, and active uptake and exsorption processes. Various distribution properties dictate whether peptide and protein therapeutics can reach optimum target site exposure to exert the intended pharmacological response. This poses a potential problem, especially for large protein drugs, with their typically limited distribution space. Binding phenomena and receptormediated cellular uptake may further complicate this issue. Elimination processesa critical determinant for the drugs systemic exposuremay follow a combination of numerous pathways, including renal and hepatic metabolism routes as well as generalized proteolysis and receptor-mediated endocytosis. Pharmacokinetic/pharmacodynamic (PK/PD) correlations for peptide and protein-based drugs are frequently convoluted by their close interaction with endogenous substances and physiologic regulatory feedback mechanisms. Extensive use of pharmacokinetic and exposure/response concepts in all phases of drug development has in the past been identied as a crucial factor for the success of a scientically driven, evidence-based, and thus accelerated drug development process. Thus, PK/PD concepts are likely to continue and expand their role as a fundamental factor in the successful development of biotechnologically derived drug products in the future. 2004 Wiley-Liss, Inc. and the American Pharmacists Association J
Pharm Sci 93:21842204, 2004

Keywords: pharmacokinetics; biotechnology; peptides; proteins; macromolecule drug delivery; disposition; PK/PD models
Correspondence to: Bernd Meibohm (Telephone: 901-4481206; Fax: 901-448-6940; E-mail: bmeibohm@utmem.edu)
Journal of Pharmaceutical Sciences, Vol. 93, 21842204 (2004) 2004 Wiley-Liss, Inc. and the American Pharmacists Association

INTRODUCTION
In recent years, biotechnologically derived drugs (biotech drugs) including proteins, peptides,

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monoclonal antibodies, and antibody fragments, as well as antisense oligonucleotides and DNA preparations for gene therapy, have been a major focus of research and development (R&D) efforts in the pharmaceutical industry. According to the PHARMA 2010 report,1 biotech products accounted for more than 35% of the 37 New Active Substances that were launched in 2001. Furthermore, it has been predicted that half of all New Active Substances developed in the next 10 to 15 years will result from research into antibodies alone.1 Epoetin-a (Epogen1, Procrit1), abciximab (ReoPro1), and trastuzumab (Herceptin1) are all examples of highly successful biotech drugs that have revolutionized pharmacotherapy in various indications. Business analysts predict success for those segments of the pharmaceutical industry with products that focus on targeted treatment in specic disease states by using knowledge based on genomics, proteomics, and metabonomics. Because the development of biotech drugs generally rests on a fundamental understanding of the related disease, their clinical development is frequently more successful than it is for drug products based on conventional small molecules. In fact, only 8% of the new molecular entities that entered a phase of clinical development between 1996 and 1998 reached the market, compared to 34% of biotech drugs.1,2 Pharmacokinetics and pharmacokinetic/pharmacodynamic (PK/PD) concepts impact every stage of the drug development process starting from lead optimization to the design of Phase III pivotal trials.3 An understanding of the concentrationeffect relationship is crucial to any drug including biotech productsbecause it lays the foundation for dosing regimen design and rational clinical application. General pharmacokinetic and pharmacodynamic principles are just as applicable to biotech agents as they are to traditional small molecule drugs. This also includes PK/PD-related recommendations for drug development such as the recently published exposure-response guidance document of the U.S. Food and Drug Administration (FDA) and the ICH E4 guideline of the International Conference on Harmonization.4,5 Pharmacokinetic and PK/PD analysis of biotech agents, however, frequently poses extra challenges and requires additional resources because most biotech drugs are identical or similar to endogenous molecules. The types of pharmacokinetics-related problems the biotech drug development team may encounter range from lack of specicity and sensitivity of

bioanalytical assays to low bioavailability and rapid drug elimination from the system. The following review provides a general discussion along with examples of the basic concepts and advances made thus far in the PK/PD evaluation of peptides and protein-based drugs. Additionally, it outlines some of the challenges and obstacles that require further attention. PK/PD-related issues for other biotech drug product such as oligonucleotides and DNA preparations are discussed elsewhere in detail.68 An overview on basic pharmacokinetic parameters of selected FDA-approved biotech drugs is presented in Table 1. Analytical Considerations Before evaluating the pharmacokinetics of biotech agents, it is essential that pharmaceutical scientists rst consider the potential problems posed by the analytical assay. Accuracy, precision, and specicity remain paramount criteria for those interpreting assay results. Unfortunately, assays for measuring and detecting peptides and proteins are often inaccurate, imprecise, and nonspecic. This lack of analytical sophistication may lead to misleading results and erroneous pharmacokinetic evaluations for the compound of interest. Moreover, an additional level of complexity may be added when analyzing glycosylated, PEGylated or other conjugated biotech drugs. Techniques commonly utilized for the analysis of small molecules (i.e., HPLC and GC) are often not applicable for the analysis of biotech products. Alternative methods for analysis of peptides and proteins usually include bioassays, immunoassays, and mass spectrometry-based approaches. In analogy to small molecules, these analytical techniques must be validated so that they meet prespecied criteria regarding accuracy, precision, selectivity, sensitivity, reproducibility, and stability, for example, those recommended by the FDA.911 Additional requirements for bioanalytical method validation for macromolecules have recently been published.12 Bioassays The utility of bioassays lies in their ability to adequately characterize the biologic product in terms of consistency, purity, stability, and potency. Bioassays may be dened as an analytical procedure measuring a biological activity of a test substance based on a specic, functional, and biological response of a test system.13,14
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Table 1. Pharmacokinetic Parameters of Selected FDA-Approved Biotech Drugs as Reported in the Prescribing Information Volume of Distribution{ 4040 1607 L 4.76.0 L

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Generic Name/Trade Name 64% 208 48 L/day 12 mL/h Nonlinear PK 268 mL/min 0.25 mL/h/kg 94 mL/kg PLV{ 380570 mL/min Praecis Abbott Genzyme Chiron Biogen Ilex/Berlex Genentech Amgen 63% (i.m.) 95% i.m. s.c. i.v. i.v. i.v., i.m. i.v. i.v. s.c.

Class

Manufacturer

Route

Bioavailability

Clearance{

Half-Life

Abarelix/Plenaxis Adalimumab/Humira Agalsidase/Fabrazyme Aldesleukin/Proleukin Alefacept/Amevive Alemtuzumab/Campath Alteplase/Activase Anakinra/Kineret

LH-RH antagonist Antirheumatic a-Galactosidase Antineoplastic (IL-2) Immunosuppressant Antineoplastic Thrombolytic Antirheumatic (IL-1Ra)

Antineoplastic Immunosuppressant LH-RH antagonist Immunosuppressant 85%

Merck Novartis Serono

i.v., i.m. i.v. s.c.

