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Diagnosing and Improving Pharmacokinetic Performance
Chapter 38
Diagnosing and Improving Pharmacokinetic Performance 38.1 INTRODUCTION In vivo animal studies are performed during drug discovery for compounds having a wide range of pharmacokinetic (PK) properties, including those whose PK parameters differ greatly from the majority of commercial drugs (i.e., not drug-like). It is often found that a drug discovery compound does not achieve sufficient in vivo PK performance to produce efficacy or meet PK advancement criteria. Doses can be increased or administered more frequently, less preferable dosing routes can be used (e.g., intravenous (I.V.)), or a customized dosage form can be administered. This may be necessary for early pharmacology proof-of-concept studies. However, there are inevitable trade-offs if such compounds move into development. Non-drug-like compounds may require IV dosing instead of the preferred oral (PO), complicated or expensive formulations for sustained release for rapidly cleared compounds, or prodrugs for insoluble or impermeable compounds. In this common scenario, it is helpful to use PK parameters to determine a list of possible underlying physicochemical, biochemical, and structural property limitations and then perform in vitro assays to confirm or rule out the possible limitating properties. This provides insights for informed decisions on specific structural modifications that can be made to improve the limiting property. Repeating the in vitro assay on the modified structures determines whether the modification has improved the property limitation. The improved compounds can be tested in vivo to determine if the PK parameters have been improved [1,2]. This mechanistic scheme is shown in Figure 38.1.
Poor in vivo PK parameter In vitro assays diagnose possible limiting properties Synthesize modified structures
Formulate for dissolution or solubility
Test new structures using in vitro assay Test improved analog or formulation for in vivo PK FIGURE 38.1 Scheme for diagnosis and improvement of a property that limits PK performance.
Drug-Like Properties. http://dx.doi.org/10.1016/B978-0-12-801076-1.00038-1 Copyright © 2016 Elsevier Inc. All rights reserved.
463
464
Drug-Like Properties
38.2
DIAGNOSING UNDERLYING PROPERTY LIMITATIONS FROM PK PERFORMANCE
This section has examples of using this mechanistic approach to diagnosing the underlying property causes when the measured PK parameters of a drug discovery lead are inadequate. The following sections list three types of PK limitations, high CL, low F, and nonlinear PK, that discovery project teams work to improve. Major possible causes of each of these limitations are provided as bullet points. Examples of in vitro assays that can be used to confirm/rule out and quantitate the inadequate property are then listed. Strategies for modifying a structure to improve the property are discussed in chapters on the individual properties. Salt forms and formulations can also be used to improve low dissolution rate or solubility inadequacies. The ideas below can be expanded to addressing other PK problems. See, for example, the diagnosis of blood– brain barrier (BBB) permeation in Chapter 10.
38.2.1
Diagnosing the Cause of High Clearance or Short Half-Life
Following are potential property limitations and in vitro or in vivo assays that can be used for diagnosis. l Liver metabolism (hepatic clearance) – Phase I metabolic stability assay ▪ Microsomal stability with NADPH (reduced nicotinamide adenine dinucleotide phosphate), indicates CYP metabolism; use of microsomes from the PK species provides a better comparison ▪ S9 or cytosol stability, indicates non-CYP metabolism (e.g., AO, hydrolases) ▪ Recombinant CYP enzymes, or microsomes with CYP-specific inhibitors, with NADPH, indicates a specific CYP enzyme involvement in metabolism – Phase II metabolic stability assay ▪ Microsomal stability with uridine diphosphate glucuronic acid (UDPGA), indicates glucuronidation (UGT) enzymes ▪ Cytosol stability with 3’-phosphoadenosine-5’-phosphosulfate (PAPS), indicates sulfotransferase (SULT) enzymes ▪ Hepatocyte stability, indicates all hepatic enzymes – Structure elucidation of metabolites from one of the above incubations, indicates the site of structure modification l Liver biliary extraction (hepatic clearance) – Extraction into bile ▪ Quantitate drug in bile-duct-cannulated animal, indicates quantity of drug extracted into bile ▪ Cell monolayer assay containing bile canalicular efflux transporter (e.g., MDR1-MDCKII P-gp), Caco-2, or other cell line transfected with human efflux transporter, or selected transporter inhibitor), measure the efflux ratio, indicates the susceptibility of the drug to the efflux transporter l Kidney excretion (renal extraction) – Quantitate drug concentration in urine after keeping animal in a “metabolic cage” that captures urine, indicates quantity of drug extracted into urine – Cell monolayer permeability assay with selected renal tubule transporter expression or selected transporter inhibitors, indicates which transporter is involved l Enzymatic hydrolysis in plasma – Plasma stability assay, indicates the rate of degradation – Structure elucidation of degradation products from plasma incubation, indicates the site of structure modification l Red blood cell (RBC) binding – RBC partition assay, indicates partitioning into blood cells that might appear as clearance If the specific metabolizing enzyme or transporter can be identified, then information on substrate specificity can be used to guide structure modifications.
