articles
nature publishing group
Pharmacokinetic and Pharmacodynamic Evaluation of the Novel CCR1 Antagonist CCX354 in Healthy Human Subjects: Implications for Selection of Clinical Dose DJ Dairaghi1, P Zhang1, Y Wang1, LC Seitz1, DA Johnson1, S Miao1, LS Ertl1, Y Zeng1, JP Powers1, AM Pennell1, P Bekker1, TJ Schall1 and JC Jaen1 The safety and pharmacokinetic (PK)/pharmacodynamic (PD) profile of the novel CCR1 antagonist CCX354 was evaluated in double-blind, placebo-controlled, single- and multiple-dose phase I studies (1–300 mg/day oral doses). CCX354 was well tolerated and displayed a linear dose–exposure profile, with half-life approaching 7 h at the 300-mg dose. The extent of CCR1 receptor blockade on blood monocytes, which correlated well with plasma concentrations of the drug, was assessed using fluorescently labeled CCL3 binding in whole blood from phase I subjects. High levels of receptor coverage at the 12-h time point were achieved after a single dose of 100 mg CCX354. Preclinical studies indicate that effective blockade of inflammatory cell infiltration into tissues requires ≥90% CCR1 inhibition on blood leukocytes at all times. The comparison of the properties of CCX354 with those published for other CCR1 antagonists has informed the dose selection for ongoing clinical development of CCX354 in rheumatoid arthritis (RA). Orchestrated temporal tissue expression of chemokines and chemokine receptors (CKRs) regulates the recruitment of leukocytes from the circulation to sites of injury and inflammation.1,2 The therapeutic potential associated with the regulation of this network has been apparent for well over a decade. There are two approved drugs in this class: the CCR5 antagonist maraviroc (Selzentry), for the treatment of HIV infection, 3 and the CXCR4 antagonist plerixafor (Mozobil), for hematopoietic stem cell mobilization.4 Importantly, neither drug works by blocking the recruitment of leukocytes to sites of inflammation; this is a goal that has yet to be achieved, and several of the early chemokine-targeted agents (monoclonal antibodies and small molecules) have been unsuccessful in the clinic.5 To date, positive reports of clinical anti-inflammatory activity have appeared with regard to two CKR antagonists. First, our group recently described the efficacy of a small-molecule CCR9 antagonist in Crohn’s disease.6,7 In another report, the CCR1 antagonist CP-481,715 showed promising clinical and histological effects in an early-phase clinical trial in rheumatoid arthritis (RA),8 although clinical efficacy was not demonstrated in a larger study in which the placebo response rate was
unusually high.9 In contrast to these successes, a list of clinical failures includes small-molecule antagonists of CCR1, 10,11 CCR2/CCR5,5 and CXCR3;12 in particular, antagonists of CCR2 (refs. 13,14) and CCR5 (ref. 15) have failed to show efficacy in phase II RA trials. These studies may now provide sufficient information to formulate a therapeutic hypothesis regarding the use of CKR antagonists in the treatment of inflammatory diseases. In RA, various chemokines are upregulated in the inflamed synovium, where they promote the recruitment of leukocytes into the joint.16,17 Analysis of synovial tissue and fluid, as well as peripheral blood, from subjects with RA has confirmed the importance of chemokines in the pathogenesis of this disease.18,19 In a comparison of subjects with RA, osteoarthritis, and reactive arthritis, abundant expression levels of CCR1, CCR5, and CXCR4 were observed in the synovial tissue in all forms of arthritis; however, the CCR1/CCR5 chemokines CCL5 and CCL15 showed a selective increase in synovial tissue only in RA.18 Furthermore, the synovial fluid in joints affected by RA contains proteolytically processed forms of chemokines with markedly enhanced activity toward CCR1.20
1ChemoCentryx, Inc., Mountain View, California, USA. Correspondence: DJ Dairaghi (
[email protected])
Received 10 December 2010; accepted 28 January 2011; advance online publication 30 March 2011. doi:10.1038/clpt.2011.33 726
VOLUME 89 NUMBER 5 | may 2011 | www.nature.com/cpt
articles
THP-1 cell chemotaxis
b
Saturation binding to CCR1 human monocytes
20,000
Vehicle 1 nmol/l CCX354 10 nmol/l CCX354 100 nmol/l CCX354 1000 nmol/l CCX354
15000 Bound/Free
15,000 15,000
10,000
10000 5000
0
15,000
5,000
10,000
10,000
5,000
0
0
Bound (cpm)
Migration signal (fluorescence)
a
Bound
5,000
CCX354: Ki = 1.5 nmol/l
10−12 10−11 10−10 10−9 10−8 10−7 10−6 10−5 10−4 0
CCX354 (mol/l)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
[125I]-CCL15 (nmol/l)
c
Human monocyte chemotaxis towards CCL3 in 100% serum 7,500
d
Control
5,000
1000 nmol/l
2,500
0 −13 10−12 10−11 10−10 10−9 10−8 10−7 10−6 10 CCL3 (mol/l)
RA patient
Migration signal (fluorescence)
250 nmol/l
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
THP-1 cell chemotaxis to RA synovial fluids
CCX354 Vehicle
0
4,000 Migration signal (fluorescence)
8,000
Figure 1 In vitro characterization of CCX354. (a) CCX354 inhibition of THP-1 cell chemotaxis toward 50 pmol/l CCL15 (n = 8 repeats per data point; the experiment was repeated 52 times). (b) CCX354 inhibits [125I]-CCL15 binding to CCR1 on human blood monocytes with a Ki of 1.5 nmol/l (n = 8 repeats per data point; the experiment was repeated 2 times). (c) Chemotaxis of human blood monocytes toward CCL3 in 100% human serum and in the presence of various concentrations of CCX354 (squares, vehicle; closed circles, 250 nmol/l; open circles, 1,000 nmol/l) (n = 8 repeats per data point; the experiment was repeated three times). (d) CCX354 (1 µmol/l) inhibition of THP-1 cell chemotaxis toward rheumatoid arthritis (RA) synovial fluid. The data shown reflect testing of one set of samples collected from 17 RA patients (each measurement was repeated three times); a separate set of 18 RA samples produced similar results (data not shown).