41 19 mL/h 1.28 mL/min/kg 57 mL/min/kg

7080% PLV 8.6 4.1 L 1.16 L/kg 35 L/kg

13.2 3.2 days 14 (1020) days 45102 min 85 min 270 h 12 days <5 min 46 h 830 h (i.v.) 3949 h (i.m.) 7.2 3.2 days 62.8 (38.2108) h

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Asparaginase/Elspar Basiliximab/Simulect Cetrorelix/Cetrotide Cyclosporine Neoral Sandimmune Daclizumab/Zenapax Novartis Novartis Roche Amgen Ligand Aventis Eli Lilly i.v. p.o., i.n i.v., s.c. i.v. 37 (3050)% p.o. i.v., p.o. i.v. 1089% 30% (p.o.)

Immunosuppressant

Darbepoetin-a/Aranesp Denileukin diftitox/Ontak

Antianemic Antineoplastic

Desmopressin/DDAVP

Antidiuretic

8.4 (518) h 19 (1027) h 15 mL/h 5.9 L 20 (1823) days 21 h (i.v.) 49 (2472) h (s.c.) 1.52.0 mL/min/kg 0.060.08 L/kg 7080 min 75.5 min (i.n.) 1.52.5 h (p.o.) 40 L/h

Drotrecogin-a/Xigris

Activated protein C

3.2% (i.n.) 0.16% (p.o.)

Epoetin-a/Epogen, Procrit Eptibatide/Integrilin Etanercept/Enbrel Amgen Serono Organon Eli Lilly AstraZeneca

Antianemic GPIIb/IIIa inhibitor Antirheumatic

Amgen, Ortho i.v., s.c. Millennium i.v. Amgen s.c. i.v., s.c. s.c.

5558 mL/h/kg 89 mL/h

Filgrastim/Neupogen Follitropin/Gonal-f

Ganirelix/Antagon Glucagon/Glucagon Goserelin/Zoladex

Hematopoietic stimulant Follicle stimulating hormone LH-RH antagonist rh Glucagon LH-RH agonist

66 39% s.c. 91% i.v., i.m, s.c. s.c.

0.50.7 mL/min/kg 150 mL/kg 0.7 0.2 L/h 10 3 L 2.4 0.2 L/h 13.5 mL/min/kg 110.5 47.5 mL/ min (men) 163.9 71.0 mL/ min (women) 43.7 11.4 L 0.25 L/kg 44.1 13.6 L (men) 20.3 4.1 L (women)

413 h (i.v.) 16.3 3.0 h (s.c.) 2.5 h 115 (98300) h 3.8 h (i.v.) 3.5 h (s.c.) 32 h 12.8 4.3 h 818 min (i.v.) 4.2 1.1 h (men) 2.3 0.6 h (women)

Iniximab/Remicade Interferon a-2a/Roferon-A Centocor Roche 2.79 (2.143.62) mL/min/kg Schering Serono Biogen Berlex/Chiron s.c. InterMune s.c. >89% 50% 9.428.9 mL/min/kg 1.4 L/min i.m. 10 h 0.252.88 L/kg 8 min4.3 h (i.v.) s.c. 3355 L/h 69 37 h i.m., s.c. i.v. i.m., s.c. >80% (i.m.) 8.09.5 days 5.1 (3.78.5) h (i.v.) 2-3 h (i.m., s.c.)

Antirheumatic Biological response modier Interferon a-2b/Intron A Biological response modier Interferon b-1a/Rebif Biological response modier Interferon b-1a/Avonex Biological response modier Interferon b-1b/Betaseron Biological response modier Interferon g-1b/Actimmune Immunomodulator

PLV{ 0.400 (0.223 0.748) L/kg

Laronidase/Aldurazyme Octreotide/Sandostatin Omalizumab/Xolair Oprelvekin/Neumega Genzyme Novartis Genentech Wyeth 100% 62% >80% MedImmune Enzon Amgen Roche 84% Centocor Berlex i.v. i.v., s.c. i.m. i.m., i.v. s.c. s.c. i.v. i.v., s.c. s.c. s.c.

38 min (i.v.) 2.9 h (i.m.) 5.9 h (s.c.) 1.72.7 mL/min/kg 0.240.6 L/kg 1.53.6 h 710 L/h 13.6 L 1.71.9 h 2.4 1.1 mL/day/kg 78 32 mL/kg 26 days 6.9 1.7 h

Nonlinear PK 94 mL/h

20 days 5.69 3.25 days 1580 h 80 (50140) h 1316 min 60 min (i.v.) 162 min (s.c.)

Lysosomal hydrolase Somatostatin analogue IgE inhibitor Thrombopoietic stimulant (IL-11) Palivizumab/Synagis Antiviral Pegaspargase/Oncaspar Antineoplastic Peglgrastim/Neulasta Hematopoietic stimulant Peginterferon a-2a/Pegasys Biological response modier Reteplase/Retavase Thrombolytic Sargramostim/Leukine Hematopoietic stimulant

Tenecteplase/TNKase Teriparatide/Forteo i.v. s.c. i.v. i.m. i.v. Genentech Pzer Abbott

Thrombolytic Genentech rh Parathyroid hormone Eli Lilly

95% 83%

PLV{

Trastuzumab/Herceptin

Antineoplastic

250450 mL/min 431 mL/min/m2 (i.v.) 549 mL/min/m2 (s.c.) 99119 mL/min 94 L/h (men) 62 L/h (women) Nonlinear PK 212 32 mL/min

Triptorelin/Trelstar Urokinase/Abbokinase

LH-RH agonist Thrombolytic

90130 min 5 min (i.v.) 0.12 L/kg (i.v.) 1 h (s.c.) PLV{ 1.7 days (10 mg) 12 days (500 mg) 3033 L 2.81 1.21 h 11.5 L 12.6 6.2 min

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PLVPlasma volume (40 mL/kg); rhrecombinant human; i.v.intravenous; i.m.intramuscular; i.n.intranasal; s.c.subcutaneous. Clearance and volume of distribution terms for drugs not administered by i.v. usually reect clearance and volume terms divided by bioavailability (i.e., CL/F or V/F).