38.2.2 l
l
l
Diagnosing Cause of Low Oral Bioavailability
High first-pass metabolism by liver – Use the same liver metabolism and biliary extraction diagnosis as above Low intestinal solubility – Solubility assay in FaSSIF, FeSSIF, FaSSGF Low intestinal permeability – Passive transcellular diffusion assay
Diagnosing and Improving Pharmacokinetic Performance Chapter 38
465
▪ PAMPA or MDCK assay, indicates apical to basolateral permeability ▪ Physicochemical properties: high TPSA, low log P, high MW, high total hydrogen bonds, indicate structural property limitations to passive diffusion – Efflux transporter assay ▪ Cell monolayer assay containing intestinal efflux transporter (MDR1-MDCKII (P-gp), Caco-2, or other cell line transfected with human efflux transporter, or selected transporter inhibitor), measure the efflux ratio, indicates the susceptibility of the drug to the efflux transporter (Intestinal efflux transporters may be saturated by the oral dose.) l
l
Enzymatic or pH hydrolysis in intestine – Stability assay in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), or pH buffer; indicates enzymatic or pH hydrolysis Intestinal first-pass metabolism – Metabolic stability assay using intestinal microsomes, S9, or cytosol
38.2.3 l l
All of the property causes listed in Sections 38.2.1 and 38.2.2 Degradation in plasma – Plasma stability assay, indicates the potential for degradation by enzymes in plasma
38.2.4 l
l
l
l
l
Diagnosing the Cause of Low AUC or Cmax
Diagnosing the Cause of Nonlinear Pharmacokinetics
Precipitation in the gastrointestinal system and slow dissolution – Solubility assay in FaSSIF, FeSSIF, FaSSGF Saturation of liver metabolic enzymes at high concentration but not at low concentration – Metabolic stability assays noted above; indicates high binding (low Km) and clearance that could saturate Saturation of intestine efflux transporters at high concentration but not at low concentration – Cell monolayer permeability assays noted above; high efflux ratio and low Km indicates susceptibility to saturation at high enterocyte concentration Saturation of uptake transporter – Transporter transfected cell monolayer to measure uptake transporter rate Autoinduction of metabolic enzymes – Chronic in vivo dosing; increased liver weight and/or metabolic enzyme activity indicates induction – In vitro hepatocyte induction assay; increase in mRNA and enzyme activity is indicative of enzyme induction
38.3 CASE STUDIES ON DIAGNOSING UNFAVORABLE PK BEHAVIOR Both transporter-mediated absorption and capacity-limited metabolism can lead to nonlinear PK profiles as illustrated by the following two case studies.
38.3.1
Pharmacokinetics of CCR5 antagonist UK-427,857
The compound in Figure 38.2 was found to have much higher dose-normalized Cmax and area under the curve (AUC) when dosed in humans at 4.3 mg/kg than when dosed at 0.43 mg/kg (Table 38.1) [3]. Thus, there was an interest in diagnosing the cause of the nonlinear PK results. P-glycoprotein (P-gp) efflux studies showed the compound is a substrate for P-gp in the in vitro Caco-2 cell monolayer permeability assay. The Papp (A > B) was measured as <1 106 cm/s and the Papp (B > A) was 12 106 cm/s, which indicated an efflux ratio (Papp, B > A/Papp, A > B) greater than 10. In follow-up studies, the P-gp inhibitor verapamil was found to reduce the efflux ratio, suggesting the compound was a P-gp substrate. The P-gp binding affinity was measured as Km ¼ 37 mM and Vmax ¼ 55 nmol/mg/min. The P-gp efflux diagnosis was further confirmed by in vivo studies using P-gp double knockout and wild-type mice. Both Cmax and AUC values were significantly higher in knockout mice than in wildtype (Table 38.2). This supported the diagnosis that the higher Cmax and AUC at higher doses were caused by saturation of
466
Drug-Like Properties
F Me F
H N
N N
N
N
O Me
Me
log D7.4 = 2.1
H-bond donor = 1
pKa = 7.3
H-bond acceptor = 6
MW = 514
CLogP = 3.11
Low permeability Good solubility FIGURE 38.2 Structure and physicochemical properties of CCR5 antagonist UK-427,857.
TABLE 38.1 Pharmacokinetic Parameters of CCR5 Antagonist UK-427,857 [3] Parameter
Human
Comments
Oral Dose (mg/kg)
0.43 (30 mg)
4.3 (300 mg)
Elimination half-life (h)
8.9
10.6
Cmax (ng/mL), dose normalized
36
144
Increased
AUC (ng.h/mL), dose normalized
272
537
Increased
Tmax (h)
2.9
1.6
Decreased
Used with permission from D.K. Walker, S. Abel, P. Comby, G.J. Muirhead, A.N.R. Nedderman, D.A. Smith, Species differences in the disposition of the CCR5 antagonist, UK-427,857, a new potential treatment for HIV, Drug Metab. Dispos. 33 (2005) 587–595. Copyright 2005 American Society for Pharmacology and Experimental Therapeutics.
TABLE 38.2 Pharmacokinetic Parameters of CCR5 Antagonist UK-427,857 in P-gp Knockout Mice [3] P.O. 16 mg/kg
Cmax ng/mL
AUC ng .h /mL
Elimin. T1/2 (h)
Wild-type fvb mice
536
440
0.7
mdr1a/1b knockout
1119
1247
1
% Increase
108%
183%
P-gp efflux in the intestine, whereas P-gp efflux was not saturated at the lower dose and it limited the absorption. Saturation at the higher dose allowed higher absorption at the higher dose. Saturation of P-gp efflux transport led to the nonlinear PK. Structure modifications can be used to reduce P-gp efflux transport.
