- Email: [email protected]
Integrative pharmacokinetic–pharmacodynamic modeling and simulation of amenamevir (ASP2151) for treatment of recurrent genital herpes
Accepted Manuscript Integrative Pharmacokinetic-Pharmacodynamic Modeling and Simulation of Amenamevir (ASP2151) for Treatment of Recurrent Genital Herpes Akitsugu Takada, Masataka Katashima, Atsunori Kaibara, Koji Chono, Kiyomitsu Katsumata, Taiji Sawamoto, Hiroshi Suzuki, Yoshitaka Yano PII:
S1347-4367(16)30029-5
DOI:
10.1016/j.dmpk.2016.05.005
Reference:
DMPK 115
To appear in:
Drug Metabolism and Pharmacokinetics
Received Date: 9 November 2015 Revised Date:
20 May 2016
Accepted Date: 27 May 2016
Please cite this article as: Takada A, Katashima M, Kaibara A, Chono K, Katsumata K, Sawamoto T, Suzuki H, Yano Y, Integrative Pharmacokinetic-Pharmacodynamic Modeling and Simulation of Amenamevir (ASP2151) for Treatment of Recurrent Genital Herpes, Drug Metabolism and Pharmacokinetics (2016), doi: 10.1016/j.dmpk.2016.05.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Integrative
Pharmacokinetic-Pharmacodynamic
Modeling
and
2
Amenamevir (ASP2151) for Treatment of Recurrent Genital Herpes
Simulation
of
3 Akitsugu Takada1, Masataka Katashima1, Atsunori Kaibara1, Koji Chono1, Kiyomitsu
5
Katsumata1, Taiji Sawamoto1, Hiroshi Suzuki1, Yoshitaka Yano2
6
RI PT
4
1
Astellas Pharma Inc., 2-5-1 Nihonbashi-Honcho, Chuo-ku, Tokyo, Japan
8
2
Kyoto Pharmaceutical University, 5 Misasagi-Nakauchicho, Yamashina-ku, Kyoto, Japan
SC
7
9 Corresponding author’s information
M AN U
10
Akitsugu Takada
12
Astellas Pharma Inc.
13
2-5-1 Nihonbashi-HonchoChuo-ku, Tokyo, 103-8411, Japan
14
E-mail: [email protected]
15
Tel: +81-3-3244-2579, Fax: +81-3-3243-5732
EP AC C
16
TE D
11
1
ACCEPTED MANUSCRIPT Abstract
18
Amenamevir is a novel drug that targets the viral helicase-primase complex. While
19
dose-dependent efficacy had been observed in non-clinical studies, no clear dose dependence
20
has been observed in humans. We therefore developed a pharmacokinetic/pharmacodynamic
21
(PK/PD) model to explain this inconsistency between species and to clarify the
22
immune-related healing of amenamevir in humans. The model consisted of a non-linear
23
kinetic model for a virtual number of virus plaques as a built-in biomarker. Lesion score was
24
defined as an endpoint of antiviral efficacy, and logit model analysis was applied to the
25
ordered-categorical lesion score. The modeling results suggested the time course profiles of
26
lesion score could be explained with the efficacy terms in the logit model, using change in
27
number of virus plaques as an indicator of the effects of amenamevir and time elapsed as an
28
indicator of the healing of the immune response. In humans, the PD effect was almost
29
dose-independent, and immune-related healing may have been the driving force behind the
30
reduction in lesion scores. Drug efficacy is occasionally masked in diseases healed by the
31
immune response, such as genital herpes. The PK/PD model proposed in the present study
32
must be useful for explanation the PK/PD relationship of such drugs.
33
EP
TE D
M AN U
SC
RI PT
17
Keywords
35
amenamevir, pharmacokinetic/pharmacodynamic, pharmacometrics, genital herpes, modeling
36
and simulation
37
AC C
34
2
ACCEPTED MANUSCRIPT 38
Introduction Recurrence of genital herpes is usually caused by HSV-2 and less commonly by HSV-1
40
[1]. Almost 50 million American adults and adolescents (20% of the total population) are
41
infected with genital herpes: it is one of the most common sexually transmitted diseases [2].
42
The recurrence of genital symptoms is very typical, with up to 80% of HSV-2-infected
43
patients experiencing recurrent outbreaks within the first 12 months following the first
44
episode.
RI PT
39
Amenamevir is a novel drug that targets the viral helicase-primase complex. This
46
protein complex is essential for herpes simplex virus (HSV) DNA synthesis, making
47
helicase-primase inhibitors such as amenamevir a potential treatment option for genital
48
herpes. A pharmacokinetic / pharmacodynamic (PK/PD) approach for anti-virus drug is
49
similar to the development of antibiotics, and in vitro IC50 is used to predict the clinical dose
50
in vivo animal model [3]. Amenamevir has demonstrated higher potency in vitro antiviral
51
activity against HSV-1 and HSV-2 than aciclovir [4].
TE D
M AN U
SC
45
A phase 2 study (ASP2151 CL-101 study) was performed in the United States to compare
53
the safety and efficacy of four different dose regimens of amenamevir with valaciclovir and
54
placebo in parallel in the acute treatment of recurrent genital HSV infection. At the first sign
55
of recurrence, patients self-initiated treatments with amenamevir 100, 200, or 400 mg daily
56
for 3 days, 1200 mg as a single dose; placebo for 3 days; or valaciclovir 500 mg twice daily
57
for 3 days. Dose and regimen were determined based on those results and PK profile of
58
amenamevir to maintain time above 200 ng/ml of plasma amenamevir concentration, T200 [5].
59
Of 695 patients randomized to the treatments, data from 437 patients with recurrent infection
60
were analyzed. The primary efficacy endpoint was time to lesion healing which was shorter
61
in all amenamevir groups and the valaciclovir group compared with placebo, but a
AC C
EP
52
3
ACCEPTED MANUSCRIPT 62
dose-response relationship was not evident. All groups except amenamevir 100mg showed a
63
higher proportion of aborted lesions compared with placebo[6]. Population PK analysis of amenamevir was conducted to estimate patients’ PK profiles
65
using 957 plasma samples in 273 genital herpes subjects in CL-101study. The plasma
66
concentration-time courses for amenamevir were described using a one-compartment model
67
with first-order absorption. Exploratory PK and PD analysis was conducted to find an
68
appropriate PK-related parameter which correlates with PD instead of finding dose-PD
69
relationship, using individual PK parameters derived from the final model by the population
70
analysis. The variable T200 was found to correlate with time to lesion healing and viral
71
shedding, which is consistent with the in vivo results [7]. However, these correlations were
72
not statistically significant, because T200 was dose dependent parameter after all.
M AN U
SC
RI PT
64
In CL-101 study[6], dose-dependent efficacy was not evident in the primary analysis
74
using time to lesion healing, although dose-dependent efficacy had previously been suggested
75
in guinea pigs [8]. To discuss address this inconsistency between clinical and non-clinical
76
results, we conducted additional PK/PD modeling and simulations using the same component
77
model to for both humans and guinea pigs have conducted in the present study with a built-in
78
biomarker—number of virus plaques, which explains the time-dependent antivirus efficacy of
79
amenamevir—to combine the PK of amenamevir and lesion scores as an efficacy endpoint.
80
Furthermore, non-drug related such as immune response was added to explain the difference.
AC C
EP
TE D
73
81
In general, screening process of new drugs is conducted by using animal to confirm the
82
efficacy, safety and pharmacokinetic. After that, these endpoints are confirmed in clinical
83
trials. Amenamevir showed effective anti-virus action dose-dependently in guinea pigs but
84
not in humans as stated above. To fully understand the phenomenon is somewhat challenging
85
theme, although many drug candidate compounds may have same theme in drug development
86
stage. In this report, we tried to fill a gap between non-clinical and clinical results with the
4
ACCEPTED MANUSCRIPT 87
same PK/PD model both animal and human, which is bi-directional translational modeling
88
and simulation approach.
90
Materials and methods
91
Analysis data from clinical and non-clinical studies
92
RI PT
89
The present study is a population PK/PD analysis using the obtained PK and PD data in the previous clinical and non-clinical studies [4,8,6]. The scheme of the PK/PD model
94
applied in this study is shown in Figure 2. As mentioned in Introduction, the results of the
95
previous virus plaque study provided the possible relationship of viral dynamics with
96
amenamevir concentration [5], and efficacy such as suppression of lesion scores was
97
expected to correlate with amenamevir concentration. The present PK/PD model assumes that
98
the time time-dependent increase in number of virus plaques is reduced by amenamevir, and
99
the increase of lesion score is dependent on the virtual number of virus plaques. Data from
M AN U
SC
93
previous studies were reanalyzed in the present study as follows:
101
1) Virus plaque assay data were used for constructing virus plaque PD model component.
102
2) Plasma concentration data of amenamevir in guinea pigs and humans (reference) were
EP
103
TE D
100
used for constructing the PK model component. 3) Lesion score data in guinea pigs and humans were used for logit model analysis.
105
The experimental conditions for the previous studies will be described briefly in subsequent
106
sections.
AC C
104
107 108
Antiviral compound and sample measurement
109
Amenamevir (molecular weight, 482.55; international non-proprietary name, amenamevir)
110
was synthesized by Astellas Pharma Inc. (Tokyo, Japan). Sample measurement was
5
ACCEPTED MANUSCRIPT conducted using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS)
112
method based on FDA validation guidance at Covance Laboratories, Ltd[7]. The lower limit
113
of quantification (LLOQ) of this assay for amenamevir was 5 ng/mL when 0.1 mL plasma
114
was used. The LLOQ data were treated as zero and not used for modeling
115
Viruses and cell lines
116
HSV strains clinically isolated in the US were kindly provided by Dr. Nancy Sawtell
117
(Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA). Other viruses and cell
118
lines were provided by Rational Drug Design Laboratories (Fukushima, Japan). Human
119
embryonic fibroblast (HEF) cells and Vero cells were grown in Eagle’s minimum essential
120
medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin G, and
121
100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). HSV-1 and HSV-2 were
122
propagated using HEF cells in maintenance medium containing 2% FBS.
123 Virus plaque reduction assay
TE D
124
M AN U
SC
RI PT
111
The antiviral activities of amenamevir against HSVs were tested using a plaque reduction
126
assay, as described previously [4,5] to determine the concentration and time dependent
127
effects of amenamevir. Briefly, HEF cells were seeded into multiwell plates and incubated
128
until they formed a monolayer. After the medium was removed, the cells were infected with
129
HSV-1, and the plates were further incubated for 1 h at 37°C. The cells were washed twice
130
with maintenance medium and then treated with the test compound until clear plaques
131
appeared. The cells were then fixed with 10% formalin in phosphate-buffered saline, stained
132
with a 0.02% crystal violet solution, and the number of plaques was determined under a light
133
microscope. Concentrations of amenamevir were 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μM.
134
Duration times of the incubation were 6, 8 and 24 h. These data were included in the present
AC C
EP
125
6
ACCEPTED MANUSCRIPT 135
study to simulate the virtual time-course profiles of virus plaques in guinea pigs and humans
136
in combination with their PK data.
137 138
PK study in guinea pigs All animal experimental procedures were approved by the Animal Ethical Committee of
140
Yamanouchi Pharmaceutical Co., Ltd. (currently known as Astellas Pharma Inc.). Female
141
Hartley guinea pigs were purchased from Charles River Laboratories (Kanagawa, Japan).
142
Amenamevir at doses of 0.3, 1.0, 3.0 mg/kg were administered as methylcellulose suspension
143
via oral to guinea pig (n = 3 for each group, 4 weeks of age). Plasma samples were obtained
144
at 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h after dosing.