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Classical bioassays may be categorized into in vivo and in vitro assays. In vivo bioassays involve the administration of the biotech agent to animals with subsequent measurement of the response in the animal. Drawbacks of this method include relatively high cost, lack of specicity, high interanimal variability, and signicant time requirements. In vitro bioassays are typically based upon a quantiable cellular response to stimulation with the test agent.15 With the advent of immortal cell lines, in vitro bioassays offer the advantage of low assay variability when compared to in vivo methods. In addition, recombinant DNA technology allows engineering cell lines that selectively express a specic drug receptor. Despite their advantages over in vivo testing, in vitro assays may be subject to interferences from numerous factors including active metabolites, the biological matrix, and environmental conditions. Furthermore, in vitro assays are only a surrogate marker for biological activity, and may not represent what actually occurs in vivo. A variation of in vitro bioassays known as the reporter gene assay identies the activation of specic gene expression induced by the test ligand. This method involves a linked reporter gene such as b-galactosidase or luciferase as the marker for gene activation. It allows an indirect quantication of ligand-to-receptor binding based on the measurement of the reporter gene expression.16 An alternative to the reporter gene-based assay is the kinase receptor activation assay (KIRA), whereby targeted receptors are tagged with an integral enzyme (i.e., phosphotyrosine). Upon ligand binding, the enzyme activation (i.e., phosphorylation) can be measured by immunoassay as a marker for ligand levels.17 A newer assay termed surface plasmon resonance (SPR) attempts to ameliorate the problem of altered receptor binding capabilities in cell cultures. In this method, the ligands bind to molecules embedded in a dextran mesh on the surface of a gold plated sensor chip. Bioactivity of samples containing drug are measured based on changes in light reection due to changes in the surface plasmon resonance. The advantage here is that the cell surfaces are trapped in a natural binding format that allows more accessible ligand binding.13,18,19 Immunoassays Because of their high sensitivity, increased specicity, precision, availability, and relative
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ease of performance, immunoassays have become the most frequently used analytical technique for quantifying peptides and proteins in biological uids. Immunoassays include enzyme-linked immunosorbant assays (ELISA) and radioimmunoassays (RIA) with poly- and monoclonal antibodies. ELISA is a standard methodology to detect and quantify biotech products. These include various human cytokines,20 bioengineered tissue plasminogen activators (t-PA) such as alteplase and tenecteplase,21 chimeric monoclonal antibodies,22 and epoetin-a.23 Despite its widespread application, the ELISA methodology does have limitations. The primary drawback is its potential lack of specicity towards active (intact), inactive (denatured), and metabolized (partially degraded) forms of the biotech drug based on the epitope specicity of the antibody.24,25 Additionally, a lack of discrimination between endogenous and exogenous compounds may also lead to an overestimation of concentration. Native antibodies directed against the biotech product may also interfere with ELISAs. A recent review regarding dening and measuring follicle-stimulating hormone (FSH) reports considerable variability between the results of different assays for gonadotropins. The authors attribute such differences to a lack of standardization of calibration between different immunoassay kits. Therapeutic products, the authors caution, should be tested based on a common methodology (i.e., pharmacopeial assay) from a regulatory perspective.15 Radioimmunoassays utilize amino acids labeled either externally with 125I or internally with 3H, 14 C, or 35S.26 125I-labeled proteins are helpful for receptor-binding studies and in elucidating the metabolism of the agent as exemplied with 125Itenecteplase. From whole-body autoradiography studies conducted in rats, radiolabeled tenecteplase has pinpointed the liver as the major organ responsible for its clearance.21 Despite its utility, external labeling with one or more iodine atoms may potentially alter the pharmacokinetics of the native agent.27 Dehalogenation of the iodinated biopharmaceutical upon administration into the system may also confound results.28 For internally labeled proteins, protein metabolism may liberate radiolabeled amino acids, which may in turn be recycled and incorporated into endogenous proteins. This has been demonstrated in a study with 14 C-labeled recombinant human interleukin-2 (rhIL-2), where comparison of the pharmacokinetic proles of two radiolabels yielded different results. The authors concluded that the 14C-rhIL-2

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was metabolized to constituent amino acids and recycled into endogenous proteins.29 Liquid Chromatography Mass Spectrometry Liquid chromatography mass spectrometry (LC/ MS)-based techniques have also been utilized in the bioanalytics of peptides and proteins. While modern LC-based assays have comparable sensitivity to immunoassays, they oftentimes possess higher selectivity.24,30 Mu ller et al., for example, used LC/MS with matrix-assisted laser desorption ionization in ex vivo pharmacokinetic studies in combination with enzyme inhibition experiments to investigate the complex metabolism of dynorphin A1-13, a peptide with opioid activity, up to the fth metabolite generation.31,32 Similarly, Feng et al. applied LC with ion-trap tandem mass spectrometry to quantify the bradykinin antagonist polypeptide B201 in plasma.33 The fact that many of the agents are endogenous substances already present in the body before drug administration adds a further level of complexity to the bioanalytics of biotech drugs. Because the analytical technique will detect a so-called baseline level prior to drug exposure, the preexisting endogenous substance must be accounted for in the pharmacokinetic analysis or must be suppressed before exogenous drug administration.34,35 Suppression via physiological regulation or feedback mechanisms is an elegant approach for the elimination of endogenous baseline levels. This methodology was, for example, used to suppress the endogenous release of insulin and growth hormone (GH; somatotropin) via infusions of glucose and somatostatin, respectively, prior to their exogenous administration for pharmacokinetic evaluation.36

cules like peptides and proteins.24 In addition, systemic availability of peptides is furthermore reduced by the intestinal functional system of cytochrome P450 3A and p-glycoprotein activity.39,40 Therefore, administration by injection or infusion, that is, intravenous (i.v.), subcutaneous (s.c.), or intramuscular (i.m.), is frequently the preferred route of delivery for these drug products. During formulation, biotech agents must be assessed individually regarding their physicochemical and biological properties to develop the optimal delivery system. Some of the major routes of administration for biotech drugs are discussed below in the order of increasing biopharmaceutic challenges to obtain adequate systemic exposure. Administration by Injection or Infusion I.v. administration of biotech drugs offers the advantage of circumventing presystemic degradation, thereby achieving the highest concentration in the biological system. FDA-approved biotech products given by the i.v. route include alteplase, tenecteplase, abciximab, rituximab, and lgrastim. i.v. administration as bolus or constant rate infusion, however, may not always provide the desired concentrationtime prole. For example, luteinizing hormone-releasing hormone (LH-RH) in bursts stimulates the release of FSH and LH, whereas a continuous baseline level will suppress the production of these hormones.41 A recent study comparing s.c. versus i.v. administration of epoetina in patients receiving hemodialysis reports that the s.c. route can maintain the hematocrit in a desired target range with a lower average weekly dose of epoetin-a.42 The drawbacks of s.c. and i.m. injections include a potentially decreased bioavailability that is secondary to variables such as local blood ow, injection trauma, protein degradation at the site of injection, and limitations of uptake into the systemic circulation related to effective capillary pore size and diffusion. Subcutaneous administration of a biotech product may result in the molecule either entering the systemic circulation via blood capillaries or through lymphatic vessels.43 Studies with recombinant human interferon a-2 (rhIFN a-2) indicate that following s.c. administration, high concentrations of the recombinant protein are found in the lymphatic system, which drains into the regional lymph nodes.44 Furthermore, there appears to be a linear relationship between the molecular weight of a biotech agent (for a given range) and the proportion of the dose absorbed by the lymphatics
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ADMINISTRATION PATHWAYS
Peptides and proteins, unlike conventional small molecule drugs, are generally not therapeutically active upon oral administration.24,37,38 This lack of systemic bioavailability is mainly caused by two factors, high gastrointestinal enzyme activity and the function of the gastrointestinal mucosa as an absorption barrier. There is substantial peptidase and protease activity in the gastrointestinal tract, making it the most efcient body compartment for peptide and protein metabolism. Furthermore, the gastrointestinal mucosa presents a major absorption barrier for water-soluble macromole-