38.3.2
Pharmacokinetics of Triazole Antifungal Voriconazole
Voriconazole (Figure 38.3) has good solubility and excellent oral absorption. In humans, less than 7% voriconazole is eliminated through feces. It is mostly eliminated by hepatic clearance. Oral bioavailability of the drug is greater than 70% in humans. Voriconazole produced an unusual nonlinear PK profile (Figure 38.4) following the PO or IV administration in rat, which has been termed a “hockey-stick” profile [4]. The PK characteristics are gender dependent. The analog compound in Figure 38.5 does not have the nonlinear PK characteristics, due to the low log D (0.5), and is eliminated mostly by kidney.
Diagnosing and Improving Pharmacokinetic Performance Chapter 38
467
N OH
N
N
R
F S
F
N
N
F Aq. solubility = 0.7 mg/mL, log D7.4 = 1.8 Excellent absorption, < 7% in feces unchanged oral bioavailability > 70% FIGURE 38.3 Structure and physicochemical properties of triazole antifungal voriconazole.
Female rat
Male rat 100
Concentration (mg/mL)
100
Concentration (mg/mL)
FIGURE 38.4 Nonlinear PK of voriconazole in rat [4]. (Used with permission from Roffey, S.J., Cole, S., Comby, P., Gibson, D., Jezequel, S.G., Nedderman, A.N.R., Smith, D.A., Walker, D.K., Wood, N. (2003). The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog, and human. Drug Metab. Dispos., 31, 731–741. Copyright 2003 American Society for Pharmacology and Experimental Therapeutics).
Oral
10 1
IV
0.1
10
Oral 1
IV
0.1 0.01
0.01 0
6
12
18
0
24
6
12
FIGURE 38.5 Effects of log D on clearance and PK.
N N
OH
F
N
N R
30
36
N
N
HO
S
F
24
N
N N
18
Time (h)
Time (h)
N
N
F
F
F
log D7.4 = 1.8
log D7.4 = 0.5
Hepatic clearance
Renal clearance of unchanged drug
Capacity-limited nonlinear PK
Linear PK
Table 38.3 lists the gender-dependent PK parameters of voriconazole. The oral AUC (dose normalized) at 30 mg/kg was higher than IV AUC at 10 mg/kg, resulting in greater than 100% oral bioavailability (%F ¼ 159%) in male rat. This was diagnosed as capacity-limited elimination, due to saturation of metabolizing enzymes. The high absorption of the drug produced high exposure of the compound in systemic circulation. Another issue was that during multiple dosing by IV or oral administration, the AUC was lower than that during single dosing. This was diagnosed as being due to autoinduction of CYP450 metabolic enzyme by voriconazole. Consistent with
468
Drug-Like Properties
TABLE 38.3 Voriconazole: PK Data Interpretation in Rat [4] Sex
Male
Female
Plasma protein binding (%)
66
66
Dose (mg/kg)
10
10
Single dose AUCt (ug h/mL)
18.6
81.6
Gender dependent
Multiple dose AUCt (ug h/mL)
6.7
13.9
Dose (mg/kg)
30
30
Single dose Cmax (ug/mL)
9.5
16.7
Gender dependent
Single dose Tmax (h)
6
1
Gender dependent
Single dose AUCt (ug h/mL)
90
215.6
>IV, capacity-limited elimination
Multiple dose AUCt (ug h/mL)
32.3
57.4
Apparent bioavailability F (%)
159
88
Capacity-limited elimin. Good absorption
IV
Oral
Used with permission from S.J. Roffey, S. Cole, P. Comby, D. Gibson, S.G. Jezequel, A.N.R. Nedderman, D.A. Smith, D.K. Walker, N. Wood, The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog and human, Drug Metab. Dispos. 31 (2003) 731–741. Copyright 2003 American Society for Pharmacology and Experimental Therapeutics.
TABLE 38.4 Voriconazole: Autoinduction of CYP450s [4] Hepatic Microsomal Cytochrome P450 (nmol P450/mg protein)
Relative Liver Weight
Voriconazole Cmax (ug/mL)
Dose (mg/kg)
Male
Female
Male
Female
Male
Female
Control
0.88
0.51
3.71
3.7
None
None
3
0.85
0.65
3.86
4.04
0.61
1.32
10
1.21
0.68
4.17
4.26
3.64
6.14
30
1.77
0.79
4.38
5.04
9.69
14.6
80
2.08
0.92
5.57
6.26
28.4
30.4
Used with permission from S.J. Roffey, S. Cole, P. Comby, D. Gibson, S.G. Jezequel, A.N.R. Nedderman, D.A. Smith, D.K. Walker, N. Wood, The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog and human, Drug Metab. Dispos. 31 (2003) 731–741. Copyright 2003 American Society for Pharmacology and Experimental Therapeutics.
this diagnosis, there was an increase in liver weight and CYP450 enzyme activity with the multiple doses (Table 38.4). As animals were exposed to voriconazole, more CYP450 enzyme was induced, which metabolized voriconazole at a higher rate and the drug was eliminated faster. Hence, multiple dosing generated lower AUC than single dosing.