Monocytes are the most important among the leukocytes that infiltrate joints in RA. A strong correlation exists between the numbers of synovial tissue monocytes and the severity of joint pain.21 Furthermore, drug-induced changes in the numbers of synovial sublining macrophages correlate well with the degree of the drugs’ clinical efficacy.22 Monocytes express high levels of CCR1.23,24 Therefore, CCR1-mediated monocyte recruitment into the synovium is considered critical in maintaining inflammation in RA. CCR1 may also play a direct role in inflammatory osteolytic bone loss in RA, as CCR1 activation is required for osteoclast maturation.25,26 CCX354 is a novel, orally bioavailable CCR1 antagonist27,28 that has recently completed phase I clinical evaluation.29,30 In this report, we describe its biochemical characterization, safety, and pharmacokinetic (PK) profile after single and multiple doses in healthy human volunteers and the extent of CCR1 blockade (pharmacodynamic (PD) assessment) on blood monocytes from selected subjects in the phase I program. We also describe how this information has been integrated with a detailed preclinical PK/PD analysis of receptor coverage requirements for optimal efficacy. Based on this knowledge, our analysis of published clinical information on other CCR1 antagonists suggests that at least some of the reported failures of other agents might have been related to insufficient levels of CCR1 blockade. Clinical pharmacology & Therapeutics | VOLUME 89 NUMBER 5 | may 2011
Results In vitro assessment of potency and selectivity
CCL15 mediates THP-1 cell chemotaxis through CCR1; this process was potently inhibited by CCX354 (half-maximal inhibitory concentration (IC50) 1.4 ± 0.2 nmol/l) (Figure 1a). No inhibition of other chemotactic receptors was observed at concentrations up to 10 µmol/l (Supplementary Table S1 online). Saturation binding experiments with [125I]-CCL15 on human monocytes identified CCX354 as a competitive CCR1 antagonist (Ki = 1.5 ± 0.1 nmol/l) (Figure 1b). Chemotaxis assays with human monocytes in 100% human serum displayed a typical bell-shaped dose–response curve with respect to the CCR1 chemokines CCL3, CCL5, CCL15*, and CCL23* (where * denotes the superagonist forms of these chemokines previously identified in synovial fluid in RA)20 (Figure 1c and Supplementary Figure S1b–d online). A marked right shift (~10-fold) and a general reduction in the height of the dose– response curves were observed in the presence of 250 nmol/l CCX354 in human serum; this 10-fold shift represents ~90% CCR1 blockade, which, as detailed below, is a critical threshold for inhibition of CCR1-mediated effects in vivo. THP-1 chemotaxis toward synovial fluid from 35 subjects with RA was also significantly blocked by CCX354 (Figure 1d) but not by the CCR2 antagonist MK0812 (Supplementary Figure S1a online). 727
articles a
Rabbit peritonitis
2.5 × 108
b
Rat plasma CCR1 coverage
2.0 × 108
1.5 × 108
% CCR1 inhibition
Peritoneal lavage (cells/animal)
100
*
80
90%
60
100 mg/kg 10 mg/kg 1 mg/kg
40 20 0
8
0
100 mg/kg
10 mg/kg
1 mg/kg
Vehicle
1.0 × 10
4
8
12
16
20
24
Time (h)
CCX354
c
4.0 × 107
Rabbit synovitis
d
*
7
100 mg/kg
20 mg/kg
4 mg/kg
Dex.
1.0 × 10
% CCR1 inhibition
*
Vehicle
Synovial lavage (cells/joint)
7
2.0 × 10
0
Rabbit plasma CCR1 coverage 100
3.0 × 107
90%
80 60
100 mg/kg 20 mg/kg 4 mg/kg
40 20 0 0
4
8 Time (h)
12
16
CCX354
Figure 2 Sustained CCR1 blockade in plasma in excess of 90% functional inhibition (chemotaxis) correlates with effective anti-inflammatory response in various animal models. (a) CCX354 was administered in oral dosages of 1, 10, or 100 mg/kg twice daily in a rat thioglycollate-induced peritonitis assay, using cell count in the peritoneal lavage as the primary readout. (b) Functional inhibition of rat CCR1 by CCX354 over a 24-h period, calculated from pharmacokinetic (PK) measurements taken in satellite animals and in vitro CCX354 dose–response inhibition of CCR1-mediated chemotaxis of rat leukocytes in 100% rat serum. (c) CCX354 was administered in oral dosages of 4, 20, or 100 mg/kg twice (−2 h and +4 h) in this 16-h assay of lipopolysaccharide-induced synovial inflammation in rabbits, using total cell count in the synovial lavage as the primary readout. (d) Functional inhibition of rabbit CCR1 over a 16-h period, calculated from PK measurements taken in satellite animals and in vitro CCX354 dose–response inhibition of CCR1-mediated chemotaxis of rabbit leukocytes in 100% rabbit serum.
Synovial fluid samples (n = 4) from subjects with osteoarthritis did not induce any THP-1 chemotactic activity (data not shown).
recruitment required >90% CCR1 blockade on blood leukocytes at all times (Figure 2d).
Plasma drug concentration levels required for in vivo effects
Pharmacokinetics and pharmacodynamics of CCX354 in humans
CCX354 was evaluated in two animal models of inflammatory leukocyte trafficking (thioglycollate-induced peritonitis in rats and lipopolysaccharide (LPS)-induced synovitis in rabbits). The rats receiving 100 mg/kg CCX354 had significantly reduced inflammatory cell counts as compared to vehicle-treated animals (Figure 2a), and these animals also experienced >90% CCR1 coverage on blood leukocytes at all times during the study (Figure 2b). At 10 mg/kg, 90% blockade of blood leukocytes was attained only ~10% of the time; these animals (and those dosed at 1 mg/kg) displayed no reduction in the rate of recruitment of inflammatory cells. Likewise, in a rabbit model of joint inflammation, total cell counts were performed on knee synovial lavage samples collected 16 h after the administration of LPS injection (Figure 2c). Rabbits receiving 100 mg/kg or 20 mg/kg CCX354 showed significant reductions in inflammatory cell counts as compared to vehicle-treated animals, whereas those receiving 4 mg/kg CCX354 showed no difference from vehicle-treated animals. As in the rat model, efficient blockade of inflammatory cell
CCX354 was well tolerated and displayed a linear dose– exposure profile at all doses (1–300 mg) in the single-ascending-dose phase I study (Table 1). Average CCX354 plasma concentration-vs.-time curves after single doses of CCX354 are shown in Figure 3a. Mean plasma CCX354 levels of slightly less than 5,000 ng/ml (11,000 ± 1,300 nmol/l) were attained in this study. The half-life of the drug in plasma approached 7 h at the 300-mg dose. After 7 days of administration of 100mg CCX354 b.i.d. (Figure 3b), steady-state CCX354 plasma levels fluctuated from a peak of 1,300 ± 140 ng/ml (2,900 ± 300 nmol/l), attained at 2 h after the dose, to a trough level of 270 ± 38 ng/ml (590 ± 84 nmol/l), reflecting an accumulation ratio of 1.35. No increases were observed in the levels of the CCR1 chemokines CCL3 or CCL5 in plasma after CCX354 administration (Supplementary Table S2 online). None of the subjects experienced any dose-limiting adverse events, and no serious adverse events were reported or observed at any of the dose levels or dosing regimens used in this study.