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Figure 1. Correlation between molecular weight and the cumulative recovery (mean SD) of rINF a-2a (MW 19,000), cytochrome c (MW 12,300), inulin (MW 5200) and 5-uoro-20 -deoxyuridine (FUDR) (MW 246) in the efferent lymph from the right popliteal lymph node following SC administration into the lower part of the right hind leg in sheep (n 3). The linear regression line has a correlation coefcient r of 0.998 (p < 0.01). From ref. 45, reproduced with permission.

(Fig. 1). Macromolecules larger than 16 kDA are predominantly absorbed into the lymphatics whereas those under 1 kDA are mostly absorbed into the blood circulation.45 This is of particular importance for those agents whose therapeutic targets are lymphoid cells (i.e., interferons and interleukins).44 Clinical studies show that palliative low to intermediate-dose s.c. recombinant interleukin-2 (rIL-2) in combination with rIFN-a can be administered to patients in the ambulatory setting with efcacy and safety proles comparable to the most aggressive i.v. rIL-2 protocol against metastatic renal cell cancer.46 Inhalation Administration Inhalation delivery of biotech agents offers the advantage of ease of administration, the presence of a large surface area (75 m2) available for absorption, high vascularity, and bypass of rst-pass metabolism. Pulmonary delivery has been extensively studied to continually improve the deposition of aerosolized drug into the highly perfused alveolar region to facilitate maximal absorption of the drug. Drawbacks of inhalation delivery include presence of certain proteases in the lung and potential local effects of the inhaled agents on the lung tissues (i.e., growth factors and cytokines). Limitations of molecular weight for systemic bioavailability after pulmonary delivery have also been discussed, but are not conclusive (Fig. 2).47,48
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Figure 2. Plasma bioavailability of therapeutic peptides versus molecular weight (MW) after pulmonary administration. Bioavailability is expressed as percentage of the dose deposited in the lungs relative to SC administration in humans (open symbols) and various animal species (solid symbols; square and circle: rodents; triangle: monkey). From ref. 47, reproduced with permission.

A recent study involving 26 patients with type II diabetes mellitus exemplies the success of inhalation insulin delivery. Results reveal that inhalation insulin treatment for 3 months signicantly improves glycemic control when compared to baseline as assessed by hemoglobin A1c levels.49 Another successful biotech drug administered through the inhalation route is dornase-a, indicated for the treatment of cystic brosis. In a multicenter 2-year clinical study, inhaled dornasea has shown to signicantly improve lung function and reduce the risk of respiratory exacerbations in pediatric cystic brosis patients.50 Intranasal Administration Similar to the inhalation route, intranasal administration of biotech agents offers the advantage of ease of administration, delivery to a surface area rich in its vascular and lymphatic network, and the bypass of rst-pass metabolism.51 Intranasal absorption of a variety of biotech drugs including calcitonin, oxytocin, LH-RH, growth hormone, interferons, and even vaccines has been extensively investigated over the last decade. In general, polypeptides with a molecular weight

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up to 2 kDa have been found to be pharmacologically active via the intranasal route. In contrast, pharmaceutical peptides with molecular weights of 26 kDa (i.e., insulin, calcitonin, and LH-RH) require the addition of absorption enhancers to reach adequate bioavailability.41 Limitations that may preclude the use of the intranasal route of administration include high variability associated with the site of deposition, the type of delivery system, changes in the mucous secretion and mucociliary clearance, as well as the presence of allergy, hay fever, or the common cold in targeted subjects.52 Recent research interest focuses on utilizing the intranasal route as means to deliver protein therapeutics directly into the central nervous system, thereby bypassing the bloodbrain barrier.53 In particular, one study reports achieving a higher concentration of 125I-labeled recombinant human insulin-like growth factor-I (125I-rh-IGF-I) in the CNS after intranasal administration rather than intravenous administration.54 The effectiveness of intranasal delivery of IGF-1 for the treatment of stroke has been reported in a more recent study through a rat model. The investigators report that 150 mg of intranasal IGF-1 can effectively reduce induced infarct size and neurologic decits as compared to controls.55 Transdermal Administration Transdermal drug delivery offers the advantages of bypassing metabolic as well as chemical degradation in the gastrointestinal tract. It also circumvents rst-pass metabolism by the liver, and provides sustained release of the biotech agent. Methods for transdermal delivery include sonophoration (application of low-frequency ultrasound) and iontophoresis (application of a low-level electric current), both increasing skin permeability to ionic therapeutic agents. Therapeutic doses of insulin, interferon-g, and epoetin-a have all been successfully delivered transdermally via sonophoresis.56 Additionally, transdermal iontophoresis has been applied in delivery of a host of proteins and peptides including leuprolide,57 insulin,58 growth hormone releasing factor,59 calcitonin,60 and parathyroid hormone.61 Peroral Administration Most biotech products are currently formulated as parenteral formulations because of their poor oral bioavailability. Oral delivery of peptides and