M AN U
145 146
SC
RI PT
139
PK study in humans
In the present study, the estimated population pharmacokinetic parameters for
148
amenamevir concentration data, which were collected at the time for screening and at the
149
clinical visits once daily through Days 1 to 4 in study CL-101[6], were used.
151
Lesion score measurement in guinea pigs
EP
150
TE D
147
Antiviral activity of ASP2151 against HSVs in guinea pigs was tested by the evaluation
153
of lesion score as described previously [8]. Briefly, Female guinea pigs (Hartley, aged 4
154
weeks at the time of viral infection) were intravaginally infected (designated as Day 0
155
post-infection) with a cotton swab saturated with PBS containing HSV-2 strain G, as
156
described previously [9]. For HSV-2 strain G, the virus pool contained 1.25 × 105 pfU/mL
157
and caused lesions in nearly 100% of control animals. Amenamevir at doses of 0 (placebo), 1,
158
3, 10, or 30 mg/kg was orally administered twice daily for 5 days starting 3 h after viral
159
inoculation as a prophylactic treatment, or 4 days after viral inoculation as a therapeutic
AC C
152
7
ACCEPTED MANUSCRIPT 160
treatment (n = 10 for each group). The disease profile was monitored daily for 21 days and
161
was scored on a 0-6 composite scale based on the severity of vaginitis and neurological
162
symptoms according to the following criteria: Score 0: no signs of infection
164
Score 1: localized, barely perceptible small vesicles
165
Score 2: small or large vesicles involving 10% to 50% of the area
166
Score 3: small or large vesicles involving 50% to 100% of the area
167
Score 4: small ulcers involving 10% to 50% of the area
168
Score 5: severe ulcers involving 50% to 100% of the area
169
Score 6: hind limb paralysis or death
170
The scores of genital herpes in guinea pigs were determined daily. In the PK/PD analysis,
171
the scores on Day 3 were used as the baseline scores to make the modeling easier because of
172
no lesion symptoms until Day 3 after virus allocation. We assumed that disease did not alter
173
the PK of amenamevir.
174
Lesion score measurement in humans
TE D
M AN U
SC
RI PT
163
The data for times to healing of all lesions were obtained as the primary efficacy endpoint
176
of Study CL-101 [6]. Genital herpes recurrence was defined as herpes recurrence below the
177
umbilicus and above the knees. Times to healing (h) of all lesions were determined by the
178
study investigator as the times from therapy initiation to re-epithelialization of all lesions,
179
excluding aborted lesions. Aborted lesions were defined by the presence of prodromal
180
symptoms including pain, tingling, itching, and burning, but lesions failed to develop beyond
181
the macule/papule stage to the vesicular/ulcerative stage. Healed lesions were defined as the
182
absence of crusts, depressions, erosions or ulcerations. Residual erythema in absence of the
183
preceding is defined as healed. Existing symptom lesions were classified by the site clinician
184
during the genital examinations on Days 1 - 6, and also on Days 8 and 10 only if lesions had
AC C
EP
175
8
ACCEPTED MANUSCRIPT not healed by Day 6 and Day 8, respectively. If a patient had existing symptom lesions at the
186
visit on Day 17, an additional genital examination for lesion classification was performed.
187
The classification of the existing symptom lesions were categorized in the similar way for
188
guinea pig as follows for a purpose of logit analysis in this study: Score 0: healed lesion
190
Score 1: crust
191
Score 2: vesicle / pustule / ulcer
192
Score 3: macule / papule
SC
189
RI PT
185
The changes of the scores are unidirectional in the order of 0, 3, 2, 1 and 0. Patients
194
whose scores were maintained to be 0 during the study period were treated as aborted lesions.
195
Mean time course profiles of observed lesion scores are shown in Fig. 1a for guinea pigs and
196
Fig. 1b for humans. The present study was conducted in accordance with the ethical
197
principles stated in the Declaration of Helsinki, Good Clinical Practice, International
198
Conference on Harmonization guidelines, and applicable laws and regulations.
TE D
M AN U
193
199
201
Analysis models and simulation data
The analysis conditions in this study are described briefly in this section.
EP
200
Population modeling
203
Logit model analysis was performed via the non-linear mixed effects model [10] using
204
NONMEM Version VI Level 1.0 (Icon Clinical Research, North Wales, PA, USA).
205
Graphical processing of the NONMEM output was performed with SAS Version 8.2, Release
206
8.02 (SAS Institute Inc., USA). Model selection was based on the goodness-of-fit criteria
207
(log-likelihood difference (l.l.d.) calculated as a difference of objective functions) with visual
208
inspection of the diagnosis plots. A value of l.l.d. more than 3.84 between two models with a
209
1 degree of freedom difference was considered to be significant (p<0.05). First-order
AC C
202
9
ACCEPTED MANUSCRIPT 210
conditional estimation with interaction (FOCE-INTERACTION) [11] was used in
211
NONMEM execution.
212 Model validation
214
Visual predictive check [12] was performed using the final models for guinea pig PK and
215
virus plaque profiles, where 1,000 hypothetical amenamevir concentrations and the numbers
216
of virus plaques were simulated at each time point. The median and 95% prediction interval
217
were constructed from the resulting predictions at each time point. Plots overlaying the
218
medians, prediction interval with observed amenamevir concentrations or observed the
219
numbers of virus plaques were created.
M AN U
220
SC
RI PT
213
PK model in guinea pigs
222
We used a linear single-compartment model with an absorption lag time and first-order
223
absorption as a guinea pig PK model by visual inspection of the observed data. The
224
pharmacokinetic parameters of ka (absorption rate constant), V/F (apparent distribution
225
volume) and CL/F (apparent total clearance) were defined, where F is the oral bioavailability
226
fraction. Mixed-effect modeling was applied using NONMEM, where log-normal distribution
227
for the inter-individual variability was assumed for each pharmacokinetic parameter, in
228
Equation 1:
AC C
EP
TE D
221
229
Pj = θ j ⋅ exp(ηj)
230
where Pj is a parameter of interest in jth subject, θj is the population mean of the
231
corresponding parameter, ηj is the random variable which gives the inter-individual
232
variability of Pj from θj; ηj was assumed to be normally distributed with a mean 0 and a
233
variance ω2 . Residual error was assumed to be described by a proportional error model
234
given by Equation. 2;
Eq. 1
10
ACCEPTED MANUSCRIPT 235
Yij = Cij (1 + εij)
Eq. 2
236
where Yij is the ith observed concentration in jth subject, Cij is the predicted concentration
237
by the model, and εij was the random variable for residual error which is assumed to be
238
normally distributed with a mean 0 and a variance σ 2 .
RI PT
239 PK model in humans
241
PK model analysis of amenamevir in patients with genital herpes had already been performed
242
using a single-compartment model with first-order absorption[7]. As there were some
243
extremely low concentration data points (LCPs) during the absorption phase in the plasma
244
concentration data, another first-order absorption rate constant (ka,LCP) was used in addition to
245
ka to explain the profile of these LCPs. On NONMEM analysis, the estimated mean
246
(and %RSE) values of apparent clearance (CL/F) was found to be 13.8 (4.28%) L/h, apparent
247
distribution volume (V/F) was 143 (4.31%) L, absorption rate constant (ka) was 0.874
248
(15.70%) h-1, and the absorption rate constant for LCP (ka,LCP) was 0.00107 (63.30%) h-1.
249
Relative bioavailability (assuming a bioavailability of 1.0 at 100 mg dose) at a dose of
250
200 mg was 0.982 (6.02%), 400 mg was 0.874 (5.74%), and 1200 mg was 0.706 (6.15%).
M AN U
TE D
EP
251
SC
240
Simulation of PK profiles in guinea pigs and humans
253
Simulations of plasma amenamevir profiles in guinea pigs and humans were conducted using
254
the population mean PK parameters for guinea pigs and the post-hoc PK parameter estimates
255
for humans to simulate time course profiles of the numbers of virus plaques in those species.
256
The data point treated as LCP were not included in the simulation processes in this study
257
because our modeling method using LCP concept was to explain the outliers (i.e. LCP) and
258
obtain a better estimate for Ka using non-LCP data only. The numbers of LCP data point was
259
33 in totally 928 points (3.56% total), and we think the effect of omitting the LCP on
AC C
252
11
ACCEPTED MANUSCRIPT 260
simulation results is negligible. Time-courses of simulated concentration are shown in
261
Figures 5a and 6a, and simulation conditions were as follows: Guinea pig = 0, 1, 3, 10, and
262
30 mg/kg BID for 5 days;
263
Human = 0, 100, 200, and 400 mg QD for 3 days or 1200 mg as a single dose.
RI PT
264 PD model for virus plaque
266
A two-compartment model was applied to explain the concentration and time-dependent
267
changes in the number of virus plaques, as shown in Figure 2. A virus cycle was incorporated
268
by defining the amenamevir effective/non-effective compartments, which are connected by
269
first-order rate constants kinact and kact [13]. Increase of virus plaque was assumed as the
270
first-order rate constant (kin), it was fixed to 0.0569 h-1 (= ln(60) / 72), which gives the
271
number of virus plaques to be 60 pFU at 72 h based on the study setting. A non-linear drug
272
effect was assumed using the Michaelis-Menten form with maximum drug effect (Emax) and a
273
Michaelis constant (EC50).
TE D
M AN U
SC
265
The mass-balance equations for the number of virus plaques are given as follows.
275
= k × V1 − k × V2 + k × V3 −
Eq. 3
276
= k × V2 − k × V3
Eq. 4
277
In Eq. 3-4, V(1) represents the number of virus plaques in a hypothetical input
278
compartment, V(2) is the amenamevir effective compartment, V(3) is the amenamevir
279
ineffective compartment; the initial value of V(1) is 60, while those of V(2) and V(3) are 0.
280
Plasma concentration of amenamevir is given by CP. No inter-individual variability was
281
assumed in this case and an additive error model for residual variability was assumed as
282
given in Equation 5;
283
Yij = Cij + εij
×
AC C
EP
274
Eq. 5
12
ACCEPTED MANUSCRIPT 284
where Yij and Cij are the observed and model predicted numbers of plaques for the ith
285
sample in jth subject, εij is the residual error which is assumed to be normally distributed with
286
a mean 0 and a variance σ 2 .
287 Simulation of virus plaque profile
289
Simulations of virtual numbers of virus plaque profiles in guinea pigs and humans were
290
conducted using the simulated amenamevir concentration and the estimated virus kinetic
291
parameters. Simulation settings were as follows.
SC
RI PT
288
- Initial administration amount of number of virus plaques was set to 60 pFU
293
- Time of virus plaque administration was defined as Day 0 (0 h)
294
- Starting time of amenamevir administration was Day 1 (24 h)
295
Lesion scores in human on Day 1 were regarded as the initial scores, this is because the
296
Phase 2 study (CL-101) was designed as the a self-initiation study, where patients
297
self-initiated treatment and returned to the clinic within 24 h after the initial dose of study
298
drug (Day 1). Time-course of virus plaques is shown in Figures 5b and 6b.
299
TE D
M AN U
292
PK/PD Model
301
Logit model analysis of lesion score
AC C
EP
300
302
Logit model analysis was applied to the ordered-categorical lesion score data. Let Yijk =
303
Y(tijk) be the lesion score at the kth time point tijk, in the ith individual of the jth treatment group,
304
and Yijk takes the values of 0, 1, 2, 3, or 4 in the guinea pig study, and 0, 1, 2, or 3 in the
305
human study. The scores 5 and 6 were not included in the analysis for guinea pig because of
306
no observed data for these scores. A logit model for the probability of having a lesion score
307
Yijk that was equal to or less than a given score yijk = 0, 1,.., n can be expressed as a
308
cumulative distribution function as follows [14,15].