728
VOLUME 89 NUMBER 5 | may 2011 | www.nature.com/cpt
articles Table 1 Summary of pharmacokinetic parameters for CCX354 after oral administration to healthy (fasted) human volunteers CCX354 dose (dosing solution) Parameter
CCX354 dose (powder in capsule)
1 mg (n = 5)
3 mg (n = 5)
10 mg (n = 5)
30 mg (n = 2)
30 mg (n = 3)
100 mg (n = 5)
300 mg (n = 5)
37 (5)
84 (37)
266 (60)
875 (64)
608 (39)
2,061 (342)
4,989 (602)
Cmax (ng/ml) Tmax (h)
0.5 (0.0)
1.0 (0.5)
1.0 (0.4)
0.8 (0.4)
0.8 (0.3)
1.2 (0.6)
1.9 (0.6)
λz (h−1)
0.19 (0.02)
0.20 (0.07)
0.17 (0.03)
0.19 (0.0)
0.21 (0.04)
0.14 (0.03)
0.11 (0.03)
t1/2 (h)
3.7 (0.5)
3.7 (1.1)
4.1 (0.7)
3.6 (0.1)
3.4 (0.7)
5.2 (0.9)
6.5 (1.6)
CL/F (l/h)
6.4 (1.9)
9.0 (2.9)
8.3 (3.2)
9.2 (2.9)
13.0 (3.0)
9.1 (0.9)
12.0 (6.0)
34 (10)
46 (15)
47 (14)
61 (4)
AUC0–24 (ng·h/ml)
Vz/F (l)
164 (44)
380 (196)
1,332 (489)
47 (11)
3,400 (1,046)
2,415 (581)
10,755 (918)
68 (16)
26,407 (7,992)
115 (54)
AUCinf (ng·h/ml)
166 (44)
385 (201)
1,360 (509)
3,423 (1,064)
2,437 (610)
11,117 (1,035)
27,541 (8,497)
Values shown are mean values (SD). AUC, area under the curve; CL/F, apparent oral clearance; Cmax, peak plasma concentration; t1/2, half-life; Tmax, time to achieve Cmax; Vz/F, apparent volume of distribution; λz terminal phase rate constant.
a
CCX354 single ascending dose phase I study
Plasma CCX354 (nmol/l)
10,000
300 mg 100 mg 30 mg 10 mg 3 mg 1 mg
1,000 100 10 1
0
12
24
36
48
60
72
Time (h)
mg single-dose cohort. The average extent of CCR1 blockade (94%) at 12 h after the dose (Figure 4e) was consistent with expectations based on the average plasma CCX354 concentration (200 ± 31 ng/ml; 440 ± 68 nmol/l; n = 5) in those samples and the in vitro potency of the drug against Alexa647-CCL3 (Ki = 15 nmol/l in serum). Plasma CCX354 concentrations generally predicted the effects measured in this assay (Supplementary Figure S4 online). Comparison of CCX354 with other CCR1 antagonists
A whole-blood ex vivo assay was used to assess blood monocyte CCR1 blockade in selected phase I study subjects. Two time points were assessed—before the dose and at 12 h after the dose—using specific Alexa647-CCL3 binding as a measure of free CCR1 (Supplementary Figure S3 online). Twelve hours after a single 100-mg dose of CCX354, monocyte CCR1 receptors remained markedly blocked in all subjects in the cohort (Figure 4a,b), whereas no CCR1 blockade was seen in the subjects receiving placebo (Figure 4c,d). Similar levels of CCR1 inhibition were consistently measured in all subjects in the 100-
Two CCR1-targeted therapeutic agents have been evaluated in RA clinical trials: CP-481,715 (refs. 8, 9) and MLN3897.11 We compared the CCR1 potencies of both these compounds with those of CCX354 in various assays on human monocytes. In the whole-blood CCL3-induced CD11b upregulation assay, CP-481,715, MLN3897, and CCX354 had average IC50 values of 160 (ref. 31), 210, and 200 nmol/l, respectively (Figure 5a). MLN3897 and CCX354 displayed IC 50 values of 45 and 130 nmol/l, respectively, against CCR1-mediated internalization of Alexa647-CCL3 in human whole blood (Figure 5b). In a third assay, CCX354 blocked CCR1-directed human monocyte chemotaxis in 100% human serum with an IC50 value of 25 nmol/l, whereas the corresponding value for MLN3897 was 2 nmol/l (Figure 5c). CP-481,715 blocked CCL3-mediated chemotaxis of human monocytes in buffer with an IC50 of 55 nmol/l31 and has been described as having a relatively low level of nonspecific binding to plasma proteins.9 We compared the steady-state plasma concentration levels achieved in the clinic for CP-481,715, MLN3897, and CCX354 after oral administration at doses considered to be therapeutically relevant, namely, 300 mg three times daily (t.i.d.), 10 mg once daily (q.d.), and 100 mg twice daily (b.i.d.), respectively (Figure 6a). Given that various PK profiles have been described for CP-481,715, we integrated all the publicly available information into an estimate of exposure achieved in its first RA trial.8 We used the information available regarding the functional potency of these molecules in human blood or serum to estimate the degree of blood leukocyte CCR1 coverage associated with those drug levels in plasma. For this purpose, we relied on the
Clinical pharmacology & Therapeutics | VOLUME 89 NUMBER 5 | may 2011
729
b
100 mg CCX354 dose b.i.d. (day 7)
Plasma CCX354 (nmol/l)
10,000 1,000 100 10 1
0
4
8
12
16
20
24
Time (h)
Figure 3 Plasma concentration-vs.-time profile of CCX354 in healthy human volunteers after single oral doses (a) of CCX354 (open squares, 1 mg; triangles, 3 mg; inverted triangles, 10 mg; diamonds, 30 mg; circles, 100 mg; closed squares, 300 mg) and (b) after receiving 100 mg CCX354 twice daily for 7 days. The points represent the experimental data (mean) of each cohort (n = 6), and the error bars represent the SE. Pharmacodynamic measurements described in Figure 4 were conducted on samples from the 100-mg cohort at the 12-h time point (identified by a circle).