38.3.3
Optimization of a PDE5 Inhibitor
UK-343,664 (Figure 38.6) is a PDE5 inhibitor with favorable selectivity over PDE6. It progressed into development. However, it had dose-dependent nonproportional PK. AUC and Cmax increased as the oral dose level increased. UK-343,664 was diagnosed as having high P-gp efflux. At higher doses the P-gp was likely saturated, producing nonproportional exposure. Thus, the follow-on studies focused on reducing P-gp efflux. The optimized analog was UK-369,003, which had low P-gp efflux in the assay and produced proportional PK [5,6].
Diagnosing and Improving Pharmacokinetic Performance Chapter 38
FIGURE 38.6 PDE5 inhibitor UK-343, 664 was diagnosed as having high P-gp efflux, leading to nonlinear pharmacokinetics in human. Synthesis of analogs focused on reducing P-gp efflux using Caco-2 efflux ratio and yielded UK-369,003, which has favorable PK parameters [5,6].
O
N N
HN
O
469
N N
O
S
O
N
N
UK-343664
UK-369003
Non-proportional PK P-gp Km = 7.3 µM log P = 3.4 IC50 PDE5 = 1.1 nM PDE5/PDE6 = 103 MW = 565
Proportional PK Caco-2 ER = 1.5 log P = 1.3 IC50 PDE5 = 1.2 nM PDE5/PDE6 = 117 MW = 519 Caco-2 = 16 x 10−6 HLM t1/2 = 28 min CL = 12.8 mL/min/kg Vd = 2.8 t1/2 = 2.6 h Tmax = 1.0 h F = 34%
PROBLEMS (1) What dosing approaches can be tried to administer compounds that have poor absorption, short PK half-life, or low bioavailability after oral dosing? (2) What approach is preferable for enhancing absorption, PK half-life, or bioavailability? (3) What physicochemical or biochemical properties will lead to poor bioavailability, if they are low? (4) Which of the following properties might be a significant contributor to an observed high CL in a PK study using an IV dose: (a) low metabolic stability (liver), (b) low CYP inhibition, (c) high biliary excretion, (d) high plasma protein binding, (e) low renal extraction, (f) low plasma stability, (g) high red blood cell binding, (h) neutral pKa, (i) hERG binding, (j) low stability at pH 4, (k) high Phase I metabolism in intestinal epithelium, (l) high BBB permeability, (m) high renal extraction, (n) high metabolic stability (liver). (5) Which of the following properties might be a significant contributor to an observed low oral bioavailability in a PK study using a PO dose: (a) low metabolic stability (liver), (b) low CYP inhibition, (c) high biliary excretion, (d) high plasma protein binding, (e) low renal extraction, (f) low plasma stability, (g) high red blood cell binding, (h) neutral pKa, (i) hERG binding, (j) low stability at pH 4, (k) high Phase I metabolism in intestinal epithelium, (l) high BBB permeability, (m) high renal extraction, (n) high metabolic stability (liver), (o) P-gp efflux. (6) What effect might be observed if a compound is highly effluxed by P-gp in the intestine: (a) high CL, (b) oral dosedependent Cmax, (c) low Vd, (d) higher AUC at higher oral dose, (e) high AUC.
REFERENCES [1] L.-S.L. Gan, D.R. Thakker, Applications of the Caco-2 model in the design and development of orally active drugs: elucidation of biochemical and physical barriers posed by the intestinal epithelium, Adv. Drug Delivery Rev. 23 (1997) 77–98.
470
Drug-Like Properties
[2] L. Di, E.H. Kerns, Application of pharmaceutical profiling assays for optimization of drug-like properties, Curr. Opin. Drug Discovery Dev. 8 (2005) 495–504. [3] D.K. Walker, S. Abel, P. Comby, G.J. Muirhead, A.N.R. Nedderman, D.A. Smith, Species differences in the disposition of the CCR5 antagonist, UK427,857, a new potential treatment for HIV, Drug Metab. Dispos. 33 (2005) 587–595. [4] S.J. Roffey, S. Cole, P. Comby, D. Gibson, S.G. Jezequel, A.N.R. Nedderman, D.A. Smith, D.K. Walker, N. Wood, The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog and human, Drug Metab. Dispos. 31 (2003) 731–741. [5] D.J. Rawson, S. Ballard, C. Barber, L. Barker, K. Beaumont, M. Bunnage, S. Cole, M. Corless, S. Denton, D. Ellis, M. Floc’h, L. Foster, J. Gosset, F. Holmwood, C. Lane, D. Leahy, J. Mathias, G. Maw, W. Million, C. Poinsard, J. Price, R. Russel, S. Street, L. Watson, The discovery of UK-369003, a novel PDE5 inhibitor with the potential for oral bioavailability and dose-proportional pharmacokinetics, Bioorg. Med. Chem. 20 (2012) 498–509. [6] S. Abel, K.C. Beaumont, C.L. Crespi, M.D. Eve, L. Fox, R. Hyland, B.C. Jones, G.J. Muirhead, D.A. Smith, R.F. Venn, D.K. Walker, Potential role for P-glycoprotein in the non-proportional pharmacokinetics of UK-343,664 in man, Xenobiotica 31 (2001) 665–676.