13
ACCEPTED MANUSCRIPT 309 310 311
Fy; θ, η = Pr&Y ≤ y; θ, η) =
*+,-./;0,12
3 *+,-./;0,12
Eq. 6
where θ is a vector of parameters and η is a random variable. Based on the cumulative probability function, the probability of having a single lesion score was given by Equation 7: Pr&Y = y) = Pr&Y ≤ y) − Pr&Y ≤ y − 1),
313
where Pr&Y < 0) = 0 and Pr&Y ≤ n) = 1.
314
The logit, g(yijk; θ, η) was defined as follows:
315
gy; θ, η = ∑ :<3 θ: × Q: y + Eff
316
for yijk = n, where Q: ?y @A B = C
317
In guinea pigs, lesion scores increased almost monotonically in the placebo group,
318
however it began to decrease after Day 2 in amenamevir groups and clear dose dependence
319
was observed as shown in Figure 1a. In contrast, lesion scores in humans decreased
320
monotonically over time in both amenamevir and placebo groups, and dose dependence was
321
not clear as shown in Figure 1b. These data suggest that the decrease in lesion scores could
322
not be explained only by the changes of virus plaques reflecting healing of amenamevir
323
(Virus Plaque component) and thus the elapsed time reflecting the healing of the immune
324
response (Time component) was included into the model in this study. For the term of
325
describing drug efficacy (Eff), following model was tested. The term Eff was expressed by
326
the addition of Virus Plaque component and Time component.
RI PT
312
AC C
EP
TE D
M AN U
1, y @A ≥ m − 1 . 0, otherwise
SC
Eq. 7
327
Eff = β3 × Virus Plaque + β × Time+η
328
In these models, number of virus plaques (given by ‘Virus Plaque’) and the elapsed time
329
from virus plaque increase (given by ‘Time’) were incorporated as fixed effects. This model
330
was built theoretically, no covariates step was conducted. Maximum Likelihood Estimation
331
was used for the fitting and posterior prediction, and individual estimated lesion scores for ith
Eq. 8
14
ACCEPTED MANUSCRIPT 332
subject at time t (Lesion scorei,t) were calculated based on the estimated parameters by
333
following equation. Lesion score , = ∑ :
335
Results
336
PK model in guinea pigs
RI PT
334
As shown in Figure. 3, result of VPC shows that most of the observed values were within
338
95% prediction interval. Estimated population mean parameters of the PK model for guinea
339
pig PK are summarized in Table 1.
SC
337
340 PD model for virus plaques
342
Estimated population mean parameters of the PD model for the virus plaque profiles are
343
summarized in Table 1. The observed plaque counts versus amenamevir concentration are
344
plotted with the 95% prediction interval in Figure 4. The estimated EC50 was 127 ng/mL,
345
which is a little smaller than the in vitro expected effective concentration of 200 ng/mL [5].
346
As the result, in vitro anti-virus effect of amenamevir was consistent with the virus kinetic
347
model.
348
Logit model for lesion score
TE D
EP
Final models for the term logit lesion score in guinea pigs and humans are obtained as
AC C
349
M AN U
341
350
follows:
351
Guinea pig:
For Lesion score =
0; Logit = 6.76 + Eff.[ * , . 1; Logit = 6.76 + 3.11 + Eff.[ * , . = 9.87 + Eff.[ * , . 2; Logit = 6.76 + 3.11 + 4.17 + Eff.[ * , . = 14.04 + Eff.[ * , .
15
ACCEPTED MANUSCRIPT 3; Logit = 6.76 + 3.11 + 4.17 + 5.89 + Eff.[ * , . = 19.93 + Eff.[ * , . 352 353
Where Eff.[ * , . = −0.265 × Virus Plaque − 0.0334 × Time
Eq.10
Human:
0; Logit = −5.21 + Eff`[:
1; Logit = −5.21 + 1.86 + Eff`[: = −3.35 + Eff`[:
RI PT
For Lesion score =
2; Logit = −5.21 + 1.86 + 3.34 + Eff`[: = −0.01 + Eff`[:
356
SC
355
Where Eff`[: = −0.0247 × Virus Plaque + 0.0424 × Time
Eq.11
In these models, a beneficial effect means a decrease of lesion scores while a worsening
M AN U
354
effect means an increase of lesion scores.
While the fixed effect in Eff of Virus Plaque was negative in both species, the fixed
358
effect in Eff of the Time component by the immune system was negative in guinea pigs but
359
positive in humans. In guinea pigs, both the Virus Plaque and Time components worsened the
360
lesion score. In humans, Virus Plaque worsened the lesion score, whereas immune system
361
improved. Estimated profiles of mean lesion scores were consistent with the observed values
362
in both species (Fig. 5(c) and Fig. 6(c)). Some simulations were performed to show the
363
probabilities above each lesion score in guinea pig (Fig. 5(d)-(g)) and in human (Fig. 6(d)-(f)),
364
where dose and time dependent profiles are shown. Fitting results are summarized in Table 2,
365
and no 95% confidence intervals (CIs) for any fixed effects included 0, indicating that all
366
parameters were significant.
367
Discussion
AC C
EP
TE D
357
368
In the present study, an empirical PK/PD model, as shown in Figure 2 for the
369
helicase-primase inhibitor in genital herpes patients was developed. In this model, the time
370
course profiles of lesion scores was not directly dependent on the amenamevir PK, but it was
16
ACCEPTED MANUSCRIPT 371
mainly dependent on the virtual number of virus plaques which suggests a time-dependent
372
anti-virus mechanism of amenamevir. The PK analysis of amenamevir concentration in guinea pig suggested liner PK profile as
374
shown in Table 1 and Figure 3. In contrast, the CL/F estimated in our previous study in
375
humans [7] suggested dose-dependent bioavailability as F decreased as dose increased. A
376
possible reason is the difference of the dosage form between the species; amenamevir was
377
administered in methylcellulose solution to guinea pigs and in tablet form to humans.
378
Amenamevir is poorly soluble, which strongly affects its passive diffusivity (data not shown).
379
When an allometric scaling [16] was applied, CL/F in humans was estimated about 0.16
380
L/h/kg, smaller than in guinea pig (2.04 L/h/kg) although the reason of the difference
381
between species was unclear. We focused on the relationship between amenamevir
382
concentrations and virus plaque data in the present study.
M AN U
SC
RI PT
373
We used the virtual number of virus plaques obtained via plaque reduction assay as a
384
marker to explain amenamevir PK/PD mechanism. Goodness of fit of the virus plaque
385
modeling were not enough acceptable especially in the higher concentration range at 6 or 8
386
hrs exposure. Several models to explain this, i.e. some models including not only the time
387
dependent component but also the dose (concentration) dependent component were tested to
388
try to improve the modeling result, however, no clear improvement was obtained(Data not
389
shown). We could not yet find the reason of this discrepancy at the higher concentration, but
390
we conclude that the current model is sufficient to simulate the efficacy of twice a day or
391
daily dosing of amenamevir because some acceptable result was obtained in the condition of
392
24hrs exposure which showed the threshold concentration to maintain the amenamevir
393
efficacy throughout the day (Fig.4c). In this our analysis, the virus plaque time profile in vivo
394
was simulated based on the virus plaque kinetic model, which was built based on in vitro data.
395
Several points remain unclear, however—namely the utility of the same viral kinetic model
AC C
EP
TE D
383
17
ACCEPTED MANUSCRIPT 396
between species, whether or not plasma concentration is the best surrogate marker in these
397
species, and differences in amenamevir efficacy for viral kinetics between species. The categories for lesion scores in human were not determined in the clinical study
399
protocol, and we originally defined four categories as shown in the present study. Scores in
400
humans showed monotonical change, i.e. the scores started from 3 and decreased along with
401
the lesion healing. Assuming that most patients showed lesion healing without recurrence and
402
therefore the lesion scores tended to monotonically decrease, the definition of lesion scores
403
seem acceptable for modeling purposes. We did not include data from patients whose lesion
404
scores remained 0 throughout the study period (aborted lesion) in the model analysis because
405
for a patient whose lesion score was 0 during the study, as we were unable to determine
406
whether or not the lack of any symptoms was due to the drug’s effects. Therefore, the PD
407
model of the present analysis was built only for patients who developed the symptoms.
M AN U
SC
RI PT
398
Finally, the lesion scores in guinea pig and human could be explained by the similar
409
models including with two fixed-effect parameters, i.e. amount of virus plaques and the
410
elapsed time. Our previous study regarding virus plaques showed that the continual exposure
411
of amenamevir above a certain concentration is necessary to prevent the virus re-production
412
[5], and this was confirmed by another study with multiple dose design which is usually used
413
in the antibiotics area [17]. During the clinical development of amenamevir, the value for
414
EC50 obtained in the non-clinical studies could be directly extrapolated into the clinical study
415
and is used for the dose rationale [5,6] As results, the non-clinical EC50 without a cure effect
416
was under-estimated, we were unable to detect a clear dose relationship in the clinical study.
AC C
EP
TE D
408
417
In the present study, we developed similar PK/PD models in both guinea pigs and
418
humans with Virus Plaque and Time components to explain the time-course profiles of lesion
419
scores. A virtual kinetic profile of virus plaque was incorporated into the model in order to
420
connect the PK profile of amenamevir with the lesion score profiles, and the terms in the
18
ACCEPTED MANUSCRIPT 421
logistic model consisting of the number of virus plaques and the elapsed time well explained
422
the dose- and time-dependent PD profiles. These results suggest that the virtual number of
423
virus plaques can be used as a built-in biomarker. While the fixed effect in Eff of Virus Plaque was negative in both species, the fixed
425
effect in Eff of the Time component for the immune system was negative in guinea pigs but
426
positive in humans. HSV-2 damages the central nervous system, which in turn affects the
427
immune system [18]. Our present findings suggest that the immune system might be
428
weakened by virus infection in guinea pigs, although evidence for this is insufficient at
429
present. The differences in results for the Time Component between species may have been
430
due to the different experimental conditions and different responses of immune systems. In
431
the guinea pig study, animals were infected with a lethal amount of HSV to ensure herpes
432
infection, and lesion severity increased with time. In humans, the immune system may work
433
adequately to reduce lesion severity.
M AN U
SC
RI PT
424
The effect of the Virus Plaque component was deemed to be large in guinea pigs, as
435
efficacy was clearly dose-dependent in the amenamevir groups while the effect was saturated
436
in the placebo group. In contrast, the effect of the Virus Plaque component was relatively
437
small in humans, and the drug effect for lesion scores was smaller than in guinea pigs.
EP
TE D
434
In humans, the PD effect was almost dose-independent, and immune system-related
439
healing was likely the driving force behind reductions in lesion scores. These findings
440
suggest that the drug effect may be masked in diseases healed by the immune response, such
441
as genital herpes. Therefore, the PK/PD model proposed in the present study will be
442
particularly useful for explaining the PK/PD relationship of drugs used to treat self-cured
443
diseases. In antibiotics and antiviral drug kinetic analyses, drug-bacteria (or virus) interaction
444
is assumed to be independent of in vivo conditions, such as host species. Here, we assumed
445
that the kinetic parameters for virus plaque data obtained in in vitro experiments could be
AC C
438
19
ACCEPTED MANUSCRIPT applied to both guinea pigs and humans. In addition, as a practical problem, we cannot obtain
447
the virus kinetic data in human and the difference of it between the species is difficult to be
448
evaluated.
449
In the development of drugs for diseases with natural healing, if healing does not happen in
450
the animal disease model, its efficacy may differ from clinical efficacy.
451
In the non-clinical studies from the perspective of the prediction of efficacy, animal model
452
without natural healing which can confirm the drug power clearly is suitable, however, it may
453
misjudge the clinical endpoint. For example, even when the development of animal models,
454
natural healing should be considered.