articles b
Subject A: 12 h after 100 mg CCX354
350
350
300
300
250
150
125
200
0
1
2
150
3
0
Alexa647-CCL3 (nmol/l)
d
2
3
Subject B: 12 h after placebo
350
350
300
300
250 200
100 75 50 25 0 Baseline
Subject B: before dosing
150
1
Alexa647-CCL3 (nmol/l)
MFI
Mean fluorescence intensity (MFI)
c
**
e Free CCR1 (%) on blood monocytes
200
250
250
CCX354 (440 nmol/l)
Subject A: before dosing
MFI
Mean fluorescence intensity (MFI)
a
12 h postdose
200
0
1
2
3
150
0
Alexa647-CCL3 (nmol/l)
1 2 Alexa647-CCL3 (nmol/l)
3
Figure 4 Assessment of free CCR1 receptors on human blood monocytes. Monocytes were first identified by flow cytometry light scatter properties and then by CD14 staining. The CCR1 signal on these cells was determined by the difference between Alexa647-CCL3 mean fluorescence in the sample (closed circles) and the mean fluorescence in the presence of a large excess (200 nmol/l) of the CCR1-specific chemokine CCL23 (open circles). Blood samples from healthy volunteers receiving doses of either CCX354 or placebo were treated with 0, 0.3, 1, or 3 nmol/l Alexa647-CCL3 for the determination of both total and nonspecific signal. The data from representative subjects are shown. Subject A: blood taken (a) immediately before and (b) 12 h after receiving a 100-mg oral dose of CCX354. Subject B: blood taken (c) immediately before and (d) 12 h after receiving a placebo. (e) Reduction in blood monocyte free CCR1, relative to predose baseline, 12 h after treatment with a single dose of 100 mg CCX354 (n = 4 subjects); n = 3 replicates per measurement. The values were derived using the Cheng– Prusoff correction of the measured displacement of specific Alexa647-CCL3 binding; **P < 0.01, Student’s t-test.
inhibition of human monocyte chemotaxis performed in 100% human serum (Figure 6b). Discussion
The biological profile of CCX354, a novel small-molecule antagonist of the human CCR1 receptor, is described here for the first time. When evaluated using human monocyte chemotaxis in 100% human serum, CCX354 displayed similar potency against all CCR1 chemokines (CCL3, CCL5, CCL15, and CCL23). These experiments also assessed the effects of nonspecific protein binding in serum on the potency of the compound, an important consideration for defining target drug plasma concentrations in clinical trials. Under these conditions, a concentration of ~250 nmol/l CCX354 was able to produce a ~10-fold right shift in the dose response to all CCR1 chemokines. The therapeutic rationale for evaluating CCX354 in subjects with RA was strengthened by the demonstrated ability of CCX354 to block most of the monocyte chemotactic activity found in the synovial fluid taken from 35 subjects with RA. Although upregulation of CCR1 and CCR2 chemokines has been described in the literature,16 the chemotactic activity found in our survey of dozens of RA samples was effectively inhibited by CCX354 but was not affected by a CCR2 antagonist. This observation is consistent with the reported clinical failure of 730
CCR2-targeted therapeutics in RA.5,13,14 We and others have previously shown the presence of high levels of CCR1 chemokines in similar RA samples, in particular the activated and potent forms of the CCR1 ligands CCL23 and CCL15.18,20 Ex vivo assessment of human blood monocyte CCR1 coverage was performed using selected time points in the singleascending-dose phase I study. On average, 94% of the CCR1 receptor sites were blocked at 12 h after a single 100-mg dose of CCX354. A similar potency for whole-blood CCR1 blockade was obtained in vitro when CCX354 was added to blank fresh blood, thereby indicating the absence of pharmacologically active plasma metabolites in humans after oral administration of CCX354. In contrast to the increased plasma chemokine concentrations reported after administration of other CKR inhibitors,32,33 we observed no increases in the levels of the CCR1 chemokines CCL3 and CCL5 under conditions of high CCR1 blockade. Given that the mechanism underlying those reported increases is not well defined, it is difficult to speculate regarding our findings. Nevertheless, the absence of increased chemokine levels is a positive feature, from a therapeutic perspective. Having established the safety, pharmacokinetics, and receptor coverage profile of CCX354 in healthy volunteers, we sought to interpret the receptor coverage profile in the light of the successful and unsuccessful clinical trials of earlier CCR1 compounds VOLUME 89 NUMBER 5 | may 2011 | www.nature.com/cpt
articles
Mean fluorescence intensity (MFI)
Inhibition of CD11b upregulation in human whole blood 225 200
MLN3897 CCX354
175 150 125
b
100 10−11 10−10 10−9 10−8 10−7 10−6 10−5 10−4 Compound (mol/l)
c
Human monocyte chemotaxis in 100% serum
Alexa647-CCL3 internalization in human whole blood
Mean fluorescence intensity (MFI)
a
1,500
CCX354 1,000
500
0 10−12 10−11 10−10 10−9 10−8 10−7 10−6 10−5 Compound (mol/l)
d CP-481,715
MLN3897
CCX354
CD11b upregulation in whole blood
160 nmol/l1
210 nmol/l
200 nmol/l
Alexa647-CCL3 internalization in whole blood
nd
45 nmol/l
130 nmol/l
Monocyte chemotaxis in 100% serum
55 nmol/l1,2
2 nmol/l
25 nmol/l
7,500
Migration signal (fluorescence)
MLN3897 5,000
CCX354
2,500
0 10−12 10−11 10−10 10−9 10−8 10−7 10−6 10−5 10−4 Compound (mol/l)
MLN3897
Figure 5 Comparison of the potencies of various clinical-stage CCR1 antagonists: CCX354 (circles) and MLN3897 (squares). (a) Inhibition of CCL3-induced upregulation of CD11b on human whole-blood monocytes; n = 4 repeats per data point; experiments were conducted three times. (b) Inhibition of Alexa647CCL3 internalization in human whole blood; n = 2 repeats per data point; experiments were conducted three times. (c) Inhibition of CCL15-induced chemotaxis of freshly isolated human monocytes in 100% human serum; n = 8 repeats per data point; experiments were conducted three times. (d) Summary of results with MLN3897 and CCX354, as compared to published results, for CP-481,715; the IC50 values are shown. nd, not determined. 1ref. 31; 2assay conducted in buffer.