proteins, however, offers the advantages of convenient, pain-free administration and signicant healthcare cost savings. Although various factors such as permeability, stability and gastrointestinal transit time can affect the rate and extent of orally administered peptides and proteins, molecular size is generally considered the ultimate obstacle.62 Nevertheless, some polypeptide drugs, for example cyclosporine and desmopressin, are available in oral dosage forms and thus indicate that these obstacles can be overcome. Several promising strategies have emerged from the intensive recent research efforts into the delivery oral peptides and proteins.38,62,63 Absorption enhancers may be used to either temporarily disrupt the intestinal barrier so that drug penetration is increased or to serve as transport carriers for the protein via complex formation. The coadministration of parathyroid hormone, an 84 amino acid protein, with N-[8-(2hydroxy-4-methoxy)bensoyl]amino caprylic acid, a transport carrier, was found to result in bioactivity as demonstrated in a rodent model of osteoporosis.64 Although parathyroid hormone has no oral bioavailability when administered alone, coadministration of this absorption enhancer resulted in 2.1% oral bioavailability relative to s.c. administration in monkeys.64 Increasing intestinal paracellular absorption was demonstrated in a study involving insulin and immunoglobulins coadministered with Zonula occulens toxin (Zot), a permeation enhancer. In animal models, Zot reversibly increased the intestinal absorption of both insulin and immunoglobulins in a time-dependent manner.37 Encapsulation in microparticles or nanoparticles may be used to shield peptides and proteins from enzymatic degradation. These solid particles may be taken up via endocytosis by the intestinal cells or passage through paracellular tight junctions. Due to their stability in the GI tract, they appear more favorable for oral delivery than liposomes.38 In particular, the gut-associated lymphoid tissue organized in Peyers patches has been suggested as an useful oral delivery target for encapsulated peptides and proteins due to its high phagocytotic activity and limited lysosomal degradation. The concept has successfully been demonstrated by oral delivery of glucagon-like peptide-1 (GLP-1) in PLGA microspheres to diabetic mice. Mice treated with the microsphere preparation had indeed a lower glycemic response to oral glucose challenge than mice treated with GLP-1 without encapsulation into microspheres.65
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Other strategies of oral peptide and protein delivery include amino acid backbone modications and chemical conjugation to improve their resistance to degradation and their permeability, and inhibition of enzymatic degradation by coadministration of protease inhibitors.38,66 Administration Route and Immunogenicity Antibody formation is a frequently observed phenomenon during chronic dosing with biotech drugs, especially if the drugs were derived from animal proteins. The presence of antibodies can obliterate the biological activity of a peptide or protein drug. Also, proteinantibody complexation may modify its pharmacokinetic prole. Most monoclonal antibodies, for example, are murine in nature. Their systemic administration can lead to the development of human antimouse immunoglobulin antibody (HAMA) response, which is in most cases directed against the constant regions of the monoclonal antibody. Genetically engineered mousehuman chimeric antibodies try to minimize this immunogenicity in humans by joining variable domains of the mouse to the constant regions of human immunoglobulins.67 The administration route may affect immunogenicity. Extravascular injection is known to stimulate antibody formation more than intravenous application. This is most likely due to the increased immunogenicity of protein aggregates and precipitates formed at the injection site.68 A recent study investigated the effect of administration route of INF-b preparations in inducing anti-INF-b antibodies in multiple sclerosis patients. The results indicated that i.m. injection was less immunogenic compared to s.c. injection, resulting in lower serum levels of anti-IFN-b antibodies that were also signicantly delayed in their appearance.69 Another approach to reduce the immunogenicity of biotech drugs involved their conjugation with polyethylene glycol (PEG), called PEGylation.70,71 PEGylation can shield antigenic determinants on the drug through steric hindrance from detection by the immune system.72 The development of pegaspargase is a successful example for overcoming the high rate of allergic reactions towards L-asparaginase using PEGylation technology.73 Other major advantages of PEGylation, however, include its ability to manipulate the pharmacokinetics and physicochemical properties of the protein drug. Conjugation of biotech drugs with
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PEG chains increases their hydrodynamic volume, which in turn, can result in a reduced renal clearance, restricted biodistribution, and prolonged residence time. PEGylation can also protect against proteolytic degradation and increase the drugs solubility.71 Peglgrastim is a PEGylated version of the granulocyte colony-stimulating factor lgrastim, which is administered for the management of chemotherapy-induced neutropenia. PEGylation minimizes lgrastims renal clearance by glomerular ltration, thereby making neutrophil-mediated clearance the predominant route of elimination. In a neutropenic setting, peglgrastim has a reduced clearance and thus prolonged half-life and more sustained duration of action because few mature neutrophils are available to mediate its elimination. Thus, PEGylation of lgrastim results in so-called self-regulating pharmacokinetics.74

DISTRIBUTION
The volume of distribution of a drug is determined largely by its physiochemical properties (e.g., charge, lipophilicity), protein binding, and its dependency on active transport processes. Much of the studies regarding biodistribution of therapeutic biotech agents are elucidated with radiolabeled compounds. For example, radiolabeled proteins are typically used to assess specic drug targets or major organs of elimination. Because most of the therapeutic proteins are large in size, their apparent volume of distribution is usually small, and limited to the volume of the extracellular space due to their limited mobility secondary to impaired passage through biomembranes.51,75 Active tissue uptake and binding to intra- and extravascular proteins, however, can substantially increase the volume of distribution of peptide and protein drugs, as for example, observed with atrial natriuretic peptide (ANP).76 After intravenous application, peptides and proteins usually follow a biexponential plasma concentrationtime prole that can best be described by a two-compartment pharmacokinetic model.77 This has, for example, been described for leuprorelin, a synthetic agonist analog of LHRH,78 for clenoliximab, a macaquehuman chimeric monoclonal antibody specic to the CD4 molecule on the surface of T lymphocytes,79 and for AJW200, a humanized monoclonal antibody to the von Willebrand factor.80 The central compartment in this model primarily represents the vascular

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space and the interstitial space of well-perfused organs with permeable capillary walls, especially the liver and kidneys. The peripheral compartment is more reective of concentrationtime proles in the interstitial space of poorly perfused tissues like skin and inactive muscle. Thus, the volume of distribution of the central compartment (Vc) in which peptides and proteins initially distribute after intravenous administration is typically 38 L, approximately equal or slightly higher than the plasma volume. The steady-state volume of distribution (Vss) frequently comprises with 1420 L, not more than twice the volume of distribution of the central compartment.77,80,81 This distribution pattern has, for example, been described for the somatostatin analogue octreotide (Vc 5.210.2 L, Vss 1830 L), and the glycoprotein IIb//IIIa inhibitor eptibatide (Vc 9.2 L).82,83 It has also been demonstrated for the t-PA analog 125I-labeled tenecteplase, which revealed a Vc of 4.26.3 L and a Vss 6.19.9 L with liver as the only organ that had a signicant uptake of radioactivity. The authors concluded that the small volume of distribution suggests primarily intravascular distribution for tenecteplase, consistent with the drugs large molecular weight (65 kDa).21 It should be stressed that pharmacokinetic calculations of Vss may be problematic for some biotech drugs. Noncompartmental determination of Vss using statistical moment theory assumes rst-order disposition processes with elimination occurring from the rapidly equilibrating or central compartment.8486 This basic assumption, however, is not fullled for numerous biotech agents, as proteolysis in peripheral tissues may constitute a substantial fraction of the overall elimination process for peptide and protein drugs. Thus, Vss values reported for biotech drugs in the literature should be interpreted with caution. Another factor that can inuence the distribution of biotech agents is protein binding. Physiologically active, endogenous peptides and proteins frequently interact with specic binding proteins involved in their transport and regulation. A wide range of biotech drugs including growth hormone,25 recombinant human DNases for use as mucolytics in cystic brosis,87 and recombinant human vascular endothelial growth factor (rhVEGF)88 have all been identied to associate with specic binding proteins. Protein binding not only affects whether the biotech agent will exert any pharmacological activity, but many times it also may have an inhibitory or stimulatory

