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Echocardiography in nonclinical studies: Where are we?
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Regulatory Toxicology and Pharmacology
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Please cite this article as: Hausner, E.A., Chi, X., Echocardiography in nonclinical studies: Where are we?, Regulatory Toxicology and Pharmacology (2020), doi: https://doi.org/10.1016/j.yrtph.2020.104615. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Inc.
Echocardiography in Nonclinical Studies: Where are we? Elizabeth A. Hausner* and Xuan Chi Running title: Nonclinical Echocardiography Division of Cardiovascular and Renal Products, Center for Drug Evaluation and Research (CDER), United States Food and Drug Administration (FDA), USA *
Corresponding author
Elizabeth A. Hausner 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 [email protected] Telephone: 301-796-1084 Fax: 301-796-9841 Word count Abstract: 178 Text: 3172 References: 10 references, 330 words
Hausner and Chi, Regulatory Echocardiography
2|Page
Hausner and Chi, Regulatory Echocardiography
Echocardiography in Nonclinical Studies: Where are we? Elizabeth A. Hausner and Xuan Chi
Keywords: echocardiography, nonclinical, reproducibility, regulatory, cardiovascular
3|Page
1
Abstract
2
Echocardiography is a powerful, noninvasive tool used both in clinical and nonclinical
3
settings, including in drug development. When used appropriately, it can provide valuable
4
translational information about pharmacodynamics, safety pharmacology, or toxicology,
5
helping to define no-observed-adverse-effect levels and providing guidance for clinical
6
monitoring and dose selection. Echocardiography is advantageous in conducting longitudinal
7
studies and reducing the number of animals used in safety assessments. To this end, there has
8
been no clear enunciation of what constitutes appropriate use of this imaging technology in a
9
nonclinical drug development setting. In this review, we describe the use of echocardiography
10
in nonclinical studies in regulatory submissions to the US Food and Drug Administration
11
Center for Drug Evaluation and Research. In addition, we discuss three main areas: the
12
operator, image acquisition, and image analysis, where variability may affect the reliability of
13
information generated in an echocardiography study. As a path forward, our recommendation
14
is for a multi-disciplinary expert working group to establish guidelines for education and
15
credentialing of nonclinical echocardiographers as well as quality assurance standards for
16
nonclinical echocardiography labs.
17
Keywords: echocardiography, translational, cardiovascular, safety, toxicology
18
19
20
21
Hausner and Chi, Regulatory Echocardiography 22 23
1.
Introduction
24
Echocardiography has evolved into a powerful noninvasive tool for both clinical medicine and
25
research. Nonclinical echocardiography is an area in need of input from the pharmaceutical
26
industry, contract research organizations, and regulatory agencies in a collaborative manner to
27
develop a standardized approach to image attainment, data collection, and to the extent
28
possible, interpretation of results. In this manuscript, we discuss the parameters we consider
29
important for translational use of echocardiography in nonclinical drug development studies,
30
based on our experience with nonclinical echocardiography data. In regulatory submissions,
31
most nonclinical echocardiography data are generated to demonstrate either an efficacy or
32
safety endpoint, including cardiac contractility, cardiac wall function, and blood flow and
33
valve performance.
34
The advantages nonclinical echocardiography offers include dynamic, longitudinal monitoring
35
of both functional and morphological changes, ease of use, and relatively low cost. Image
36
acquisition, measurement or data collection, and interpretation are operator-dependent with a
37
potential for significant intra- and inter-observer variability. This variability along with
38
relatively low spatial resolution as compared to histopathology contributes to potentially
39
relatively low sensitivity of echocardiography to detect functional and morphological cardiac
40
changes in nonclinical studies. The degree of change that can reliably be detected may depend
41
upon the species, homogeneity of the subjects, sonographic equipment and analytical skill as
42
well as the parameter examined. An important question to be considered during our review is
43
- what standards should be applied to echocardiography data to optimize reproducibility, data
44
rigor, and translational value? Moreover, what is the framework of training, design and
45
conduct of studies, analysis, and reporting, that defines an informative, reliable study? The 6|Page
Hausner and Chi, Regulatory Echocardiography 46
“design of studies” includes components such as how many prestudy exams,logistics of exams
47
including timing relative to pharmacokinetics parameters, how often and at what time points
48
should echocardiography data be collected. Design of studies, is highly dependent on the
49
overall goals for the specific study and is beyond the scope of this manuscript.