M AN U
455
SC
RI PT
446
Conclusions
457
This PK/PD modeling approach based on bi-directional translational approach is useful for
458
not only new candidate exploration in the non-clinical stage but also further application in
459
clinical data analysis. We believe that this kind of modeling and simulation approach will
460
give some suggestions especially as a unique PK/PD modeling approach connecting the
461
non-clinical and clinical data during the HSV drug development.
EP
462
TE D
456
Conflicts of interest
464
Financial support for this study was provided by Astellas Pharma Inc. The authors report no
465
conflicts of interest.
466
AC C
463
20
ACCEPTED MANUSCRIPT 467 References:
469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514
1. Stanberry L, Cunningham A, Mertz G, Mindel A, Peters B, Reitano M, Sacks S, Wald A, Wassilew S, Woolley P (1999) New developments in the epidemiology, natural history and management of genital herpes. Antiviral Res 42 (1):1-14. doi:S0166-3542(99)00004-2 [pii] 2. Snoeck R, De Clercq E (2002) New treatments for genital herpes. Curr Opin Infect Dis 15 (1):49-55 3. Reddy MB, Morcos PN, Le Pogam S, Ou Y, Frank K, Lave T, Smith P (2012) Pharmacokinetic/Pharmacodynamic predictors of clinical potency for hepatitis C virus nonnucleoside polymerase and protease inhibitors. Antimicrob Agents Chemother 56 (6):3144-3156. doi:10.1128/AAC.06283-11 AAC.06283-11 [pii] 4. Chono K, Katsumata K, Kontani T, Kobayashi M, Sudo K, Yokota T, Konno K, Shimizu Y, Suzuki H (2010) ASP2151, a novel helicase-primase inhibitor, possesses antiviral activity against varicella-zoster virus and herpes simplex virus types 1 and 2. J Antimicrob Chemother 65 (8):1733-1741. doi:10.1093/jac/dkq198 dkq198 [pii] 5. Katsumata K, Chono K, Kato K, Ohtsu Y, Takakura S, Kontani T, Suzuki H (2013) Pharmacokinetics and pharmacodynamics of ASP2151, a helicase-primase inhibitor, in a murine model of herpes simplex virus infection. Antimicrob Agents Chemother 57 (3):1339-1346. doi:10.1128/AAC.01803-12 AAC.01803-12 [pii] 6. Tyring S, Wald A, Zadeikis N, Dhadda S, Takenouchi K, Rorig R (2012) ASP2151 for the treatment of genital herpes: a randomized, double-blind, placebo- and valacyclovir-controlled, dose-finding study. J Infect Dis 205 (7):1100-1110. doi:10.1093/infdis/jis019 jis019 [pii] 7. Takada A, Katashima M, Kaibara A, Sawamoto T, Zhang W, Keirns J (2014) Statistical analysis of Amenamevir (ASP2151) between pharmacokinetics and clinical efficacies with non-linear effect model for the treatment of Genital Herpes. Clin Pharmacol Drug Dev. doi:DOI: 10.1002/cpdd.108 8. Katsumata K, Chono K, Sudo K, Shimizu Y, Kontani T, Suzuki H (2011) Effect of ASP2151, a herpesvirus helicase-primase inhibitor, in a guinea pig model of genital herpes. Molecules 16 (9):7210-7223. doi:10.3390/molecules16097210 molecules16097210 [pii] 9. Miller RL, Imbertson LM, Reiter MJ, Gerster JF (1999) Treatment of primary herpes simplex virus infection in guinea pigs by imiquimod. Antiviral Res 44 (1):31-42. doi:S0166-3542(99)00052-2 [pii] 10. Beal L, Sheiner B (1989) NONMEM Users Guide NONMEM Project Group, UCSF. 11. Wang Y (2007) Derivation of various NONMEM estimation methods. J Pharmacokinet Pharmacodyn 34 (5):575-593. doi:10.1007/s10928-007-9060-6 12. Post TM, Freijer JI, Ploeger BA, Danhof M (2008) Extensions to the visual predictive check to facilitate model performance evaluation. J Pharmacokinet Pharmacodyn 35 (2):185-202. doi:10.1007/s10928-007-9081-1 13. Lobo ED, Balthasar JP (2002) Pharmacodynamic modeling of chemotherapeutic effects: application of a transit compartment model to characterize methotrexate effects in vitro. AAPS PharmSci 4 (4):E42. doi:10.1208/ps040442 14. Sheiner LB (1994) A new approach to the analysis of analgesic drug trials, illustrated with bromfenac data. Clin Pharmacol Ther 56 (3):309-322
AC C
EP
TE D
M AN U
SC
RI PT
468
21
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
15. Liu CY, Sambol NC (1999) Pharmacodynamic analysis of analgesic clinical trials: nonlinear mixed-effects logistic models. J Biopharm Stat 9 (2):253-270. doi:10.1081/BIP-100101175 16. Holford N, Heo YA, Anderson B (2013) A pharmacokinetic standard for babies and adults. J Pharm Sci 102 (9):2941-2952. doi:10.1002/jps.23574 17. Frimodt-Moller N (2002) How predictive is PK/PD for antibacterial agents? Int J Antimicrob Agents 19 (4):333-339. doi:S0924857902000298 [pii] 18. Black PH (1994) Central nervous system-immune system interactions: psychoneuroendocrinology of stress and its immune consequences. Antimicrob Agents Chemother 38 (1):1-6
AC C
515 516 517 518 519 520 521 522 523 524 525 526
22
ACCEPTED MANUSCRIPT Tables Table 1 Estimated Population parameters of amenamevir in a guinea pig PK model and Virus Plaque PD model
PD model for Virus Plaque （Common parameters in Guinea pigs and humans） ）
95% CIb (Lower – Upper) 1.93 – 2.15 3.05 – 4.73 1.02 – 2.50 0.154 – 0.204 5.9 – 14.0 8.61 – 18.6 9.2 – 28.9 5.3 – 12.7 13.6 – 45.4 112 –246 – 0.764 – 0.984 65.3 – 189 7.0 – 10.2
RI PT
CL/F (L/h/kg) V/F (L/kg) ka (h-1) Lag Time (h) ηCL/F (CV%) ηka (CV%) ηLagTime (CV%) εc kinact (h-1) kact (h-1) kin (h-1) Emax (pFU) EC50 (ng/mL) εc
Estimate (%RSEa) 2.04 (2.76%) 3.89 (11.1%) 1.76 (21.4%) 0.179 (7.15%) 10.7 (35.3%) 14.5 (33.1%) 21.4 (41.6%) 9.76 (35.9%) 29.5 (27.6%) 179 (19.0%) 0.0569 (Fixed) 0.874 (6.40%) 127 (24.8%) 8.75 (18.7%)
SC
PK model for Guinea pig
Parameter
M AN U
Model
AC C
EP
TE D
CI: confidence interval, CL/F: oral clearance, V/F: volume of distribution, ka: absorption rate, η: inter-individual variability, ε: intra-individual variability, kinact: inactivation ratio , kact: activation ratio, kin: increase ratio , Emax: maximum drug effect, EC50: Michaelis constant a: %RSE is percent relative standard error (100% × Standard Error / Estimate) b: 95% CI = T ± 1.96 × Standard Error c: given as standard deviation
ACCEPTED MANUSCRIPT Table 2 Estimated parameters of the logistic regression model for lesion score in guinea pigs and humans
θScore=0 θScore=1 θScore=2 θScore=3 ηc
RI PT
β2
Estimate (%RSEa) -0.0247 (29.5%) 0.0424 (6.0%) -5.21 (6.8%) 1.86 (7.3%) 3.34 (6.0%)
Human 95% CIb (Lower – Upper) -0.0390, -0.0104 0.0374, 0.0474 -5.90, -4.52 1.60, 2.12 2.94, 3.74
SC
β1
Estimate (%RSEa) -0.265 (10.6%) -0.0334 (14.6%) 6.76 (15.4%) 3.11 (13.2%) 4.17 (11.2%) 5.89 (12.4%) 1.62 (31.7%)
M AN U
Parameter
Guina pig 95% CIb (Lower – Upper) -0.320 -0.210 -0.0430, -0.0238 4.72, 8.80 2.30, 3.92 3.25, 5.09 4.46, 7.32 0.996, 2.07
1.36 (16.2%)
1.12, 1.56
AC C
EP
TE D
CI: confidence interval, β1: effect of virus plaque, β2: time effect for healing, θScore=x: logit value for score x, η: inter-individual variability a: %RSE is percent relative standard error (100% × Standard Error / Estimate) b: 95% CI = T ± 1.96 × Standard Error c: given as standard deviation
ACCEPTED MANUSCRIPT Figure captions Figure 1: Time course profiles of mean observed lesion scores in guinea pigs (a) and humans (b). (a) Closed circle: Placebo, open triangle: 1 mg/kg, closed triangle: 3 mg/kg, open square: 10 mg/kg, closed square: 30 mg/kg; treatment duration was 5 days from Day 1.
RI PT
(b) Closed circle: Placebo, open triangle: 100 mg, closed triangle: 200 mg, open square: 400 mg, closed square: 1200 mg; treatment duration was 3 days from Day 1.
SC
Figure 2: Overview of the PK/PD model
M AN U
Figure 3: Results of population PK modeling and visual predictive check in guinea pigs. (a) Dose = 0.3 mg/kg, (b) 1.0 mg/kg, (c) 3.0 mg/kg. Solid line: median, filled region: 95% prediction interval.
TE D
Figure 4: Results of population PD modeling and visual predictive check for virus plaque data. (a) Amenamevir duration time = 6 h, (b) 8 h, (c) 24 h. Solid line: median, filled
EP
region: 95% prediction interval.
AC C
Figure 5: Results of model predicted time course profiles in guinea pigs. Closed circle: Placebo, open triangle: 1 mg/kg, closed triangle: 3 mg/kg, open square: 10 mg/kg, closed square: 30 mg/kg; treatment duration was 5 days from Day 1. (a), (b) Simulated time-course profiles of plasma concentration and virus plaque. (c) Observed (plots) and model-predicted (lines) time-course profiles of lesion scores in guinea pigs. Predictions are given as surface of lesion scores (z-axis) as a function of time (x-axis) and dose (y-axis). Symbols show the observed values. (d) to (g) Predicted probability surfaces for lesion scores (z-axis) as a
ACCEPTED MANUSCRIPT function of day (x-axis) and dose (y-axis) in guinea pigs. (d) Pr{Y>=1}, (e) Pr{Y>=2}, (f) Pr{Y>=3}, (g) Pr{Y=4}.
Figure 6: Results of model predicted time course profiles in humans. Closed circle:
RI PT
Placebo, open triangle: 100 mg, closed triangle: 200 mg, open square: 400 mg, closed square: 1200 mg; treatment duration was 3 days from Day 1. (a), (b) Simulated time-course profiles of plasma concentration and virus plaque. (c) Observed (plots) and model-predicted (lines)
SC
time-course profiles of lesion scores in humans. Predictions are given as surface of lesion scores (z-axis) as a function of time (x-axis) and dose (y-axis). Symbols show the observed
M AN U
values. (d) to (f) Predicted probability surfaces for lesion scores (z-axis) as a function of day
AC C
EP
TE D
(x-axis) and dose (y-axis) in human. (b) Pr{Y>=1}, (c) Pr{Y>=2}, (d) Pr{Y=3}.