effect on the biological activity of the agent.25 Recombinant cytokines, when injected into the bloodstream, may encounter various cytokine binding proteins, including soluble cytokine receptors and anticytokine antibodies. In either case, the binding protein may either prolong the cytokine circulation time by acting as a storage depot or it may enhance the cytokine clearance.20 In the case of rhVEGF, administration of high doses of the protein results in nonlinear pharmacokinetics. The investigators attribute the nonlinearity to saturable binding, internalization, and degradation of rhVEGF mediated by high-afnity receptors that line the vasculature.88 Aside from physicochemical properties and protein binding of biotech agents, site-specic and target-oriented receptor mediated uptake can also inuence biodistribution. Avid, ongoing research explores the use of the transferrin/ transferrinreceptor system for drug delivery and targeting. Transferrins are a family of ironbinding proteins that transport iron for absorption, storage, and utilization by the body. The family of transport proteins and their cell surface receptors mediate intracellular uptake via endocytosis. This has been utilized for the delivery of proteins with antitumor activity into proliferating malignant cells that overexpress tranferrin receptors.89 Additionally, an abundance of transferrin receptors can be found on the brain capillary endothelium. The antitransferrin receptor monoclonal antibody OX26 undergoes receptor-mediated transcytosis through the bloodbrain barrier, allowing it to serve as a bloodbrain barrier delivery vector for biotech agents. Promising results show successful delivery of therapeutic proteins across the bloodbrain barrier such as human basic broblast growth factor (bFGF)90 and brain-derived neurotrophic factor (BDNF)91 when conjugated to the OX26 antibody.

ELIMINATION
General tendencies in the in vivo disposition of proteins and peptides may often be predicted from their physiological function. Peptides, for example, frequently have hormone activity and usually have short elimination half-lives. This is desirable for a close regulation of their endogenous levels and thus function. Contrary to that, transport proteins like albumin or antibodies have elimination halflives of several days, which enables and ensures the continuous maintenance of necessary concenJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 9, SEPTEMBER 2004

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trations in the blood stream. The reported terminal half-life for SB209763, a humanized monoclonal antibody against respiratory syncytial virus, for example, was reported as 2250 days.92 Peptide and protein drugs are nearly exclusively metabolized via the same catabolic pathways as endogenous or dietary proteins, leading to amino acids that are reutilized in the endogenous amino acid pool for the de novo biosynthesis of structural or functional body proteins. Nonmetabolic elimination pathways such as renal or biliary excretion are negligible for most peptides and proteins. Some peptides and proteins such as immunoglobulin A and octreotide, however, are excreted into the bile.24,83 If biliary excretion of peptides and proteins occurs, it generally results in subsequent metabolism of these compounds in the gastrointestinal tract.77 Proteolysis Proteolytic enzymes such as proteases and peptidases are ubiquitously available throughout the body. Thus, locations of intensive peptide and protein metabolism are not only limited to liver, kidneys, and gastrointestinal tissue, but also include blood and other body tissues. Peripheral blood mediated elimination may affect a substantial portion of peptide and protein drugs. Endo-oligopeptidase was originally isolated and characterized in nerve tissues to hydrolyze several neuropeptides including enkephalin-containing peptides. Immunocytochemical studies also show the presence of endo-oligopeptidase in rat blood cells including T-lymphocytes and other leukocytes, possibly indicating its participation in neuroimmunomodulation.93 Renal Elimination Renal metabolism of biotech agents has been extensively studied in the past. In particular, metabolism of small proteins and peptides appears to occur mainly in the kidneys through one of three routes. The rst route involves glomerular ltration of complex proteins followed by reabsorption into endocytic vesicles in the proximal tubule and subsequent hydrolysis into small peptide fragments and amino acids.94 This method of elimination has been described for IL-2,95 IL-11,96 growth hormone,97 and insulin.98 Controversy exists surrounding the glomerular ltration selectivity regarding size, molecular
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conformation, and charge of the biotech agent. Complex mathematical models have been developed to calculate the sieving coefcient, or the average ltrate-to-plasma concentration ratio along the length of a representative capillary.99 Glomerular size-selectivity studies have used dextran and Ficoll, a copolymer of sucrose and epichlorohydrin, as test macromolecules over a wide range of molecular sizes.100 Although the ultrastructural model for size selectivity in glomerular ltration does provide a framework in relating structure to actual functional properties of the glomerular barrier, future studies are still warranted.99 In addition to size-selectivity, glomerular charge-selectivity has also been observed where anionic polymers pass through the capillary wall less readily than neutral polymers, which in turn, pass through less readily than cationic polymers. Much of the chargeselectivity studies have utilized combinations of dextran (neutral)/dextran sulfate (anionic) and neutral horseradish peroxidase/anionic horseradish peroxidase.101 The second major route of renal elimination of peptide and protein drugs is through hydrolysis by brush border enzymes located on the luminal membrane followed by reabsorption of the metabolites. This route of elimination applies to small linear peptides such as angiotensin I and II, bradykinin, glucagon, and LH-RH.94,102,103 In vivo inhibition studies with excess peptides reveal that hydrolysis at the luminal membrane appears to be specic for small linear proteins, whereas larger and more complex proteins and peptides are endocytosed and degraded in lysozymes.103 Furthermore, recent studies implicate the protondriven peptide transporters PEPT1 and PEPT2 as the main route of cellular uptake of small peptides and peptide-like drugs from the glomerular ltrates.104 These high-afnity transport proteins seem to exhibit selective uptake of di- and tripeptides, which implicates their role in renal amino acid homeostasis.105 The third route of renal elimination is peritubular extraction of biotech products from postglomerular capillaries and intracellular metabolism. Experiments using radio-iodinated growth hormone (125I-rGH) have demonstrated that although reabsorption into endocytic vesicles at the proximal tubule is still the dominant route of disposition, a small percentage of the hormone may be extracted from the peritubular capillaries.97,106 Peritubular transport of proteins and peptides from the basolateral membrane has also