Diagnosing and Improving Pharmacokinetic Performance 38.1 INTRODUCTION In vivo animal studies are performed during drug discovery for compounds having a wide range of pharmacokinetic (PK) properties, including those whose PK parameters differ greatly from the majority of commercial drugs (i.e., not drug-like). It is often found that a drug discovery compound does not achieve sufficient in vivo PK performance to produce efficacy or meet PK advancement criteria. Doses can be increased or administered more frequently, less preferable dosing routes can be used (e.g., intravenous (I.V.)), or a customized dosage form can be administered. This may be necessary for early pharmacology proof-of-concept studies. However, there are inevitable trade-offs if such compounds move into development. Non-drug-like compounds may require IV dosing instead of the preferred oral (PO), complicated or expensive formulations for sustained release for rapidly cleared compounds, or prodrugs for insoluble or impermeable compounds. In this common scenario, it is helpful to use PK parameters to determine a list of possible underlying physicochemical, biochemical, and structural property limitations and then perform in vitro assays to confirm or rule out the possible limitating properties. This provides insights for informed decisions on specific structural modifications that can be made to improve the limiting property. Repeating the in vitro assay on the modified structures determines whether the modification has improved the property limitation. The improved compounds can be tested in vivo to determine if the PK parameters have been improved [1,2]. This mechanistic scheme is shown in Figure 38.1.
Poor in vivo PK parameter In vitro assays diagnose possible limiting properties Synthesize modified structures
Formulate for dissolution or solubility
Test new structures using in vitro assay Test improved analog or formulation for in vivo PK FIGURE 38.1 Scheme for diagnosis and improvement of a property that limits PK performance.
Drug-Like Properties. http://dx.doi.org/10.1016/B978-0-12-801076-1.00038-1 Copyright © 2016 Elsevier Inc. All rights reserved.
463
464
Drug-Like Properties
38.2
DIAGNOSING UNDERLYING PROPERTY LIMITATIONS FROM PK PERFORMANCE
This section has examples of using this mechanistic approach to diagnosing the underlying property causes when the measured PK parameters of a drug discovery lead are inadequate. The following sections list three types of PK limitations, high CL, low F, and nonlinear PK, that discovery project teams work to improve. Major possible causes of each of these limitations are provided as bullet points. Examples of in vitro assays that can be used to confirm/rule out and quantitate the inadequate property are then listed. Strategies for modifying a structure to improve the property are discussed in chapters on the individual properties. Salt forms and formulations can also be used to improve low dissolution rate or solubility inadequacies. The ideas below can be expanded to addressing other PK problems. See, for example, the diagnosis of blood– brain barrier (BBB) permeation in Chapter 10.
38.2.1
Diagnosing the Cause of High Clearance or Short Half-Life
Following are potential property limitations and in vitro or in vivo assays that can be used for diagnosis. l Liver metabolism (hepatic clearance) – Phase I metabolic stability assay ▪ Microsomal stability with NADPH (reduced nicotinamide adenine dinucleotide phosphate), indicates CYP metabolism; use of microsomes from the PK species provides a better comparison ▪ S9 or cytosol stability, indicates non-CYP metabolism (e.g., AO, hydrolases) ▪ Recombinant CYP enzymes, or microsomes with CYP-specific inhibitors, with NADPH, indicates a specific CYP enzyme involvement in metabolism – Phase II metabolic stability assay ▪ Microsomal stability with uridine diphosphate glucuronic acid (UDPGA), indicates glucuronidation (UGT) enzymes ▪ Cytosol stability with 3’-phosphoadenosine-5’-phosphosulfate (PAPS), indicates sulfotransferase (SULT) enzymes ▪ Hepatocyte stability, indicates all hepatic enzymes – Structure elucidation of metabolites from one of the above incubations, indicates the site of structure modification l Liver biliary extraction (hepatic clearance) – Extraction into bile ▪ Quantitate drug in bile-duct-cannulated animal, indicates quantity of drug extracted into bile ▪ Cell monolayer assay containing bile canalicular efflux transporter (e.g., MDR1-MDCKII P-gp), Caco-2, or other cell line transfected with human efflux transporter, or selected transporter inhibitor), measure the efflux ratio, indicates the susceptibility of the drug to the efflux transporter l Kidney excretion (renal extraction) – Quantitate drug concentration in urine after keeping animal in a “metabolic cage” that captures urine, indicates quantity of drug extracted into urine – Cell monolayer permeability assay with selected renal tubule transporter expression or selected transporter inhibitors, indicates which transporter is involved l Enzymatic hydrolysis in plasma – Plasma stability assay, indicates the rate of degradation – Structure elucidation of degradation products from plasma incubation, indicates the site of structure modification l Red blood cell (RBC) binding – RBC partition assay, indicates partitioning into blood cells that might appear as clearance If the specific metabolizing enzyme or transporter can be identified, then information on substrate specificity can be used to guide structure modifications.