50 51
2.
How has echocardiography been used in nonclinical studies to date?
52
We searched the US Food and Drug Administration (FDA) Center for Drug Evaluation and
53
Research (CDER) electronic document archival database, which houses all CDER electronic
54
reviews and correspondence for drugs and biologics in clinical development since 2001. Our
55
search consisted of several steps. We identified nonclinical reviews that reported or discussed
56
the use of echocardiography. These reviews were retrieved and separated into those that
57
assessed nonclinical echocardiography and those that referenced clinical use. For reviews of
58
nonclinical echocardiography, we retrieved original study reports to better understand the
59
utility of echocardiography in regulatory nonclinical studies. With the implementation of the
60
Standard for Exchange of Nonclinical Data (SEND) and data warehouse tools, future
61
echocardiography data in safety pharmacology or toxicology studies may be modeled in
62
SEND. These standardized echocardiography data will be more amenable to cross-study
63
analyses.
64
2.1
Results of our search
65
We sorted Investigational New Drug (IND) applications involving nonclinical
66
echocardiography studies by CDER review divisions. The submissions were distributed across
67
twelve review divisions with the Division of Cardiovascular and Renal Products (DCRP) and
68
the Division of Metabolic and Endocrinology Products (DMEP) receiving the largest number 7|Page
Hausner and Chi, Regulatory Echocardiography 69
of submissions (Table 1). In addition, we categorized studies by the following types: support of
70
pharmacodynamic evaluation, cardiovascular safety pharmacology, and general toxicology.
71
72
Table 1: Distribution of Nonclinical Echocardiography Studies Across CDER Review
73
Divisions and by the purpose of studies (From January, 2001through June, 2019 ) Type of Study CDER Review
# of INDs with Nonclinical
Division
Echo Reviews
Pharmacology
DCRP
24
Safety Toxicology
(Pharmacodynamics)
Pharmacology
22 (rat; dog; goat)
1 (dog)
2 (rat, dog) 15 (mice, rat, dog,
18 DMEP
2 (mouse; rat)
DAP
4
OHOP
3
DAIP
2
DBRUP
2
1 (NHP)
monkey) 4 (dog, monkey)
1 (dog and monkey)
2 (monkey) 2 (dog and monkey) 1 (dog)
DPARP
1 (NHP) 1 (juvenile NHP)
2 1 (dog) DTOP
2
DNP
2
DGIEP
1
DDDP
1
DAAAP
1
8|Page
1 (dog)
1 (rat)
1 (rat) 1 (DNP)
1 (rat) 1 (juvenile rat) 1 (dog)
Hausner and Chi, Regulatory Echocardiography 74
NHP = non-human primate; DGIEP = Division of Gastroenterology and Inborn Errors Products; DDDP =
75
Division of Dermatology and Dental Products; DBRUP: = Division of Bone, Reproductive and Urologic
76
Products; DPARP = Division of Pulmonary, Allergy, and Rheumatology Products; DAAAP = Division of
77
Anesthesia, Analgesia, and Addiction Products; DNP = Division of Neurology Products; DCRP = Division of
78
Cardiovascular and Renal Products; DMEP = Division of Metabolism and Endocrinology Products; OHOP =
79
Office of Hematology and Oncology Products; DAP = Division of Antiviral Products; DAIP = Division of Anti-
80
Infective Products; DTOP = Division of Transplant and Ophthalmology Products
81
82
Nonclinical echocardiography studies submitted to DCRP were primarily in vivo
83
pharmacology studies to show efficacy in animal models for drugs in development for cardiac
84
indications. In contrast, nonclinical echocardiography studies submitted to other divisions
85
were primarily included as safety studies to evaluate potential cardiac toxicities (cardiac
86
contractility or morphological changes). In some submissions, nonclinical echocardiography
87
data provided critical proof-of-concept information such as a dose-response relationship for
88
contractility or safety and tolerability data. These data helped determine first-in-human doses.
89
Since similar investigations and measurements can be done in laboratory animals and humans,
90
nonclinical echocardiography data can help advise the clinical monitoring plan. In our review
91
of submissions containing nonclinical echocardiography data, we observed a lack of details of
92
how these studies were conducted, thereby limiting data interpretability. As echocardiography
93
is a highly operator- and instrument-dependent modality, technical details are crucial for
94
regulatory confidence.