in RA. For this purpose, we compared the potency of CCX354 with that of MLN3897 and CP-481,715 in physiologically relevant media (human blood or serum) using several assays described in the literature for the latter. CP-481,715 blocked CCL3-induced whole-blood monocyte CD11b upregulation and monocyte chemotaxis (buffer), with IC50 values of 160 and 55 nmol/l, respectively.31 CCX354 and MLN3897 blocked CD11b upregulation in the whole-blood assay, with IC50 values of 200 and 210 nmol/l, respectively. They blocked human monocyte chemotaxis in 100% human serum, with IC50 values of 25 and 2 nmol/l, respectively. Both compounds displayed greater potency in the serum chemotaxis assay than in the whole-blood CD11b assay, although the difference was larger for MLN3897. The potential explanations for this disconnect range from accumulation of the compound in erythrocytes (although CCX354 accumulation in erythrocytes is minimal; data not shown) to differences in the experimental methods used in the two assays (such as the type and concentration of chemokine: 1 nmol/l CCL3 in the CD11b assay and 0.1 nmol/l CCL15 for chemotaxis). The ability of MLN3897 to block CCR1mediated internalization of Alexa488-CCL3 has been described previously;11 we used a similar assay (Alexa647-CCL3 internalization in human whole blood) to compare, side by side, the potencies of MLN3897 and CCX354. In this assay, both compounds displayed potencies that were intermediate between those displayed in the previous two assays. A more potent value has been implied in the literature with regard to the potency of MLN3897;11 differences in the labeled CCL3 protein might account for this discrepancy (Alexa488-labeled recombinant CCL3 vs. C-terminal
Alexa647-modified synthetic CCL3). Caution is warranted in describing the potency of a molecule in terms of a single value because the value will depend on the exact conditions under which the assay is performed. We have nevertheless compared the three clinical-stage CCR1 antagonists across a range of assay formats carried out in whole blood or in 100% human serum; our findings are that CCX354 and CP-481,715 are approximately equipotent, whereas MLN3897 is equipotent with the other two compounds in the whole-blood assay but significantly more potent when evaluated in plain serum. Also remarkable, although not entirely unusual, is the degree to which the potency of a compound such as CCX354 (~1 nmol/l in buffer) needs to be adjusted when assays are conducted in physiologically relevant media (in this case, its IC50 increases to 25–200 nmol/l in serum/blood, depending on the assay). In order to estimate CCR1 coverage achieved on blood monocytes with the various CCR1 antagonists tested clinically, we relied on the monocyte chemotaxis assay, which provides the highest estimation of receptor coverage for MLN3897 and thus best challenges the contention that CCR1 inhibition has already been adequately tested in the clinic. A comparison of plasma levels achieved in the clinic with oral doses of CP-481,715, MLN3897, and CCX354 clearly shows that much higher levels of CP-481,715 and CCX354 have been achieved relative to MLN3897 (~200-fold higher, based on comparisons of plasma area under the curve (AUC)). Furthermore, even after taking into account the 10- to 20-fold greater potency of MLN3897, the estimated extent of human blood leukocyte CCR1 inhibition associated with a dosage
Clinical pharmacology & Therapeutics | VOLUME 89 NUMBER 5 | may 2011
731
articles
Plasma conc. (nmol/l)
a
Human plasma concentrations 10,000
CP-481,715 (300 mg t.i.d.)
1,000
CCX354 (100 mg b.i.d.) MLN3897 (10 mg q.d.)
100 10 1 0
b
4
8
12 Time (h)
16
20
24
Inhibition of CCR1 in human RA studies
% CCR1 inhibition
100
CCX354 (100 mg b.i.d.) CP-481,715 (300 mg t.i.d.)
90 90%
MLN3897 (10 mg q.d.)
80 70 60 0
4
8
12 Time (h)
16
20
24
Figure 6 Time course comparison of the (a) pharmacokinetic and (b) calculated pharmacodynamic levels of CCR1 blockade achieved with three different CCR1 antagonists in humans. The pharmacokinetic profiles of CP-481,715 and MLN3897 were obtained from published reports. CP-481,715 was administered at a dosage of 300 mg once every 8 h, MLN3897 was administered at 10 mg once daily, and CCX354 was administered at 100 mg twice daily. The potencies of the various compounds, measured using CCR1-mediated human monocyte chemotaxis in 100% human serum, were used in the CCR1 inhibition calculations. conc., concentration; RA, rheumatoid arthritis.