38.2.2 l
l
l
Diagnosing Cause of Low Oral Bioavailability
High first-pass metabolism by liver – Use the same liver metabolism and biliary extraction diagnosis as above Low intestinal solubility – Solubility assay in FaSSIF, FeSSIF, FaSSGF Low intestinal permeability – Passive transcellular diffusion assay
Diagnosing and Improving Pharmacokinetic Performance Chapter 38
465
▪ PAMPA or MDCK assay, indicates apical to basolateral permeability ▪ Physicochemical properties: high TPSA, low log P, high MW, high total hydrogen bonds, indicate structural property limitations to passive diffusion – Efflux transporter assay ▪ Cell monolayer assay containing intestinal efflux transporter (MDR1-MDCKII (P-gp), Caco-2, or other cell line transfected with human efflux transporter, or selected transporter inhibitor), measure the efflux ratio, indicates the susceptibility of the drug to the efflux transporter (Intestinal efflux transporters may be saturated by the oral dose.) l
l
Enzymatic or pH hydrolysis in intestine – Stability assay in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), or pH buffer; indicates enzymatic or pH hydrolysis Intestinal first-pass metabolism – Metabolic stability assay using intestinal microsomes, S9, or cytosol
38.2.3 l l
All of the property causes listed in Sections 38.2.1 and 38.2.2 Degradation in plasma – Plasma stability assay, indicates the potential for degradation by enzymes in plasma
38.2.4 l
l
l
l
l
Diagnosing the Cause of Low AUC or Cmax
Diagnosing the Cause of Nonlinear Pharmacokinetics
Precipitation in the gastrointestinal system and slow dissolution – Solubility assay in FaSSIF, FeSSIF, FaSSGF Saturation of liver metabolic enzymes at high concentration but not at low concentration – Metabolic stability assays noted above; indicates high binding (low Km) and clearance that could saturate Saturation of intestine efflux transporters at high concentration but not at low concentration – Cell monolayer permeability assays noted above; high efflux ratio and low Km indicates susceptibility to saturation at high enterocyte concentration Saturation of uptake transporter – Transporter transfected cell monolayer to measure uptake transporter rate Autoinduction of metabolic enzymes – Chronic in vivo dosing; increased liver weight and/or metabolic enzyme activity indicates induction – In vitro hepatocyte induction assay; increase in mRNA and enzyme activity is indicative of enzyme induction
38.3 CASE STUDIES ON DIAGNOSING UNFAVORABLE PK BEHAVIOR Both transporter-mediated absorption and capacity-limited metabolism can lead to nonlinear PK profiles as illustrated by the following two case studies.
38.3.1
Pharmacokinetics of CCR5 antagonist UK-427,857
The compound in Figure 38.2 was found to have much higher dose-normalized Cmax and area under the curve (AUC) when dosed in humans at 4.3 mg/kg than when dosed at 0.43 mg/kg (Table 38.1) [3]. Thus, there was an interest in diagnosing the cause of the nonlinear PK results. P-glycoprotein (P-gp) efflux studies showed the compound is a substrate for P-gp in the in vitro Caco-2 cell monolayer permeability assay. The Papp (A > B) was measured as <1 106 cm/s and the Papp (B > A) was 12 106 cm/s, which indicated an efflux ratio (Papp, B > A/Papp, A > B) greater than 10. In follow-up studies, the P-gp inhibitor verapamil was found to reduce the efflux ratio, suggesting the compound was a P-gp substrate. The P-gp binding affinity was measured as Km ¼ 37 mM and Vmax ¼ 55 nmol/mg/min. The P-gp efflux diagnosis was further confirmed by in vivo studies using P-gp double knockout and wild-type mice. Both Cmax and AUC values were significantly higher in knockout mice than in wildtype (Table 38.2). This supported the diagnosis that the higher Cmax and AUC at higher doses were caused by saturation of
466
Drug-Like Properties
F Me F
H N
N N
N
N
O Me
Me
log D7.4 = 2.1
H-bond donor = 1
pKa = 7.3
H-bond acceptor = 6
MW = 514
CLogP = 3.11
Low permeability Good solubility FIGURE 38.2 Structure and physicochemical properties of CCR5 antagonist UK-427,857.
TABLE 38.1 Pharmacokinetic Parameters of CCR5 Antagonist UK-427,857 [3] Parameter
Human
Comments
Oral Dose (mg/kg)
0.43 (30 mg)
4.3 (300 mg)
Elimination half-life (h)
8.9
10.6
Cmax (ng/mL), dose normalized
36
144
Increased
AUC (ng.h/mL), dose normalized
272
537
Increased
Tmax (h)
2.9
1.6
Decreased
Used with permission from D.K. Walker, S. Abel, P. Comby, G.J. Muirhead, A.N.R. Nedderman, D.A. Smith, Species differences in the disposition of the CCR5 antagonist, UK-427,857, a new potential treatment for HIV, Drug Metab. Dispos. 33 (2005) 587–595. Copyright 2005 American Society for Pharmacology and Experimental Therapeutics.
TABLE 38.2 Pharmacokinetic Parameters of CCR5 Antagonist UK-427,857 in P-gp Knockout Mice [3] P.O. 16 mg/kg
Cmax ng/mL
AUC ng .h /mL
Elimin. T1/2 (h)
Wild-type fvb mice
536
440
0.7
mdr1a/1b knockout
1119
1247
1
% Increase
108%
183%
P-gp efflux in the intestine, whereas P-gp efflux was not saturated at the lower dose and it limited the absorption. Saturation at the higher dose allowed higher absorption at the higher dose. Saturation of P-gp efflux transport led to the nonlinear PK. Structure modifications can be used to reduce P-gp efflux transport.