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been shown for insulin107 and the mycotoxin ochratoxin A.108 Hepatic Elimination Aside from renal metabolism, the liver may also play a major role in the metabolism of peptides and proteins. Substrates for hepatic metabolism include insulin, glucagon, epidermal growth factor (EGF), antibodies, and t-PAs.109,110 For insulin, an acidic endopeptidase termed endosomal acidic insulinase appears to mediate internalized insulin proteolysis at a number of sites.111 Specically, the endosomal activity is the result of an aspartic acid protease named cathepsin D.112 Similarly, proteolysis of glucagon has also been attributed to membrane-bound forms of cathepsins B and D.113 For larger proteins, such as t-PA (65 kDa), various receptor mediated transport processes in the liver can facilitate their uptake and subsequent metabolism. Radio-iodinated t-PA (125I-t-PA) results implicate the role of mannose and asialoglycoprotein receptors in the liver for facilitating t-PA uptake and clearance.110 Additionally, evidence also points to another hepatic membrane receptorthe low density lipoprotein receptor-related protein (LRP)for contributing to overall t-PA clearance.110,114 Receptor-Mediated Endocytosis Because many therapeutically used peptides and proteins are endogenous molecules, organs that express receptors for these agents can also serve as potential sources for their overall elimination via receptor-mediated uptake and subsequent intracellular metabolism. The endocytosis process is not limited to hepatocytes, but can occur in other cells as well, including the therapeutic target cells. The binding and subsequent degradation via interaction with these generally highafnity, low-capacity binding sites is a typical example for a pharmacologic target-mediated drug disposition, where binding to the pharmacodynamic target structure affects drug disposition.115 Because the number of receptors is limited, their binding and the related drug uptake can usually be saturated within therapeutic concentrations, or more specically, at relatively low molar ratios between the protein drug and the receptor. As a consequence, pharmacokinetics for these drugs frequently does not follow the rule of superposition, that is, clearance and potentially other pharmacokinetic parameters

are dose-dependent. Thus, receptor-mediated elimination constitutes a major source for nonlinear pharmacokinetic behavior of numerous peptide and protein drugs, resulting in a lack of dose proportionality.6 Nonlinearity in pharmacokinetics based on receptor-mediated drug disposition has been frequently observed for monoclonal antibody drugs. The antiimmunoglobulin E antibody CGP 51901 for the treatment of seasonal allergic rhinitis, for example, has been characterized by a decreasing elimination rate constant in the serum concentrationtime prole with increasing dose.116 A similar observation has been made for the humanized anti-IgE antibody omalizumab for the treatment of allergic asthma.117,118 The anti-HER2 humanized monoclonal antibody trastuzumab, approved for the combination treatment of HER2 protein overexpressing metastatic breast cancer, has been characterized by dose-dependent pharmacokinetics as well (Fig. 3).119 With increasing dose level, the mean half-life increases and the clearance decreases. Because trastuzumab is rapidly internalized after binding to its target structure on the cell surface, saturation of this elimination pathway is a likely factor for this nonlinearity.120

Figure 3. Serum concentrationtime proles (mean SD) of the humanized monoclonal antibody trastuzumab after rst administration in patients with HER2-overexpressing metastatic breast cancer. Dose levels are 1 mg/kg (open circles), 2 mg/kg (closed circles), 4 mg/kg (open triangles), and 8 mg/kg (closed triangles). Like many other monoclonal antibodies, trastuzumab is characterized by dose-dependent pharmacokinetics, with increasing half-life and decreasing clearance if

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Exogenous administration of radiolabeled epidermal growth factor (125I-hEGF) shows saturation of the receptor-mediated uptake mechanism into hepatocytes at higher doses.121 Similarly, a mechanism-based target-mediated drug distribution model has been developed to accurately describe the nonlinear pharmacokinetics of rhVEGF in patients with coronary artery disease.88 Nonlinearity was caused by elimination of rhVEGF by binding to saturable high-afnity receptors followed by internalization and degradation. Kinetic analysis of the in vivo tissue distribution of the granulocyte colony stimulating factor (G-CSF) derivative nartograstim revealed a nonlinear clearance process by the bone marrow and spleen with increasing doses of nartogastrim.122 Further studies with nartogastrim suggest that nonlinearity in the early-phase bone marrow clearance might be due to the downregulation of G-CSF receptors on the cell surface.123

ing of protein drugs have recently been published in this journal.129

EXPOSURE/RESPONSE CORRELATIONS
Pharmacokinetic characteristics and systemic exposure of drugs, conventional small molecule drugs as well as peptides and proteins, are meaningful for applied pharmacotherapy and dosage selection only if they can be related to desired and/or undesired therapeutic outcomes, such as efcacy and/or toxicity. Integrated pharmacokinetic/pharmacodynamic (PK/PD) modeling concepts are a particularly useful tool in the characterization of the frequently complex exposure/response relationships of peptide and protein drugs.130,131 The application of PK/PD-modeling has been identied as benecial in all phases of preclinical and clinical drug development. This is especially the case when there is a focus on dosage optimization and identication of covariates that are causal for between- and within-subject differences in drug response and/or toxicity.3 The widespread application of PK/PD-modeling during the drug development process has in the past repeatedly been promoted by industry, academia, and regulatory authorities,130,132134 and has recently further been endorsed by FDAs publication of the Exposure-Response Guidance document.4 Due to their inherent structural similarity to endogenous compounds, the molecular mechanism of action of both peptides and proteins is generally well understood. Hence, mechanismbased PK/PD-modeling is frequently applied for peptide and protein drugs, thereby appreciating the physiological events involved in the elaboration of the observed effect. The major advantage of mechanism-based modeling compared to empirical modeling comes from improved predictive capabilities in extrapolating the model to other clinical situations.135 Application of PK/PD-modeling concepts for peptides and proteins includes relatively simple as well as very sophisticated and elaborate modeling approaches that are dependent on the response system modeled and the intended use of the model. Elaborate models have, for example, been developed for situations where the effect is not mediated via direct interaction between drug concentration and response system; the effect rather involves several transduction processes that include at their rate-limiting step the stimulation or inhibition of a