of 10-mg MLN3897 q.d. remains well below the degree of inhibition associated with either 300-mg CP-481,715 t.i.d. or 100-mg CCX354 b.i.d. At these doses, we estimate that the degree of CCR1 blockade produced by CP-481,715 ranged from 94% (trough) to 97% (peak) throughout the day. Similarly, CCX354 produced blood CCR1 blockade ranging from 96% (trough) to 99% (peak). On the other hand, MLN3897 did not achieve anywhere near this level of CCR1 coverage; the estimated values ranged from 74% (trough) to 83% (peak). That is to say, free CCR1 levels for CP-481,715 and CCX354 would be 3–6% and 1–4%, respectively, whereas for MLN3897 these values would be 17–26%. Finally, we used several preclinical models to estimate the extent of blood leukocyte CCR1 inhibition required to effectively block leukocyte recruitment into sites of inflammation. Because of the decreased potency of CCX354 toward rat and rabbit CCR1, doses of CCX354 were selected for these experiments such that we could define ineffective and maximally effective doses. Blood leukocyte CCR1 blockade in these studies was estimated from in vitro evaluation of CCX354 potency on rat and rabbit leukocyte chemotaxis (in 100% rat and rabbit serum, respectively). Robust pharmacological effects were seen only at concentration levels that blocked ≥90% of the blood leukocyte CCR1 at all times. This observation is hardly surprising, given that the whole field of chemokine-based therapeutics has gradually moved toward the realization that very high receptor coverage may be required at all times to elicit a clear biological/therapeutic effect.9 The reasons for this requirement are not entirely clear, but it might suggest that the in vitro chemotaxis assays do not fully recapitulate the complexity of the in vivo system, with the resulting underestimation of target drug plasma levels in clinical trials. This possible underestimation is, of course, less of an issue when similar assays are used 732
preclinically and clinically to define the PK/PD relationship for the drug. In our case, we relied on inhibition of CCR1-mediated primary cell chemotaxis, performed in 100% serum, to establish this cross-species bridge. For CCX354, this analysis predicts that, even with an affinity of ~1 nmol/l for its target receptor, plasma levels in excess of ~120 ng/ml (250 nmol/l; 90% inhibition of monocyte chemotaxis in serum) should be maintained in patients to ensure a full therapeutic benefit. We recognize that a potential source of uncertainty in the assessment of therapeutically relevant receptor coverage arises from the use of monocytes from healthy individuals, as we and others31 have done. Some differences have been described regarding CCR1 expression on blood monocytes from RA patients.18 Looking back to the demonstrated evidence of biological/ therapeutic effects of CP-481,715 in subjects with RA8 and the failed phase II clinical trial with MLN389711 in the context of our estimation of relative blood leukocyte CCR1 coverage, we see that CP-481,715 most likely exceeded the 90% receptor coverage threshold at all times in that study, despite its poor PK and weaker potency relative to MLN3897, whereas the latter appears to have fallen short of such a threshold at all times. Taking into account the phase I data related to tolerability, PK, and receptor coverage for CCX354, and the PK/PD requirements described above, we recently initiated a multinational phase II trial in RA subjects. In this study, called CARAT-2,34 we are evaluating a dosage of 100-mg CCX354 b.i.d., which should provide >96% blood leukocyte CCR1 coverage throughout the day. Methods Phase I clinical trials
Double-blind, randomized, placebo-controlled, single-ascending-dose and multiple-ascending-dose phase I studies were conducted in order VOLUME 89 NUMBER 5 | may 2011 | www.nature.com/cpt
articles to evaluate the safety, tolerability, PK and PD properties of CCX354 in healthy subjects. Single-ascending-dose study: Within each of six serial cohorts, healthy subjects (n = 8) were randomized to receive either a single dose of CCX354 (n = 5) or placebo (n = 3). The doses tested were 1, 3, 10, 30, 100, and 300 mg. A total of 48 subjects (29 men; 19 women) participated (mean age: 36 years; range: 22–45); 46 of the subjects were Caucasian and 2 were black. Of these subjects, 13 were former smokers and 35 had never smoked. Multiple-ascending-dose: Within each of six serial cohorts, healthy subjects (n = 6) were randomized to CCX354 (n = 5) or placebo (n = 1) daily for 7 days. The dosages tested were 3 mg q.d., 10 mg b.i.d., 30 mg q.d., 30 mg b.i.d., 100 mg b.i.d., and 300 mg q.d. The subjects received study medication 1 h after a regular breakfast in the morning and, in the b.i.d. groups, 1 h after a regular dinner in the evening. A total of 36 subjects participated (20 men; 16 women; mean age: 37 years; range: 24–48); all the subjects were Caucasian. Ten subjects were former smokers, and 26 had never smoked. All subjects in both studies were in good general health prior to enrolling, and all gave written informed consent to participate before any study procedures were initiated. Study protocols were approved by the Ethics Committee of the Two Basels and by Swissmedic. The studies were conducted at Covance (Basel, Switzerland) in accordance with the Declaration of Helsinki principles, the good clinical practice guidelines of the US Food and Drug Administration, and International Conference on Harmonisation guidelines. Safety and tolerability assessments: Standard clinical methods were used to assess safety and tolerability, including physical examinations, vital signs, 12-lead electrocardiogram, and clinical laboratory measurements, throughout both studies. Adverse events were recorded for the entire study duration. Investigators assessed all adverse events for severity, duration, outcome, and possible relationship to the study medication. PK assessment: Plasma samples were collected at predetermined time points. Blood samples (8 ml) were collected into EDTA tubes, gently mixed, and kept on wet ice until centrifuged (within 30 min) at 2,000g, in a refrigerated centrifuge, for 10 min. The resulting plasma was stored