ACCEPTED MANUSCRIPT Figure 1
M AN U
SC
RI PT
a)
AC C
EP
TE D
b)
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figure 2
ACCEPTED MANUSCRIPT Figure 3
SC
RI PT
a)
EP AC C
c)
TE D
M AN U
b)
ACCEPTED MANUSCRIPT Figure 4
SC
RI PT
a)
EP AC C
c)
TE D
M AN U
b)
ACCEPTED MANUSCRIPT Figure 5 a)
SC
RI PT
b)
EP AC C
d)
TE D
M AN U
c)
e)
ACCEPTED MANUSCRIPT g)
AC C
EP
TE D
M AN U
SC
RI PT
f)
ACCEPTED MANUSCRIPT Figure 6 b)
SC
RI PT
a)
AC C
d)
EP
TE D
M AN U
c)
e)
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
f)
S1347-4367(16)30029-5
DOI:
10.1016/j.dmpk.2016.05.005
Reference:
DMPK 115
To appear in:
Drug Metabolism and Pharmacokinetics
Received Date: 9 November 2015 Revised Date:
20 May 2016
Accepted Date: 27 May 2016
Please cite this article as: Takada A, Katashima M, Kaibara A, Chono K, Katsumata K, Sawamoto T, Suzuki H, Yano Y, Integrative Pharmacokinetic-Pharmacodynamic Modeling and Simulation of Amenamevir (ASP2151) for Treatment of Recurrent Genital Herpes, Drug Metabolism and Pharmacokinetics (2016), doi: 10.1016/j.dmpk.2016.05.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Integrative
Pharmacokinetic-Pharmacodynamic
Modeling
and
2
Amenamevir (ASP2151) for Treatment of Recurrent Genital Herpes
Simulation
of
3 Akitsugu Takada1, Masataka Katashima1, Atsunori Kaibara1, Koji Chono1, Kiyomitsu
5
Katsumata1, Taiji Sawamoto1, Hiroshi Suzuki1, Yoshitaka Yano2
6
RI PT
4
1
Astellas Pharma Inc., 2-5-1 Nihonbashi-Honcho, Chuo-ku, Tokyo, Japan
8
2
Kyoto Pharmaceutical University, 5 Misasagi-Nakauchicho, Yamashina-ku, Kyoto, Japan
SC
7
9 Corresponding author’s information
M AN U
10
Akitsugu Takada
12
Astellas Pharma Inc.
13
2-5-1 Nihonbashi-HonchoChuo-ku, Tokyo, 103-8411, Japan
14
E-mail: [email protected]
15
Tel: +81-3-3244-2579, Fax: +81-3-3243-5732
EP AC C
16
TE D
11
1
ACCEPTED MANUSCRIPT Abstract
18
Amenamevir is a novel drug that targets the viral helicase-primase complex. While
19
dose-dependent efficacy had been observed in non-clinical studies, no clear dose dependence
20
has been observed in humans. We therefore developed a pharmacokinetic/pharmacodynamic
21
(PK/PD) model to explain this inconsistency between species and to clarify the
22
immune-related healing of amenamevir in humans. The model consisted of a non-linear
23
kinetic model for a virtual number of virus plaques as a built-in biomarker. Lesion score was
24
defined as an endpoint of antiviral efficacy, and logit model analysis was applied to the
25
ordered-categorical lesion score. The modeling results suggested the time course profiles of
26
lesion score could be explained with the efficacy terms in the logit model, using change in
27
number of virus plaques as an indicator of the effects of amenamevir and time elapsed as an
28
indicator of the healing of the immune response. In humans, the PD effect was almost
29
dose-independent, and immune-related healing may have been the driving force behind the
30
reduction in lesion scores. Drug efficacy is occasionally masked in diseases healed by the
31
immune response, such as genital herpes. The PK/PD model proposed in the present study
32
must be useful for explanation the PK/PD relationship of such drugs.
33
EP
TE D
M AN U
SC
RI PT
17
Keywords
35
amenamevir, pharmacokinetic/pharmacodynamic, pharmacometrics, genital herpes, modeling
36
and simulation
37
AC C
34
2
ACCEPTED MANUSCRIPT 38
Introduction Recurrence of genital herpes is usually caused by HSV-2 and less commonly by HSV-1
40
[1]. Almost 50 million American adults and adolescents (20% of the total population) are
41
infected with genital herpes: it is one of the most common sexually transmitted diseases [2].
42
The recurrence of genital symptoms is very typical, with up to 80% of HSV-2-infected
43
patients experiencing recurrent outbreaks within the first 12 months following the first
44
episode.
RI PT
39
Amenamevir is a novel drug that targets the viral helicase-primase complex. This
46
protein complex is essential for herpes simplex virus (HSV) DNA synthesis, making
47
helicase-primase inhibitors such as amenamevir a potential treatment option for genital
48
herpes. A pharmacokinetic / pharmacodynamic (PK/PD) approach for anti-virus drug is
49
similar to the development of antibiotics, and in vitro IC50 is used to predict the clinical dose
50
in vivo animal model [3]. Amenamevir has demonstrated higher potency in vitro antiviral
51
activity against HSV-1 and HSV-2 than aciclovir [4].
TE D
M AN U
SC
45
A phase 2 study (ASP2151 CL-101 study) was performed in the United States to compare
53
the safety and efficacy of four different dose regimens of amenamevir with valaciclovir and
54
placebo in parallel in the acute treatment of recurrent genital HSV infection. At the first sign
55
of recurrence, patients self-initiated treatments with amenamevir 100, 200, or 400 mg daily
56
for 3 days, 1200 mg as a single dose; placebo for 3 days; or valaciclovir 500 mg twice daily
57
for 3 days. Dose and regimen were determined based on those results and PK profile of
58
amenamevir to maintain time above 200 ng/ml of plasma amenamevir concentration, T200 [5].
59
Of 695 patients randomized to the treatments, data from 437 patients with recurrent infection
60
were analyzed. The primary efficacy endpoint was time to lesion healing which was shorter
61
in all amenamevir groups and the valaciclovir group compared with placebo, but a
AC C
EP
52
3
ACCEPTED MANUSCRIPT 62
dose-response relationship was not evident. All groups except amenamevir 100mg showed a
63
higher proportion of aborted lesions compared with placebo[6]. Population PK analysis of amenamevir was conducted to estimate patients’ PK profiles
65
using 957 plasma samples in 273 genital herpes subjects in CL-101study. The plasma
66
concentration-time courses for amenamevir were described using a one-compartment model
67
with first-order absorption. Exploratory PK and PD analysis was conducted to find an
68
appropriate PK-related parameter which correlates with PD instead of finding dose-PD
69
relationship, using individual PK parameters derived from the final model by the population
70
analysis. The variable T200 was found to correlate with time to lesion healing and viral
71
shedding, which is consistent with the in vivo results [7]. However, these correlations were
72
not statistically significant, because T200 was dose dependent parameter after all.
M AN U
SC
RI PT
64
In CL-101 study[6], dose-dependent efficacy was not evident in the primary analysis
74
using time to lesion healing, although dose-dependent efficacy had previously been suggested
75
in guinea pigs [8]. To discuss address this inconsistency between clinical and non-clinical
76
results, we conducted additional PK/PD modeling and simulations using the same component
77
model to for both humans and guinea pigs have conducted in the present study with a built-in
78
biomarker—number of virus plaques, which explains the time-dependent antivirus efficacy of
79
amenamevir—to combine the PK of amenamevir and lesion scores as an efficacy endpoint.
80
Furthermore, non-drug related such as immune response was added to explain the difference.
AC C
EP
TE D
73
81
In general, screening process of new drugs is conducted by using animal to confirm the
82
efficacy, safety and pharmacokinetic. After that, these endpoints are confirmed in clinical
83
trials. Amenamevir showed effective anti-virus action dose-dependently in guinea pigs but
84
not in humans as stated above. To fully understand the phenomenon is somewhat challenging
85
theme, although many drug candidate compounds may have same theme in drug development
86
stage. In this report, we tried to fill a gap between non-clinical and clinical results with the
4
ACCEPTED MANUSCRIPT 87
same PK/PD model both animal and human, which is bi-directional translational modeling
88
and simulation approach.
90
Materials and methods
91
Analysis data from clinical and non-clinical studies
92
RI PT
89
The present study is a population PK/PD analysis using the obtained PK and PD data in the previous clinical and non-clinical studies [4,8,6]. The scheme of the PK/PD model
94
applied in this study is shown in Figure 2. As mentioned in Introduction, the results of the
95
previous virus plaque study provided the possible relationship of viral dynamics with
96
amenamevir concentration [5], and efficacy such as suppression of lesion scores was
97
expected to correlate with amenamevir concentration. The present PK/PD model assumes that
98
the time time-dependent increase in number of virus plaques is reduced by amenamevir, and
99
the increase of lesion score is dependent on the virtual number of virus plaques. Data from
M AN U
SC
93
previous studies were reanalyzed in the present study as follows:
101
1) Virus plaque assay data were used for constructing virus plaque PD model component.
102
2) Plasma concentration data of amenamevir in guinea pigs and humans (reference) were
EP
103
TE D
100
used for constructing the PK model component. 3) Lesion score data in guinea pigs and humans were used for logit model analysis.
105
The experimental conditions for the previous studies will be described briefly in subsequent
106
sections.
AC C
104
107 108
Antiviral compound and sample measurement
109
Amenamevir (molecular weight, 482.55; international non-proprietary name, amenamevir)
110
was synthesized by Astellas Pharma Inc. (Tokyo, Japan). Sample measurement was
5
ACCEPTED MANUSCRIPT conducted using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS)
112
method based on FDA validation guidance at Covance Laboratories, Ltd[7]. The lower limit
113
of quantification (LLOQ) of this assay for amenamevir was 5 ng/mL when 0.1 mL plasma
114
was used. The LLOQ data were treated as zero and not used for modeling
115
Viruses and cell lines
116
HSV strains clinically isolated in the US were kindly provided by Dr. Nancy Sawtell
117
(Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA). Other viruses and cell
118
lines were provided by Rational Drug Design Laboratories (Fukushima, Japan). Human
119
embryonic fibroblast (HEF) cells and Vero cells were grown in Eagle’s minimum essential
120
medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin G, and
121
100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). HSV-1 and HSV-2 were
122
propagated using HEF cells in maintenance medium containing 2% FBS.
123 Virus plaque reduction assay
TE D
124
M AN U
SC
RI PT
111
The antiviral activities of amenamevir against HSVs were tested using a plaque reduction
126
assay, as described previously [4,5] to determine the concentration and time dependent
127
effects of amenamevir. Briefly, HEF cells were seeded into multiwell plates and incubated
128
until they formed a monolayer. After the medium was removed, the cells were infected with
129
HSV-1, and the plates were further incubated for 1 h at 37°C. The cells were washed twice
130
with maintenance medium and then treated with the test compound until clear plaques
131
appeared. The cells were then fixed with 10% formalin in phosphate-buffered saline, stained
132
with a 0.02% crystal violet solution, and the number of plaques was determined under a light
133
microscope. Concentrations of amenamevir were 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μM.
134
Duration times of the incubation were 6, 8 and 24 h. These data were included in the present
AC C
EP
125
6
ACCEPTED MANUSCRIPT 135
study to simulate the virtual time-course profiles of virus plaques in guinea pigs and humans
136
in combination with their PK data.
137 138
PK study in guinea pigs All animal experimental procedures were approved by the Animal Ethical Committee of
140
Yamanouchi Pharmaceutical Co., Ltd. (currently known as Astellas Pharma Inc.). Female
141
Hartley guinea pigs were purchased from Charles River Laboratories (Kanagawa, Japan).
142
Amenamevir at doses of 0.3, 1.0, 3.0 mg/kg were administered as methylcellulose suspension
143
via oral to guinea pig (n = 3 for each group, 4 weeks of age). Plasma samples were obtained
144
at 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h after dosing.
M AN U
145 146
SC
RI PT
139
PK study in humans
In the present study, the estimated population pharmacokinetic parameters for
148
amenamevir concentration data, which were collected at the time for screening and at the
149
clinical visits once daily through Days 1 to 4 in study CL-101[6], were used.