38.3.2
Pharmacokinetics of Triazole Antifungal Voriconazole
Voriconazole (Figure 38.3) has good solubility and excellent oral absorption. In humans, less than 7% voriconazole is eliminated through feces. It is mostly eliminated by hepatic clearance. Oral bioavailability of the drug is greater than 70% in humans. Voriconazole produced an unusual nonlinear PK profile (Figure 38.4) following the PO or IV administration in rat, which has been termed a “hockey-stick” profile [4]. The PK characteristics are gender dependent. The analog compound in Figure 38.5 does not have the nonlinear PK characteristics, due to the low log D (0.5), and is eliminated mostly by kidney.
Diagnosing and Improving Pharmacokinetic Performance Chapter 38
467
N OH
N
N
R
F S
F
N
N
F Aq. solubility = 0.7 mg/mL, log D7.4 = 1.8 Excellent absorption, < 7% in feces unchanged oral bioavailability > 70% FIGURE 38.3 Structure and physicochemical properties of triazole antifungal voriconazole.
Female rat
Male rat 100
Concentration (mg/mL)
100
Concentration (mg/mL)
FIGURE 38.4 Nonlinear PK of voriconazole in rat [4]. (Used with permission from Roffey, S.J., Cole, S., Comby, P., Gibson, D., Jezequel, S.G., Nedderman, A.N.R., Smith, D.A., Walker, D.K., Wood, N. (2003). The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog, and human. Drug Metab. Dispos., 31, 731–741. Copyright 2003 American Society for Pharmacology and Experimental Therapeutics).
Oral
10 1
IV
0.1
10
Oral 1
IV
0.1 0.01
0.01 0
6
12
18
0
24
6
12
FIGURE 38.5 Effects of log D on clearance and PK.
N N
OH
F
N
N R
30
36
N
N
HO
S
F
24
N
N N
18
Time (h)
Time (h)
N
N
F
F
F
log D7.4 = 1.8
log D7.4 = 0.5
Hepatic clearance
Renal clearance of unchanged drug
Capacity-limited nonlinear PK
Linear PK
Table 38.3 lists the gender-dependent PK parameters of voriconazole. The oral AUC (dose normalized) at 30 mg/kg was higher than IV AUC at 10 mg/kg, resulting in greater than 100% oral bioavailability (%F ¼ 159%) in male rat. This was diagnosed as capacity-limited elimination, due to saturation of metabolizing enzymes. The high absorption of the drug produced high exposure of the compound in systemic circulation. Another issue was that during multiple dosing by IV or oral administration, the AUC was lower than that during single dosing. This was diagnosed as being due to autoinduction of CYP450 metabolic enzyme by voriconazole. Consistent with
468
Drug-Like Properties
TABLE 38.3 Voriconazole: PK Data Interpretation in Rat [4] Sex
Male
Female
Plasma protein binding (%)
66
66
Dose (mg/kg)
10
10
Single dose AUCt (ug h/mL)
18.6
81.6
Gender dependent
Multiple dose AUCt (ug h/mL)
6.7
13.9
Dose (mg/kg)
30
30
Single dose Cmax (ug/mL)
9.5
16.7
Gender dependent
Single dose Tmax (h)
6
1
Gender dependent
Single dose AUCt (ug h/mL)
90
215.6
>IV, capacity-limited elimination
Multiple dose AUCt (ug h/mL)
32.3
57.4
Apparent bioavailability F (%)
159
88
Capacity-limited elimin. Good absorption
IV
Oral
Used with permission from S.J. Roffey, S. Cole, P. Comby, D. Gibson, S.G. Jezequel, A.N.R. Nedderman, D.A. Smith, D.K. Walker, N. Wood, The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog and human, Drug Metab. Dispos. 31 (2003) 731–741. Copyright 2003 American Society for Pharmacology and Experimental Therapeutics.
TABLE 38.4 Voriconazole: Autoinduction of CYP450s [4] Hepatic Microsomal Cytochrome P450 (nmol P450/mg protein)
Relative Liver Weight
Voriconazole Cmax (ug/mL)
Dose (mg/kg)
Male
Female
Male
Female
Male
Female
Control
0.88
0.51
3.71
3.7
None
None
3
0.85
0.65
3.86
4.04
0.61
1.32
10
1.21
0.68
4.17
4.26
3.64
6.14
30
1.77
0.79
4.38
5.04
9.69
14.6
80
2.08
0.92
5.57
6.26
28.4
30.4
Used with permission from S.J. Roffey, S. Cole, P. Comby, D. Gibson, S.G. Jezequel, A.N.R. Nedderman, D.A. Smith, D.K. Walker, N. Wood, The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog and human, Drug Metab. Dispos. 31 (2003) 731–741. Copyright 2003 American Society for Pharmacology and Experimental Therapeutics.
this diagnosis, there was an increase in liver weight and CYP450 enzyme activity with the multiple doses (Table 38.4). As animals were exposed to voriconazole, more CYP450 enzyme was induced, which metabolized voriconazole at a higher rate and the drug was eliminated faster. Hence, multiple dosing generated lower AUC than single dosing.