at −80 °C until analysis. After protein precipitation, the supernatant solutions were analyzed by high-performance liquid chromatography– tandem mass spectrometry using a validated method (nominal plasma concentration range: 1–1,000 ng/ml). PK values were generated using noncompartmental analysis with WinNonlin Professional, version 5.2 (Pharsight, Mountain View, CA). PD assessment: Peripheral blood samples from selected phase I cohorts were collected into EDTA tubes at the indicated times; 0.2-ml aliquots were mixed with phycoerythrin-conjugated anti-human CD14 monoclonal antibody (BD Biosciences, San Jose, CA) and several concentrations of Alexa647-CCL3, with or without an excess of unlabeled CCL23 (a different, selective CCR1 ligand). After 30 min on ice, red blood cells were lysed using ice-cold FACS Lysing Solution (BD Biosciences), and the leukocytes were preserved in fixative. Monocytes were identified by consecutive forward/side-scatter and CD14+ gating. Levels of free CCR1 were calculated from the extent (mean fluorescence intensity) of specific Alexa647-CCL3 (3 nmol/l) binding (Supplementary Figure S2 online) using the Cheng–Prusoff correction (GraphPad Prism, San Diego, CA). PK profiles of MLN3897 and CP-481,715 in humans
MLN3897: Steady-state plasma concentrations after daily dosing of 10-mg MLN3897 have been described previously.11,35 In our analysis, we utilized parameters reflective of the average values for those published profiles, namely peak plasma concentration (Cmax: 5 ng/ml), trough plasma concentration (Cmin: 2.8 ng/ml), time to achieve Cmax (Tmax: 5 h), and AUC0–24 (90 ng·h/ml). CP-481,715: The PK profile of this compound in healthy volunteers has been described in two reports.36,37 For our analysis, we assumed steadystate PK parameters for 300 mg t.i.d. in the RA study (Cmax: 1,150 ng/ ml, AUC0–24: 19,580 ng·h/ml) to have been twofold and 3.5-fold higher, Clinical pharmacology & Therapeutics | VOLUME 89 NUMBER 5 | may 2011
respectively, than the values derived from the 300-mg single-dose data in healthy volunteers, and we used a calculated Cmin value of 450 ng/ ml. This extrapolation appears warranted in light of the indication that substantially higher plasma levels were noted in subjects with RA than in healthy volunteers.36 In vitro potency assessments
Cells and reagents: THP-1 cells were obtained from ATCC (Rockville, MD). Human monocytes were isolated from healthy volunteer buffy coats (Stanford Blood Center, Palo Alto, CA) using MACS separation reagents (Miltenyi, Germany). Samples of CCX354 (ref. 27) and MLN3897 (ref. 38) were prepared at ChemoCentryx (Mountain View, CA). Recombinant chemokines were obtained from R&D Systems (Minneapolis, MN), [125I]-CCL15 was from PerkinElmer (Boston, MA), and Alexa647-CCL3 was from Almac Sciences (Glasgow, Scotland). Serum samples from humans, RA synovial samples from humans (individual samples, HBSS 1:10 dilution), and rat and rabbit plasmas were obtained from Bioreclamation (Hicksville, NY), and phycoerythrin-conjugated anti-human CD14 monoclonal antibody was from BD Biosciences. In vitro assays: Chemotaxis and radioligand binding assays were conducted essentially in the manner previously described.39 Inhibition of CCR1 internalization using Alexa647-CCL3 was performed in the manner previously described,11 with one modification, namely the use of synthetic Alexa647-CCL3 instead of Alexa488-labeled recombinant CCL3. CD11b upregulation on monocytes in human whole blood has been previously described.36 Data analysis: Inhibition values (IC50) were calculated using nonlinear regression with a one-site competition model (GraphPad Prism). If experiments were performed more than three times, the values shown are mean values ± SE. Animal models
All animal studies were approved by the institutional animal care and use committee of ChemoCentryx. Thioglycollate-induced peritonitis: Anesthetized Wistar rats (n = 6/ group) were injected intraperitoneally with thioglycollate and dosed orally with CCX354 or vehicle every 12 h, starting 2 h before and ending 34 h after thioglycollate injection. Peritoneal cells were collected by lavage 48 h after thioglycollate injection and analyzed using flow cytometry. Satellite animals (n = 2) were used for PK measurements. LPS-induced joint inflammation: Anesthetized rabbits (n = 6/group) were administered LPS (10 ng) into both knees.40 The animals were dosed orally at −2 h and +4 h (relative to LPS administration) with either CCX354 or vehicle. Synovial cells were collected after 16 h by lavage and analyzed using flow cytometry. Satellite animals (n = 2) were used for PK measurements. Blood leukocyte CCR1 blockade was estimated on the basis of the potency of CCX354 in chemotaxis assays, using freshly isolated rat/rabbit leukocytes and conducted in 100% plasma (Supplementary Figure S2 online). CCX354 did not affect leukocyte chemotaxis toward other rat/ rabbit chemokines/chemoattractants, such as C5a, CXCL6/GCP-2, and CXCL1/KC in rats and C5a in rabbits (data not shown). SUPPLEMENTARY MATERIAL is linked to the online version of the paper at http://www.nature.com/cpt
Conflict of Interest All authors are employees of ChemoCentryx. © 2011 American Society for Clinical Pharmacology and Therapeutics
1. Charo, I.F. & Ransohoff, R.M. The many roles of chemokines and chemokine receptors in inflammation. N. Engl. J. Med. 354, 610–621 (2006). 2. Viola, A. & Luster, A.D. Chemokines and their receptors: drug targets in immunity and inflammation. Annu. Rev. Pharmacol. Toxicol. 48, 171–197 (2008). 733