151
Lesion score measurement in guinea pigs
EP
150
TE D
147
Antiviral activity of ASP2151 against HSVs in guinea pigs was tested by the evaluation
153
of lesion score as described previously [8]. Briefly, Female guinea pigs (Hartley, aged 4
154
weeks at the time of viral infection) were intravaginally infected (designated as Day 0
155
post-infection) with a cotton swab saturated with PBS containing HSV-2 strain G, as
156
described previously [9]. For HSV-2 strain G, the virus pool contained 1.25 × 105 pfU/mL
157
and caused lesions in nearly 100% of control animals. Amenamevir at doses of 0 (placebo), 1,
158
3, 10, or 30 mg/kg was orally administered twice daily for 5 days starting 3 h after viral
159
inoculation as a prophylactic treatment, or 4 days after viral inoculation as a therapeutic
AC C
152
7
ACCEPTED MANUSCRIPT 160
treatment (n = 10 for each group). The disease profile was monitored daily for 21 days and
161
was scored on a 0-6 composite scale based on the severity of vaginitis and neurological
162
symptoms according to the following criteria: Score 0: no signs of infection
164
Score 1: localized, barely perceptible small vesicles
165
Score 2: small or large vesicles involving 10% to 50% of the area
166
Score 3: small or large vesicles involving 50% to 100% of the area
167
Score 4: small ulcers involving 10% to 50% of the area
168
Score 5: severe ulcers involving 50% to 100% of the area
169
Score 6: hind limb paralysis or death
170
The scores of genital herpes in guinea pigs were determined daily. In the PK/PD analysis,
171
the scores on Day 3 were used as the baseline scores to make the modeling easier because of
172
no lesion symptoms until Day 3 after virus allocation. We assumed that disease did not alter
173
the PK of amenamevir.
174
Lesion score measurement in humans
TE D
M AN U
SC
RI PT
163
The data for times to healing of all lesions were obtained as the primary efficacy endpoint
176
of Study CL-101 [6]. Genital herpes recurrence was defined as herpes recurrence below the
177
umbilicus and above the knees. Times to healing (h) of all lesions were determined by the
178
study investigator as the times from therapy initiation to re-epithelialization of all lesions,
179
excluding aborted lesions. Aborted lesions were defined by the presence of prodromal
180
symptoms including pain, tingling, itching, and burning, but lesions failed to develop beyond
181
the macule/papule stage to the vesicular/ulcerative stage. Healed lesions were defined as the
182
absence of crusts, depressions, erosions or ulcerations. Residual erythema in absence of the
183
preceding is defined as healed. Existing symptom lesions were classified by the site clinician
184
during the genital examinations on Days 1 - 6, and also on Days 8 and 10 only if lesions had
AC C
EP
175
8
ACCEPTED MANUSCRIPT not healed by Day 6 and Day 8, respectively. If a patient had existing symptom lesions at the
186
visit on Day 17, an additional genital examination for lesion classification was performed.
187
The classification of the existing symptom lesions were categorized in the similar way for
188
guinea pig as follows for a purpose of logit analysis in this study: Score 0: healed lesion
190
Score 1: crust
191
Score 2: vesicle / pustule / ulcer
192
Score 3: macule / papule
SC
189
RI PT
185
The changes of the scores are unidirectional in the order of 0, 3, 2, 1 and 0. Patients
194
whose scores were maintained to be 0 during the study period were treated as aborted lesions.
195
Mean time course profiles of observed lesion scores are shown in Fig. 1a for guinea pigs and
196
Fig. 1b for humans. The present study was conducted in accordance with the ethical
197
principles stated in the Declaration of Helsinki, Good Clinical Practice, International
198
Conference on Harmonization guidelines, and applicable laws and regulations.
TE D
M AN U
193
199
201
Analysis models and simulation data
The analysis conditions in this study are described briefly in this section.
EP
200
Population modeling
203
Logit model analysis was performed via the non-linear mixed effects model [10] using
204
NONMEM Version VI Level 1.0 (Icon Clinical Research, North Wales, PA, USA).
205
Graphical processing of the NONMEM output was performed with SAS Version 8.2, Release
206
8.02 (SAS Institute Inc., USA). Model selection was based on the goodness-of-fit criteria
207
(log-likelihood difference (l.l.d.) calculated as a difference of objective functions) with visual
208
inspection of the diagnosis plots. A value of l.l.d. more than 3.84 between two models with a
209
1 degree of freedom difference was considered to be significant (p<0.05). First-order
AC C
202
9
ACCEPTED MANUSCRIPT 210
conditional estimation with interaction (FOCE-INTERACTION) [11] was used in
211
NONMEM execution.
212 Model validation
214
Visual predictive check [12] was performed using the final models for guinea pig PK and
215
virus plaque profiles, where 1,000 hypothetical amenamevir concentrations and the numbers
216
of virus plaques were simulated at each time point. The median and 95% prediction interval
217
were constructed from the resulting predictions at each time point. Plots overlaying the
218
medians, prediction interval with observed amenamevir concentrations or observed the
219
numbers of virus plaques were created.
M AN U
220
SC
RI PT
213
PK model in guinea pigs
222
We used a linear single-compartment model with an absorption lag time and first-order
223
absorption as a guinea pig PK model by visual inspection of the observed data. The
224
pharmacokinetic parameters of ka (absorption rate constant), V/F (apparent distribution
225
volume) and CL/F (apparent total clearance) were defined, where F is the oral bioavailability
226
fraction. Mixed-effect modeling was applied using NONMEM, where log-normal distribution
227
for the inter-individual variability was assumed for each pharmacokinetic parameter, in
228
Equation 1:
AC C
EP
TE D
221
229
Pj = θ j ⋅ exp(ηj)
230
where Pj is a parameter of interest in jth subject, θj is the population mean of the
231
corresponding parameter, ηj is the random variable which gives the inter-individual
232
variability of Pj from θj; ηj was assumed to be normally distributed with a mean 0 and a
233
variance ω2 . Residual error was assumed to be described by a proportional error model
234
given by Equation. 2;
Eq. 1
10
ACCEPTED MANUSCRIPT 235
Yij = Cij (1 + εij)
Eq. 2
236
where Yij is the ith observed concentration in jth subject, Cij is the predicted concentration
237
by the model, and εij was the random variable for residual error which is assumed to be
238
normally distributed with a mean 0 and a variance σ 2 .
RI PT
239 PK model in humans
241
PK model analysis of amenamevir in patients with genital herpes had already been performed
242
using a single-compartment model with first-order absorption[7]. As there were some
243
extremely low concentration data points (LCPs) during the absorption phase in the plasma
244
concentration data, another first-order absorption rate constant (ka,LCP) was used in addition to
245
ka to explain the profile of these LCPs. On NONMEM analysis, the estimated mean
246
(and %RSE) values of apparent clearance (CL/F) was found to be 13.8 (4.28%) L/h, apparent
247
distribution volume (V/F) was 143 (4.31%) L, absorption rate constant (ka) was 0.874
248
(15.70%) h-1, and the absorption rate constant for LCP (ka,LCP) was 0.00107 (63.30%) h-1.
249
Relative bioavailability (assuming a bioavailability of 1.0 at 100 mg dose) at a dose of
250
200 mg was 0.982 (6.02%), 400 mg was 0.874 (5.74%), and 1200 mg was 0.706 (6.15%).
M AN U
TE D
EP
251
SC
240
Simulation of PK profiles in guinea pigs and humans
253
Simulations of plasma amenamevir profiles in guinea pigs and humans were conducted using
254
the population mean PK parameters for guinea pigs and the post-hoc PK parameter estimates
255
for humans to simulate time course profiles of the numbers of virus plaques in those species.
256
The data point treated as LCP were not included in the simulation processes in this study
257
because our modeling method using LCP concept was to explain the outliers (i.e. LCP) and
258
obtain a better estimate for Ka using non-LCP data only. The numbers of LCP data point was
259
33 in totally 928 points (3.56% total), and we think the effect of omitting the LCP on
AC C
252
11
ACCEPTED MANUSCRIPT 260
simulation results is negligible. Time-courses of simulated concentration are shown in
261
Figures 5a and 6a, and simulation conditions were as follows: Guinea pig = 0, 1, 3, 10, and
262
30 mg/kg BID for 5 days;
263
Human = 0, 100, 200, and 400 mg QD for 3 days or 1200 mg as a single dose.
RI PT
264 PD model for virus plaque
266
A two-compartment model was applied to explain the concentration and time-dependent
267
changes in the number of virus plaques, as shown in Figure 2. A virus cycle was incorporated
268
by defining the amenamevir effective/non-effective compartments, which are connected by
269
first-order rate constants kinact and kact [13]. Increase of virus plaque was assumed as the
270
first-order rate constant (kin), it was fixed to 0.0569 h-1 (= ln(60) / 72), which gives the
271
number of virus plaques to be 60 pFU at 72 h based on the study setting. A non-linear drug
272
effect was assumed using the Michaelis-Menten form with maximum drug effect (Emax) and a
273
Michaelis constant (EC50).
TE D
M AN U
SC
265
The mass-balance equations for the number of virus plaques are given as follows.
275
= k × V1 − k × V2 + k × V3 −
Eq. 3
276
= k × V2 − k × V3
Eq. 4
277
In Eq. 3-4, V(1) represents the number of virus plaques in a hypothetical input
278
compartment, V(2) is the amenamevir effective compartment, V(3) is the amenamevir
279
ineffective compartment; the initial value of V(1) is 60, while those of V(2) and V(3) are 0.
280
Plasma concentration of amenamevir is given by CP. No inter-individual variability was
281
assumed in this case and an additive error model for residual variability was assumed as
282
given in Equation 5;
283
Yij = Cij + εij
×
AC C
EP
274
Eq. 5
12
ACCEPTED MANUSCRIPT 284
where Yij and Cij are the observed and model predicted numbers of plaques for the ith
285
sample in jth subject, εij is the residual error which is assumed to be normally distributed with
286
a mean 0 and a variance σ 2 .
287 Simulation of virus plaque profile
289
Simulations of virtual numbers of virus plaque profiles in guinea pigs and humans were
290
conducted using the simulated amenamevir concentration and the estimated virus kinetic
291
parameters. Simulation settings were as follows.
SC
RI PT
288
- Initial administration amount of number of virus plaques was set to 60 pFU
293
- Time of virus plaque administration was defined as Day 0 (0 h)
294
- Starting time of amenamevir administration was Day 1 (24 h)
295
Lesion scores in human on Day 1 were regarded as the initial scores, this is because the
296
Phase 2 study (CL-101) was designed as the a self-initiation study, where patients
297
self-initiated treatment and returned to the clinic within 24 h after the initial dose of study
298
drug (Day 1). Time-course of virus plaques is shown in Figures 5b and 6b.
299
TE D
M AN U
292
PK/PD Model
301
Logit model analysis of lesion score
AC C
EP
300
302
Logit model analysis was applied to the ordered-categorical lesion score data. Let Yijk =
303
Y(tijk) be the lesion score at the kth time point tijk, in the ith individual of the jth treatment group,
304
and Yijk takes the values of 0, 1, 2, 3, or 4 in the guinea pig study, and 0, 1, 2, or 3 in the
305
human study. The scores 5 and 6 were not included in the analysis for guinea pig because of
306
no observed data for these scores. A logit model for the probability of having a lesion score
307
Yijk that was equal to or less than a given score yijk = 0, 1,.., n can be expressed as a
308
cumulative distribution function as follows [14,15].
13
ACCEPTED MANUSCRIPT 309 310 311
Fy; θ, η = Pr&Y ≤ y; θ, η) =
*+,-./;0,12
3 *+,-./;0,12
Eq. 6
where θ is a vector of parameters and η is a random variable. Based on the cumulative probability function, the probability of having a single lesion score was given by Equation 7: Pr&Y = y) = Pr&Y ≤ y) − Pr&Y ≤ y − 1),
313
where Pr&Y < 0) = 0 and Pr&Y ≤ n) = 1.