38.3.3
Optimization of a PDE5 Inhibitor
UK-343,664 (Figure 38.6) is a PDE5 inhibitor with favorable selectivity over PDE6. It progressed into development. However, it had dose-dependent nonproportional PK. AUC and Cmax increased as the oral dose level increased. UK-343,664 was diagnosed as having high P-gp efflux. At higher doses the P-gp was likely saturated, producing nonproportional exposure. Thus, the follow-on studies focused on reducing P-gp efflux. The optimized analog was UK-369,003, which had low P-gp efflux in the assay and produced proportional PK [5,6].
Diagnosing and Improving Pharmacokinetic Performance Chapter 38
FIGURE 38.6 PDE5 inhibitor UK-343, 664 was diagnosed as having high P-gp efflux, leading to nonlinear pharmacokinetics in human. Synthesis of analogs focused on reducing P-gp efflux using Caco-2 efflux ratio and yielded UK-369,003, which has favorable PK parameters [5,6].
O
N N
HN
O
469
N N
O
S
O
N
N
UK-343664
UK-369003
Non-proportional PK P-gp Km = 7.3 µM log P = 3.4 IC50 PDE5 = 1.1 nM PDE5/PDE6 = 103 MW = 565
Proportional PK Caco-2 ER = 1.5 log P = 1.3 IC50 PDE5 = 1.2 nM PDE5/PDE6 = 117 MW = 519 Caco-2 = 16 x 10−6 HLM t1/2 = 28 min CL = 12.8 mL/min/kg Vd = 2.8 t1/2 = 2.6 h Tmax = 1.0 h F = 34%
PROBLEMS (1) What dosing approaches can be tried to administer compounds that have poor absorption, short PK half-life, or low bioavailability after oral dosing? (2) What approach is preferable for enhancing absorption, PK half-life, or bioavailability? (3) What physicochemical or biochemical properties will lead to poor bioavailability, if they are low? (4) Which of the following properties might be a significant contributor to an observed high CL in a PK study using an IV dose: (a) low metabolic stability (liver), (b) low CYP inhibition, (c) high biliary excretion, (d) high plasma protein binding, (e) low renal extraction, (f) low plasma stability, (g) high red blood cell binding, (h) neutral pKa, (i) hERG binding, (j) low stability at pH 4, (k) high Phase I metabolism in intestinal epithelium, (l) high BBB permeability, (m) high renal extraction, (n) high metabolic stability (liver). (5) Which of the following properties might be a significant contributor to an observed low oral bioavailability in a PK study using a PO dose: (a) low metabolic stability (liver), (b) low CYP inhibition, (c) high biliary excretion, (d) high plasma protein binding, (e) low renal extraction, (f) low plasma stability, (g) high red blood cell binding, (h) neutral pKa, (i) hERG binding, (j) low stability at pH 4, (k) high Phase I metabolism in intestinal epithelium, (l) high BBB permeability, (m) high renal extraction, (n) high metabolic stability (liver), (o) P-gp efflux. (6) What effect might be observed if a compound is highly effluxed by P-gp in the intestine: (a) high CL, (b) oral dosedependent Cmax, (c) low Vd, (d) higher AUC at higher oral dose, (e) high AUC.
REFERENCES [1] L.-S.L. Gan, D.R. Thakker, Applications of the Caco-2 model in the design and development of orally active drugs: elucidation of biochemical and physical barriers posed by the intestinal epithelium, Adv. Drug Delivery Rev. 23 (1997) 77–98.
470
Drug-Like Properties
[2] L. Di, E.H. Kerns, Application of pharmaceutical profiling assays for optimization of drug-like properties, Curr. Opin. Drug Discovery Dev. 8 (2005) 495–504. [3] D.K. Walker, S. Abel, P. Comby, G.J. Muirhead, A.N.R. Nedderman, D.A. Smith, Species differences in the disposition of the CCR5 antagonist, UK427,857, a new potential treatment for HIV, Drug Metab. Dispos. 33 (2005) 587–595. [4] S.J. Roffey, S. Cole, P. Comby, D. Gibson, S.G. Jezequel, A.N.R. Nedderman, D.A. Smith, D.K. Walker, N. Wood, The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog and human, Drug Metab. Dispos. 31 (2003) 731–741. [5] D.J. Rawson, S. Ballard, C. Barber, L. Barker, K. Beaumont, M. Bunnage, S. Cole, M. Corless, S. Denton, D. Ellis, M. Floc’h, L. Foster, J. Gosset, F. Holmwood, C. Lane, D. Leahy, J. Mathias, G. Maw, W. Million, C. Poinsard, J. Price, R. Russel, S. Street, L. Watson, The discovery of UK-369003, a novel PDE5 inhibitor with the potential for oral bioavailability and dose-proportional pharmacokinetics, Bioorg. Med. Chem. 20 (2012) 498–509. [6] S. Abel, K.C. Beaumont, C.L. Crespi, M.D. Eve, L. Fox, R. Hyland, B.C. Jones, G.J. Muirhead, D.A. Smith, R.F. Venn, D.K. Walker, Potential role for P-glycoprotein in the non-proportional pharmacokinetics of UK-343,664 in man, Xenobiotica 31 (2001) 665–676.