INTERSPECIES SCALING
In drug development, extrapolation of pharmacokinetic data from animals to humans is frequently necessary, especially at the transition of preclinical to clinical development to predict plasma concentrations in humans and select an appropriate dosage range for rst-in-man studies. Allometric scaling is a frequently used tool to make predictions on pharmacokinetic parameters in humans based on data from several animal species, with several approaches available at variable success rates.124126 For most traditional, small molecule drugs, allometric scaling is often imprecise or completely fails, especially if hepatic metabolism is a major elimination pathway. This is probably due to interspecies differences in substrate specicity and/or activity levels of drug metabolizing enzymes and drug transporters. For peptides and proteins, however, allometric scaling has frequently proven to be much more precise and reliable, probably because of the fact that the handling of peptides and proteins is well preserved between different mammalian species.26,81 Clearance and volume of distribution of numerous therapeutically used proteins like growth hormone, t-PA, and a recombinant humanized monoclonal antibody against VEGF follow a welldened, weight-dependent allometric relationship between laboratory animals and humans.127,128 Further in-depth discussions on interspecies scalJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 9, SEPTEMBER 2004

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physiologic process, or include endogenous positive or negative feedback mechanisms. In a relatively simple approach without detailed appreciation of the involved molecular and cellular processes, Radwanski et al. assessed the effect of recombinant IL-10 on the ex vivo release of the pro-inammatory cytokines TNF-a and IL-1b in LPS-stimulated leukocytes using an inhibitory Emax-model.136 IL-10 caused prolonged and substantial suppression of both, TNF-a and IL-1b. The resulting pharmacodynamic parameters were thought to help guide dosing patterns for the immunosuppressive action of IL-10 in conditions with overproduction of pro-inammatory cytokines. Similarly, Gaussem et al. related exposure to response for the tripeptide-based direct thrombin inhibitor S18326.137 Using a population PK/PDbased sigmoid Emax-model, S18326 plasma concentrations were related to antifactor IIa activity assessed via a chromogenic thrombin inhibition assay (Fig. 4). This approach indicated that thrombin inhibition plateaued at S18326 concentrations above 0.5 mM. A more mechanism-based approach appreciating the underlying physiological processes was used in the evaluation of SB-240563, a humanized monoclonal antibody directed towards IL-5 in monkeys.138 IL-5 appears to play a signicant role in the production, activation, and maturation of eosinophils. The delayed effect of SB-240563 on eosinophils is consistent with its mechanism of action via binding to and thus inactivation of IL-5. It was modeled using an indirect response model with inhibition of the production of response (eosinophils) (Fig. 5). The obtained IC50 value for

Figure 5. PK/PD-model predicted and observed plasma concentration (observed: closed circles; predicted: solid line) and eosinophil count (observed: open squares; predicted: dashed line) following SC administration of 1 mg/kg of the anti-IL-5 humanized monoclonal antibody SB-240563 in a cynomolgus monkey. A mechanism based indirect response model was used to model eosinophil count as a function of SB-240563 plasma concentration. From ref. 138, reproduced with permission.

Figure 4. Relationship between individual anti-Factor IIa activity and predicted plasma concentration based on population pharmacokinetics of the thrombin inhibitor S18326. A sigmoid Emax-model was used to relate concentration to effect. Ninety-ve percent condence limits around the mean predicted relationship

reduction of circulating eosinophils combined with a long terminal half-life of 13 days suggests the possibility of an infrequent dosing regimen for SB240563 for the pharmacotherapy of disorders with increased eosinophil function, such as asthma. Indirect response models were also used for the effect of growth hormone on endogenous IGF-1 concentration,139 as well as the effect of epoetin-a on two response parameters, free ferritin concentration, and soluble transferrin receptor concentration.140 Similarly, a modied indirect response model was used to relate the concentration of the humanized antifactor IX antibody SB-249417 to factor IX activity in Cynomolgus monkeys as well as humans.141,142 The drug effect in this model was introduced by interrupting the natural degradation of Factor IX by sequestration of Factor IX by the antibody. More sophisticated, mechanism-based PK/PD models have been developed in cases where endogenous regulatory mechanisms are involved in the response to peptide and protein drugs, for example, physiologic feedback mechanisms and/or tolerance processes as well as circadian rhythms. For modeling the effect of cetrorelix, a LH-RH antagonist, on endogenous testosterone levels, Pechstein et al. had to incorporate a cosine-based circadian rhythm for testosterone baseline levels into their modeling approach.143 Only after inclusion of this time-dependent variation in testosterJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 9, SEPTEMBER 2004

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one baseline, effects of cetrorelix could accurately be delineated from the physiologic variation in testosterone serum concentrations (Fig. 6). Fattinger et al. developed an even more extensive modeling complex relating plasma concentrations of antide, another LH-RH antagonist, to its effect on the plasma concentrations of the endogenous hormones LH and testosterone. Although LH concentration was modeled as directly affected through inhibition of LH secretion in the pituitary by antide, testosterone was indirectly affected via modulation of LH concentration. The modeling also accounted for the additional feedback inhibition of LH production via endogenous testosterone levels.144

CONCLUSION
Peptide and protein drugs are subject to the same general principles of pharmacokinetics and exposure/response correlations as conventional small drug molecules. Due to their similarity to protein nutrients and/or especially regulatory endogenous peptides and proteins, however, numerous caveats and pitfalls related to bioanalytics and pharmacokinetics have to be considered and addressed during their drug development process. Furthermore, PK/PD correlations are frequently complicated due to the close interaction of peptide and protein drugs with endogenous substances and regulatory feedback mechanisms. Additional investigations and resources are necessary to overcome some of these difculties to ensure a rapid and successful drug development process. Biotech and genomic companies currently perform nearly one-fth of all pharmaceutical R&D. This gure is expected to double within the next 10 years145 as several biotechnology companies have already become major players in the pharmaceutical industry.1 Based on these important trends and the current success of peptides and proteins in applied therapeutics, it can be predicted that biotech drugs will play a major, if not dominant, role in the drug development arena over the coming decades. The widespread application of pharmacokinetic and pharmacodynamic concepts including exposureresponse correlations in all preclinical and clinical phases of drug development is expected to result in a scientically driven, evidence-based, and more focused drug development process.3 Thus, PK/PD concepts are likely to continue and expand their role as a fundamental factor in the successful development of biotechnologically derived drug products in the future. A rapid, cost-efcient, and rational drug development process based on the efcient use of pharmacokinetic and PK/PD-based concepts will undoubtedly propel the success and future of all biotech products, and might thus provide the novel medications that may serve as the key for the aspired personalized medicine in the healthcare systems of the future.146

Figure 6. PK/PD relationship between endogenous testosterone concentrations (lled circles observed, solid line modeled) and plasma concentrations (mean SD) of the LH-RH antagonist cetrorelix (open circles observed, dashed line modeled) after i.v. dosing of 3 mg cetrorelix. Model A only includes an indirect response model with constant testosterone baseline, Model B additionally

ACKNOWLEDGMENTS
Dr. Lisa Tang is partially supported by a Rho Chi Schering-Plough Graduate Scholarship through the American Foundation for Pharmaceutical Education (AFPE).

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