314
The logit, g(yijk; θ, η) was defined as follows:
315
gy; θ, η = ∑ :<3 θ: × Q: y + Eff
316
for yijk = n, where Q: ?y @A B = C
317
In guinea pigs, lesion scores increased almost monotonically in the placebo group,
318
however it began to decrease after Day 2 in amenamevir groups and clear dose dependence
319
was observed as shown in Figure 1a. In contrast, lesion scores in humans decreased
320
monotonically over time in both amenamevir and placebo groups, and dose dependence was
321
not clear as shown in Figure 1b. These data suggest that the decrease in lesion scores could
322
not be explained only by the changes of virus plaques reflecting healing of amenamevir
323
(Virus Plaque component) and thus the elapsed time reflecting the healing of the immune
324
response (Time component) was included into the model in this study. For the term of
325
describing drug efficacy (Eff), following model was tested. The term Eff was expressed by
326
the addition of Virus Plaque component and Time component.
RI PT
312
AC C
EP
TE D
M AN U
1, y @A ≥ m − 1 . 0, otherwise
SC
Eq. 7
327
Eff = β3 × Virus Plaque + β × Time+η
328
In these models, number of virus plaques (given by ‘Virus Plaque’) and the elapsed time
329
from virus plaque increase (given by ‘Time’) were incorporated as fixed effects. This model
330
was built theoretically, no covariates step was conducted. Maximum Likelihood Estimation
331
was used for the fitting and posterior prediction, and individual estimated lesion scores for ith
Eq. 8
14
ACCEPTED MANUSCRIPT 332
subject at time t (Lesion scorei,t) were calculated based on the estimated parameters by
333
following equation. Lesion score , = ∑ :
335
Results
336
PK model in guinea pigs
RI PT
334
As shown in Figure. 3, result of VPC shows that most of the observed values were within
338
95% prediction interval. Estimated population mean parameters of the PK model for guinea
339
pig PK are summarized in Table 1.
SC
337
340 PD model for virus plaques
342
Estimated population mean parameters of the PD model for the virus plaque profiles are
343
summarized in Table 1. The observed plaque counts versus amenamevir concentration are
344
plotted with the 95% prediction interval in Figure 4. The estimated EC50 was 127 ng/mL,
345
which is a little smaller than the in vitro expected effective concentration of 200 ng/mL [5].
346
As the result, in vitro anti-virus effect of amenamevir was consistent with the virus kinetic
347
model.
348
Logit model for lesion score
TE D
EP
Final models for the term logit lesion score in guinea pigs and humans are obtained as
AC C
349
M AN U
341
350
follows:
351
Guinea pig:
For Lesion score =
0; Logit = 6.76 + Eff.[ * , . 1; Logit = 6.76 + 3.11 + Eff.[ * , . = 9.87 + Eff.[ * , . 2; Logit = 6.76 + 3.11 + 4.17 + Eff.[ * , . = 14.04 + Eff.[ * , .
15
ACCEPTED MANUSCRIPT 3; Logit = 6.76 + 3.11 + 4.17 + 5.89 + Eff.[ * , . = 19.93 + Eff.[ * , . 352 353
Where Eff.[ * , . = −0.265 × Virus Plaque − 0.0334 × Time
Eq.10
Human:
0; Logit = −5.21 + Eff`[:
1; Logit = −5.21 + 1.86 + Eff`[: = −3.35 + Eff`[:
RI PT
For Lesion score =
2; Logit = −5.21 + 1.86 + 3.34 + Eff`[: = −0.01 + Eff`[:
356
SC
355
Where Eff`[: = −0.0247 × Virus Plaque + 0.0424 × Time
Eq.11
In these models, a beneficial effect means a decrease of lesion scores while a worsening
M AN U
354
effect means an increase of lesion scores.
While the fixed effect in Eff of Virus Plaque was negative in both species, the fixed
358
effect in Eff of the Time component by the immune system was negative in guinea pigs but
359
positive in humans. In guinea pigs, both the Virus Plaque and Time components worsened the
360
lesion score. In humans, Virus Plaque worsened the lesion score, whereas immune system
361
improved. Estimated profiles of mean lesion scores were consistent with the observed values
362
in both species (Fig. 5(c) and Fig. 6(c)). Some simulations were performed to show the
363
probabilities above each lesion score in guinea pig (Fig. 5(d)-(g)) and in human (Fig. 6(d)-(f)),
364
where dose and time dependent profiles are shown. Fitting results are summarized in Table 2,
365
and no 95% confidence intervals (CIs) for any fixed effects included 0, indicating that all
366
parameters were significant.
367
Discussion
AC C
EP
TE D
357
368
In the present study, an empirical PK/PD model, as shown in Figure 2 for the
369
helicase-primase inhibitor in genital herpes patients was developed. In this model, the time
370
course profiles of lesion scores was not directly dependent on the amenamevir PK, but it was
16
ACCEPTED MANUSCRIPT 371
mainly dependent on the virtual number of virus plaques which suggests a time-dependent
372
anti-virus mechanism of amenamevir. The PK analysis of amenamevir concentration in guinea pig suggested liner PK profile as
374
shown in Table 1 and Figure 3. In contrast, the CL/F estimated in our previous study in
375
humans [7] suggested dose-dependent bioavailability as F decreased as dose increased. A
376
possible reason is the difference of the dosage form between the species; amenamevir was
377
administered in methylcellulose solution to guinea pigs and in tablet form to humans.
378
Amenamevir is poorly soluble, which strongly affects its passive diffusivity (data not shown).
379
When an allometric scaling [16] was applied, CL/F in humans was estimated about 0.16
380
L/h/kg, smaller than in guinea pig (2.04 L/h/kg) although the reason of the difference
381
between species was unclear. We focused on the relationship between amenamevir
382
concentrations and virus plaque data in the present study.
M AN U
SC
RI PT
373
We used the virtual number of virus plaques obtained via plaque reduction assay as a
384
marker to explain amenamevir PK/PD mechanism. Goodness of fit of the virus plaque
385
modeling were not enough acceptable especially in the higher concentration range at 6 or 8
386
hrs exposure. Several models to explain this, i.e. some models including not only the time
387
dependent component but also the dose (concentration) dependent component were tested to
388
try to improve the modeling result, however, no clear improvement was obtained(Data not
389
shown). We could not yet find the reason of this discrepancy at the higher concentration, but
390
we conclude that the current model is sufficient to simulate the efficacy of twice a day or
391
daily dosing of amenamevir because some acceptable result was obtained in the condition of
392
24hrs exposure which showed the threshold concentration to maintain the amenamevir
393
efficacy throughout the day (Fig.4c). In this our analysis, the virus plaque time profile in vivo
394
was simulated based on the virus plaque kinetic model, which was built based on in vitro data.
395
Several points remain unclear, however—namely the utility of the same viral kinetic model
AC C
EP
TE D
383
17
ACCEPTED MANUSCRIPT 396
between species, whether or not plasma concentration is the best surrogate marker in these
397
species, and differences in amenamevir efficacy for viral kinetics between species. The categories for lesion scores in human were not determined in the clinical study
399
protocol, and we originally defined four categories as shown in the present study. Scores in
400
humans showed monotonical change, i.e. the scores started from 3 and decreased along with
401
the lesion healing. Assuming that most patients showed lesion healing without recurrence and
402
therefore the lesion scores tended to monotonically decrease, the definition of lesion scores
403
seem acceptable for modeling purposes. We did not include data from patients whose lesion
404
scores remained 0 throughout the study period (aborted lesion) in the model analysis because
405
for a patient whose lesion score was 0 during the study, as we were unable to determine
406
whether or not the lack of any symptoms was due to the drug’s effects. Therefore, the PD
407
model of the present analysis was built only for patients who developed the symptoms.
M AN U
SC
RI PT
398
Finally, the lesion scores in guinea pig and human could be explained by the similar
409
models including with two fixed-effect parameters, i.e. amount of virus plaques and the
410
elapsed time. Our previous study regarding virus plaques showed that the continual exposure
411
of amenamevir above a certain concentration is necessary to prevent the virus re-production
412
[5], and this was confirmed by another study with multiple dose design which is usually used
413
in the antibiotics area [17]. During the clinical development of amenamevir, the value for
414
EC50 obtained in the non-clinical studies could be directly extrapolated into the clinical study
415
and is used for the dose rationale [5,6] As results, the non-clinical EC50 without a cure effect
416
was under-estimated, we were unable to detect a clear dose relationship in the clinical study.
AC C
EP
TE D
408
417
In the present study, we developed similar PK/PD models in both guinea pigs and
418
humans with Virus Plaque and Time components to explain the time-course profiles of lesion
419
scores. A virtual kinetic profile of virus plaque was incorporated into the model in order to
420
connect the PK profile of amenamevir with the lesion score profiles, and the terms in the
18
ACCEPTED MANUSCRIPT 421
logistic model consisting of the number of virus plaques and the elapsed time well explained
422
the dose- and time-dependent PD profiles. These results suggest that the virtual number of
423
virus plaques can be used as a built-in biomarker. While the fixed effect in Eff of Virus Plaque was negative in both species, the fixed
425
effect in Eff of the Time component for the immune system was negative in guinea pigs but
426
positive in humans. HSV-2 damages the central nervous system, which in turn affects the
427
immune system [18]. Our present findings suggest that the immune system might be
428
weakened by virus infection in guinea pigs, although evidence for this is insufficient at
429
present. The differences in results for the Time Component between species may have been
430
due to the different experimental conditions and different responses of immune systems. In
431
the guinea pig study, animals were infected with a lethal amount of HSV to ensure herpes
432
infection, and lesion severity increased with time. In humans, the immune system may work
433
adequately to reduce lesion severity.
M AN U
SC
RI PT
424
The effect of the Virus Plaque component was deemed to be large in guinea pigs, as
435
efficacy was clearly dose-dependent in the amenamevir groups while the effect was saturated
436
in the placebo group. In contrast, the effect of the Virus Plaque component was relatively
437
small in humans, and the drug effect for lesion scores was smaller than in guinea pigs.
EP
TE D
434
In humans, the PD effect was almost dose-independent, and immune system-related
439
healing was likely the driving force behind reductions in lesion scores. These findings
440
suggest that the drug effect may be masked in diseases healed by the immune response, such
441
as genital herpes. Therefore, the PK/PD model proposed in the present study will be
442
particularly useful for explaining the PK/PD relationship of drugs used to treat self-cured
443
diseases. In antibiotics and antiviral drug kinetic analyses, drug-bacteria (or virus) interaction
444
is assumed to be independent of in vivo conditions, such as host species. Here, we assumed
445
that the kinetic parameters for virus plaque data obtained in in vitro experiments could be
AC C
438
19
ACCEPTED MANUSCRIPT applied to both guinea pigs and humans. In addition, as a practical problem, we cannot obtain
447
the virus kinetic data in human and the difference of it between the species is difficult to be
448
evaluated.
449
In the development of drugs for diseases with natural healing, if healing does not happen in
450
the animal disease model, its efficacy may differ from clinical efficacy.
451
In the non-clinical studies from the perspective of the prediction of efficacy, animal model
452
without natural healing which can confirm the drug power clearly is suitable, however, it may
453
misjudge the clinical endpoint. For example, even when the development of animal models,
454
natural healing should be considered.
M AN U
455
SC
RI PT
446
Conclusions
457
This PK/PD modeling approach based on bi-directional translational approach is useful for
458
not only new candidate exploration in the non-clinical stage but also further application in
459
clinical data analysis. We believe that this kind of modeling and simulation approach will
460
give some suggestions especially as a unique PK/PD modeling approach connecting the
461
non-clinical and clinical data during the HSV drug development.