articles 3. Gulick, R.M. et al.; MOTIVATE Study Teams. Maraviroc for previously treated patients with R5 HIV-1 infection. N. Engl. J. Med. 359, 1429–1441 (2008). 4. Brave, M. et al. FDA review summary: Mozobil in combination with granulocyte colony-stimulating factor to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation. Oncology 78, 282–288 (2010). 5. Pease, J.E. & Horuk, R. Chemokine receptor antagonists: part 1. Expert Opin. Ther. Pat. 19, 39–58 (2009). 6. Keshav, S., Johnson, D., Bekker, P. & Schall, T.J. PROTECT-1 study demonstrated efficacy of the intestine-specific chemokine receptor antagonist CCX282-B (Traficet-EN) in treatment of patients with moderate-to-severe Crohn’s disease. Gastroenterology 136, A65 (2009). 7. Keshav, S. et al. PROTECT-1 study of intestine-specific chemokine receptor antagonist CCX282-B (TRAFICET-EN) in Crohn’s disease. Gut 58, A468 (2009). 8. Haringman, J.J., Kraan, M.C., Smeets, T.J., Zwinderman, K.H. & Tak, P.P. Chemokine blockade and chronic inflammatory disease: proof of concept in patients with rheumatoid arthritis. Ann. Rheum. Dis. 62, 715–721 (2003). 9. Gladue, R.P., Brown, M.F. & Zwillich, S.H. CCR1 antagonists: what have we learned from clinical trials. Curr. Top. Med. Chem. 10, 1268–1277 (2010). 10. Zipp, F. et al.; CCR1 Antagonist Study Group. Blockade of chemokine signaling in patients with multiple sclerosis. Neurology 67, 1880–1883 (2006). 11. Vergunst, C.E. et al. MLN3897 plus methotrexate in patients with rheumatoid arthritis: safety, efficacy, pharmacokinetics, and pharmacodynamics of an oral CCR1 antagonist in a phase IIa, double-blind, placebo-controlled, randomized, proof-of-concept study. Arthritis Rheum. 60, 3572–3581 (2009). 12. Pease, J.E. & Horuk, R. Chemokine receptor antagonists: part 2. Expert Opin. Ther. Pat. 19, 199–221 (2009). 13. Vergunst, C.E. et al. Modulation of CCR2 in rheumatoid arthritis: a doubleblind, randomized, placebo-controlled clinical trial. Arthritis Rheum. 58, 1931–1939 (2008). 14. Haringman, J.J. et al. A randomized controlled trial with an anti-CCL2 (antimonocyte chemotactic protein 1) monoclonal antibody in patients with rheumatoid arthritis. Arthritis Rheum. 54, 2387–2392 (2006). 15. Gerlag, D.M. et al.; ESCAPE Study Group. Preclinical and clinical investigation of a CCR5 antagonist, AZD5672, in patients with rheumatoid arthritis receiving methotrexate. Arthritis Rheum. 62, 3154–3160 (2010). 16. Vergunst, C.E., van de Sande, M.G., Lebre, M.C. & Tak, P.P. The role of chemokines in rheumatoid arthritis and osteoarthritis. Scand. J. Rheumatol. 34, 415–425 (2005). 17. Tarrant, T.K. & Patel, D.D. Chemokines and leukocyte trafficking in rheumatoid arthritis. Pathophysiology 13, 1–14 (2006). 18. Haringman, J.J., Smeets, T.J., Reinders-Blankert, P. & Tak, P.P. Chemokine and chemokine receptor expression in paired peripheral blood mononuclear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis. Ann. Rheum. Dis. 65, 294–300 (2006). 19. Szekanecz, Z., Kim, J. & Koch, A.E. Chemokines and chemokine receptors in rheumatoid arthritis. Semin. Immunol. 15, 15–21 (2003). 20. Berahovich, R.D., Miao, Z., Wang, Y., Premack, B., Howard, M.C. & Schall, T.J. Proteolytic activation of alternative CCR1 ligands in inflammation. J. Immunol. 174, 7341–7351 (2005). 21. Tak, P.P. et al. Analysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activity. Arthritis Rheum. 40, 217–225 (1997). 22. Wijbrandts, C.A., Vergunst, C.E., Haringman, J.J., Gerlag, D.M., Smeets, T.J. & Tak, P.P. Absence of changes in the number of synovial sublining macrophages
734
after ineffective treatment for rheumatoid arthritis: implications for use of synovial sublining macrophages as a biomarker. Arthritis Rheum. 56, 3869–3871 (2007). 23. Tsou, C.L. et al. Identification of C-C chemokine receptor 1 (CCR1) as the monocyte hemofiltrate C-C chemokine (HCC)-1 receptor. J. Exp. Med. 188, 603–608 (1998). 24. Ward, S.G., Bacon, K. & Westwick, J. Chemokines and T lymphocytes: more than an attraction. Immunity 9, 1–11 (1998). 25. Yu, X., Huang, Y., Collin-Osdoby, P. & Osdoby, P. CCR1 chemokines promote the chemotactic recruitment, RANKL development, and motility of osteoclasts and are induced by inflammatory cytokines in osteoblasts. J. Bone Miner. Res. 19, 2065–2077 (2004). 26. Vallet, S. et al. MLN3897, a novel CCR1 inhibitor, impairs osteoclastogenesis and inhibits the interaction of multiple myeloma cells and osteoclasts. Blood 110, 3744–3752 (2007). 27. Zhang, P. et al. Azaindazole compounds and methods of use. US patent 7,524,845 (2009). 28. Proudfoot, A.E., Power, C.A. & Schwarz, M.K. Anti-chemokine small molecule drugs: a promising future? Expert Opin. Investig. Drugs 19, 345–355 (2010). 29. Dairaghi, D.J. et al. A novel CCR1 antagonist CCX354-C for rheumatoid arthritis. Arthritis Rheum. 62, S460 (2010). 30. Dairaghi, D.J. et al. A novel CCR1 antagonist CCX354-C for rheumatoid arthritis. Ann. Rheum. Dis. 69, S214 (2010). 31. Gladue, R.P. et al. CP-481,715, a potent and selective CCR1 antagonist with potential therapeutic implications for inflammatory diseases. J. Biol. Chem. 278, 40473–40480 (2003). 32. Cardona, A.E. et al. Scavenging roles of chemokine receptors: chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues. Blood 112, 256–263 (2008). 33. Aiello, R.J. et al. CCR2 receptor blockade alters blood monocyte subpopulations but does not affect atherosclerotic lesions in apoE(-/-) mice. Atherosclerosis 208, 370–375 (2010). 34. CARAT-2 Trial Registry Number NCT01242917 (2010). 35. Lu, C., Balani, S.K., Qian, M.G., Prakash, S.R., Ducray, P.S. & von Moltke, L.L. Quantitative prediction and clinical observation of a CYP3A inhibitor-based drug-drug interactions with MLN3897, a potent C-C chemokine receptor-1 antagonist. J. Pharmacol. Exp. Ther. 332, 562–568 (2010). 36. Clucas, A.T., Shah, A., Zhang, Y.D., Chow, V.F. & Gladue, R.P. Phase I evaluation of the safety, pharmacokinetics and pharmacodynamics of CP-481,715. Clin. Pharmacokinet. 46, 757–766 (2007). 37. Borregaard, J. et al. Evaluation of the effect of the specific CCR1 antagonist CP-481715 on the clinical and cellular responses observed following epicutaneous nickel challenge in human subjects. Contact Derm. 59, 212–219 (2008). 38. Neves, C., Chevalier, A. & Billot, P. Solid forms of a chemokine receptor antagonist and methods of use thereof. PCT Pat. Appl. WO-2006/066200-A2 (2006). 39. Walters, M.J. et al. Characterization of CCX282-B, an orally bioavailable antagonist of the CCR9 chemokine receptor, for treatment of inflammatory bowel disease. J. Pharmacol. Exp. Ther. 335, 61–69 (2010). 40. Podolin, P.L. et al. A potent and selective nonpeptide antagonist of CXCR2 inhibits acute and chronic models of arthritis in the rabbit. J. Immunol. 169, 6435–6444 (2002).
VOLUME 89 NUMBER 5 | may 2011 | www.nature.com/cpt