EP
462
TE D
456
Conflicts of interest
464
Financial support for this study was provided by Astellas Pharma Inc. The authors report no
465
conflicts of interest.
466
AC C
463
20
ACCEPTED MANUSCRIPT 467 References:
469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514
1. Stanberry L, Cunningham A, Mertz G, Mindel A, Peters B, Reitano M, Sacks S, Wald A, Wassilew S, Woolley P (1999) New developments in the epidemiology, natural history and management of genital herpes. Antiviral Res 42 (1):1-14. doi:S0166-3542(99)00004-2 [pii] 2. Snoeck R, De Clercq E (2002) New treatments for genital herpes. Curr Opin Infect Dis 15 (1):49-55 3. Reddy MB, Morcos PN, Le Pogam S, Ou Y, Frank K, Lave T, Smith P (2012) Pharmacokinetic/Pharmacodynamic predictors of clinical potency for hepatitis C virus nonnucleoside polymerase and protease inhibitors. Antimicrob Agents Chemother 56 (6):3144-3156. doi:10.1128/AAC.06283-11 AAC.06283-11 [pii] 4. Chono K, Katsumata K, Kontani T, Kobayashi M, Sudo K, Yokota T, Konno K, Shimizu Y, Suzuki H (2010) ASP2151, a novel helicase-primase inhibitor, possesses antiviral activity against varicella-zoster virus and herpes simplex virus types 1 and 2. J Antimicrob Chemother 65 (8):1733-1741. doi:10.1093/jac/dkq198 dkq198 [pii] 5. Katsumata K, Chono K, Kato K, Ohtsu Y, Takakura S, Kontani T, Suzuki H (2013) Pharmacokinetics and pharmacodynamics of ASP2151, a helicase-primase inhibitor, in a murine model of herpes simplex virus infection. Antimicrob Agents Chemother 57 (3):1339-1346. doi:10.1128/AAC.01803-12 AAC.01803-12 [pii] 6. Tyring S, Wald A, Zadeikis N, Dhadda S, Takenouchi K, Rorig R (2012) ASP2151 for the treatment of genital herpes: a randomized, double-blind, placebo- and valacyclovir-controlled, dose-finding study. J Infect Dis 205 (7):1100-1110. doi:10.1093/infdis/jis019 jis019 [pii] 7. Takada A, Katashima M, Kaibara A, Sawamoto T, Zhang W, Keirns J (2014) Statistical analysis of Amenamevir (ASP2151) between pharmacokinetics and clinical efficacies with non-linear effect model for the treatment of Genital Herpes. Clin Pharmacol Drug Dev. doi:DOI: 10.1002/cpdd.108 8. Katsumata K, Chono K, Sudo K, Shimizu Y, Kontani T, Suzuki H (2011) Effect of ASP2151, a herpesvirus helicase-primase inhibitor, in a guinea pig model of genital herpes. Molecules 16 (9):7210-7223. doi:10.3390/molecules16097210 molecules16097210 [pii] 9. Miller RL, Imbertson LM, Reiter MJ, Gerster JF (1999) Treatment of primary herpes simplex virus infection in guinea pigs by imiquimod. Antiviral Res 44 (1):31-42. doi:S0166-3542(99)00052-2 [pii] 10. Beal L, Sheiner B (1989) NONMEM Users Guide NONMEM Project Group, UCSF. 11. Wang Y (2007) Derivation of various NONMEM estimation methods. J Pharmacokinet Pharmacodyn 34 (5):575-593. doi:10.1007/s10928-007-9060-6 12. Post TM, Freijer JI, Ploeger BA, Danhof M (2008) Extensions to the visual predictive check to facilitate model performance evaluation. J Pharmacokinet Pharmacodyn 35 (2):185-202. doi:10.1007/s10928-007-9081-1 13. Lobo ED, Balthasar JP (2002) Pharmacodynamic modeling of chemotherapeutic effects: application of a transit compartment model to characterize methotrexate effects in vitro. AAPS PharmSci 4 (4):E42. doi:10.1208/ps040442 14. Sheiner LB (1994) A new approach to the analysis of analgesic drug trials, illustrated with bromfenac data. Clin Pharmacol Ther 56 (3):309-322
AC C
EP
TE D
M AN U
SC
RI PT
468
21
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
15. Liu CY, Sambol NC (1999) Pharmacodynamic analysis of analgesic clinical trials: nonlinear mixed-effects logistic models. J Biopharm Stat 9 (2):253-270. doi:10.1081/BIP-100101175 16. Holford N, Heo YA, Anderson B (2013) A pharmacokinetic standard for babies and adults. J Pharm Sci 102 (9):2941-2952. doi:10.1002/jps.23574 17. Frimodt-Moller N (2002) How predictive is PK/PD for antibacterial agents? Int J Antimicrob Agents 19 (4):333-339. doi:S0924857902000298 [pii] 18. Black PH (1994) Central nervous system-immune system interactions: psychoneuroendocrinology of stress and its immune consequences. Antimicrob Agents Chemother 38 (1):1-6
AC C
515 516 517 518 519 520 521 522 523 524 525 526
22
ACCEPTED MANUSCRIPT Tables Table 1 Estimated Population parameters of amenamevir in a guinea pig PK model and Virus Plaque PD model
PD model for Virus Plaque （Common parameters in Guinea pigs and humans） ）
95% CIb (Lower – Upper) 1.93 – 2.15 3.05 – 4.73 1.02 – 2.50 0.154 – 0.204 5.9 – 14.0 8.61 – 18.6 9.2 – 28.9 5.3 – 12.7 13.6 – 45.4 112 –246 – 0.764 – 0.984 65.3 – 189 7.0 – 10.2
RI PT
CL/F (L/h/kg) V/F (L/kg) ka (h-1) Lag Time (h) ηCL/F (CV%) ηka (CV%) ηLagTime (CV%) εc kinact (h-1) kact (h-1) kin (h-1) Emax (pFU) EC50 (ng/mL) εc
Estimate (%RSEa) 2.04 (2.76%) 3.89 (11.1%) 1.76 (21.4%) 0.179 (7.15%) 10.7 (35.3%) 14.5 (33.1%) 21.4 (41.6%) 9.76 (35.9%) 29.5 (27.6%) 179 (19.0%) 0.0569 (Fixed) 0.874 (6.40%) 127 (24.8%) 8.75 (18.7%)
SC
PK model for Guinea pig
Parameter
M AN U
Model
AC C
EP
TE D
CI: confidence interval, CL/F: oral clearance, V/F: volume of distribution, ka: absorption rate, η: inter-individual variability, ε: intra-individual variability, kinact: inactivation ratio , kact: activation ratio, kin: increase ratio , Emax: maximum drug effect, EC50: Michaelis constant a: %RSE is percent relative standard error (100% × Standard Error / Estimate) b: 95% CI = T ± 1.96 × Standard Error c: given as standard deviation
ACCEPTED MANUSCRIPT Table 2 Estimated parameters of the logistic regression model for lesion score in guinea pigs and humans
θScore=0 θScore=1 θScore=2 θScore=3 ηc
RI PT
β2
Estimate (%RSEa) -0.0247 (29.5%) 0.0424 (6.0%) -5.21 (6.8%) 1.86 (7.3%) 3.34 (6.0%)
Human 95% CIb (Lower – Upper) -0.0390, -0.0104 0.0374, 0.0474 -5.90, -4.52 1.60, 2.12 2.94, 3.74
SC
β1
Estimate (%RSEa) -0.265 (10.6%) -0.0334 (14.6%) 6.76 (15.4%) 3.11 (13.2%) 4.17 (11.2%) 5.89 (12.4%) 1.62 (31.7%)
M AN U
Parameter
Guina pig 95% CIb (Lower – Upper) -0.320 -0.210 -0.0430, -0.0238 4.72, 8.80 2.30, 3.92 3.25, 5.09 4.46, 7.32 0.996, 2.07
1.36 (16.2%)
1.12, 1.56
AC C
EP
TE D
CI: confidence interval, β1: effect of virus plaque, β2: time effect for healing, θScore=x: logit value for score x, η: inter-individual variability a: %RSE is percent relative standard error (100% × Standard Error / Estimate) b: 95% CI = T ± 1.96 × Standard Error c: given as standard deviation
ACCEPTED MANUSCRIPT Figure captions Figure 1: Time course profiles of mean observed lesion scores in guinea pigs (a) and humans (b). (a) Closed circle: Placebo, open triangle: 1 mg/kg, closed triangle: 3 mg/kg, open square: 10 mg/kg, closed square: 30 mg/kg; treatment duration was 5 days from Day 1.
RI PT
(b) Closed circle: Placebo, open triangle: 100 mg, closed triangle: 200 mg, open square: 400 mg, closed square: 1200 mg; treatment duration was 3 days from Day 1.
SC
Figure 2: Overview of the PK/PD model
M AN U
Figure 3: Results of population PK modeling and visual predictive check in guinea pigs. (a) Dose = 0.3 mg/kg, (b) 1.0 mg/kg, (c) 3.0 mg/kg. Solid line: median, filled region: 95% prediction interval.
TE D
Figure 4: Results of population PD modeling and visual predictive check for virus plaque data. (a) Amenamevir duration time = 6 h, (b) 8 h, (c) 24 h. Solid line: median, filled
EP
region: 95% prediction interval.
AC C
Figure 5: Results of model predicted time course profiles in guinea pigs. Closed circle: Placebo, open triangle: 1 mg/kg, closed triangle: 3 mg/kg, open square: 10 mg/kg, closed square: 30 mg/kg; treatment duration was 5 days from Day 1. (a), (b) Simulated time-course profiles of plasma concentration and virus plaque. (c) Observed (plots) and model-predicted (lines) time-course profiles of lesion scores in guinea pigs. Predictions are given as surface of lesion scores (z-axis) as a function of time (x-axis) and dose (y-axis). Symbols show the observed values. (d) to (g) Predicted probability surfaces for lesion scores (z-axis) as a
ACCEPTED MANUSCRIPT function of day (x-axis) and dose (y-axis) in guinea pigs. (d) Pr{Y>=1}, (e) Pr{Y>=2}, (f) Pr{Y>=3}, (g) Pr{Y=4}.
Figure 6: Results of model predicted time course profiles in humans. Closed circle:
RI PT
Placebo, open triangle: 100 mg, closed triangle: 200 mg, open square: 400 mg, closed square: 1200 mg; treatment duration was 3 days from Day 1. (a), (b) Simulated time-course profiles of plasma concentration and virus plaque. (c) Observed (plots) and model-predicted (lines)
SC
time-course profiles of lesion scores in humans. Predictions are given as surface of lesion scores (z-axis) as a function of time (x-axis) and dose (y-axis). Symbols show the observed
M AN U
values. (d) to (f) Predicted probability surfaces for lesion scores (z-axis) as a function of day
AC C
EP
TE D
(x-axis) and dose (y-axis) in human. (b) Pr{Y>=1}, (c) Pr{Y>=2}, (d) Pr{Y=3}.
ACCEPTED MANUSCRIPT Figure 1
M AN U
SC
RI PT
a)
AC C
EP
TE D
b)
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figure 2
ACCEPTED MANUSCRIPT Figure 3
SC
RI PT
a)
EP AC C
c)
TE D
M AN U
b)
ACCEPTED MANUSCRIPT Figure 4
SC
RI PT
a)
EP AC C
c)
TE D
M AN U
b)
ACCEPTED MANUSCRIPT Figure 5 a)
SC
RI PT
b)
EP AC C
d)
TE D
M AN U
c)
e)
ACCEPTED MANUSCRIPT g)
AC C
EP
TE D
M AN U
SC
RI PT
f)
ACCEPTED MANUSCRIPT Figure 6 b)
SC
RI PT
a)
AC C
d)
EP
TE D
M AN U
c)
e)
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
f)