95
The nonclinical studies using echocardiography were primarily conducted in rodents and dogs
96
for pharmacology, and dogs and nonhuman primates (NHPs) for safety pharmacology and
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Hausner and Chi, Regulatory Echocardiography 97
toxicology, besides being conducted in pigs, goats, and sheep. Two of the studies summarized
98
in Table 1 were conducted in juvenile animals (rat and NHP).
99
2.2
Questions raised by the studies identified
100
Our search indicates that the reporting of nonclinical echocardiography for regulatory
101
purposes occurs infrequently. Variability in the frequency of nonclinical echocardiography
102
study reports submitted to specific review divisions is most likely due to the nature of the
103
therapeutic areas and the likelihood of cardiovascular involvement. Why certain divisions
104
receive echocardiography data primarily for pharmacologic support while the others receive
105
for safety assessment is beyond the scope of this manuscript.
106
Information about the technique and study design supplied in the sponsor’s reports is variable.
107
Some reports provide detailed information regarding the frequency at which studies were
108
conducted, angles of insonation, and formulas used for analysis. Other reports are
109
minimalistic and simply report the use of the technology and present summary results. This is
110
an understandable consequence of the use of a relatively new tool for which industry
111
standards and regulatory guidances have not yet been established. Currently, no regulatory
112
authority has published standards regarding the use of echocardiography in nonclinical
113
studies.
114
There are several general concerns for any new technology added to nonclinical safety
115
assessment. First, there is a learning curve for those implementing the technology and for
116
those reviewing the data. When positive controls are included in these studies, the tendency is
117
to use comparators with profound effects. Although detection of these obvious effects may be
118
somewhat reassuring, it is not indicative of more subtle effects being reliably identified. 10 | P a g e
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Second, echocardiography, a new tool in early drug development, possibly raises the bar for a
120
new drug. That is, a new drug is subjected to scrutiny not used in the past and this creates
121
more stringent expectations for a new entity. It is important to establish species-specific
122
normative data based on historical controls so that the significance of changes can be
123
interpreted. Moreover, it is critical to correlate the clinical experience from approved drugs
124
with findings from nonclinical echocardiography studies to understand what constitutes
125
acceptable background effects. Participation of veterinary cardiologists in safety assessment
126
studies may help bridge the clinical applicability of nonclinical studies and safety monitoring
127
in human clinical trials.
128
2.3
Sources of variability
129
The information available to CDER reviewers for any given nonclinical study is what is
130
provided by the sponsor, and the content and quality varies across different submissions. To
131
help determine the reliability of the echo information provided, reviewers need to know the
132
acquisition and analysis methods used. This includes information regarding who generated
133
and acquired the images, and how the analysis was conducted.
134 135
3.
The observer : who is generating the data?
136
According to the Good Laboratory Practices (GLP) (21CFR58), it is at the discretion of a
137
study director to decide if an individual involved in a GLP study has the appropriate
138
combination of education, training, and experience to perform the assigned functions. The
139
GLP regulations also direct a testing facility to maintain a current summary of the training,
140
experience, and job description for each person engaged in the conduct of a nonclinical
11 | P a g e
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laboratory investigation. This information is required to be available for audit during an FDA
142
inspection.
143
The appropriate combination of education, training, and experience for a nonclinical
144
echocardiography operator is not well defined from a regulatory standpoint. The regulations
145
do not specify credentials for various specialties involved in a nonclinical study, e.g., clinical
146
pathology or histopathology, but there are credentialing bodies for veterinary experts for these
147
and other areas. Several scientific societies have established criteria for knowledge, skills, and
148
ability in other areas of nonclinical studies (e.g. diplomates of toxicology, safety
149
pharmacology). Demonstrating competence and how best to maintain this competence may be
150
dependent on the kind of studies conducted and the species of animals studied. Improvements
151
in technology, updates to individual imaging systems, and changes in the studies (e.g.,
152
pharmacology versus safety pharmacology) all necessitate continuing education.
153
The American Society of Echocardiography (ASE) has produced several documents
154
pertaining to the initial and continuing education of a sonographer. The ASE identifies three
155
elements for demonstrating competence in a sonographer: credentialing and formal education,
156
technical competence, and continuing education. The type of training described is intended to
157
address unique imaging considerations for human subspecialty disciplines including adult,
158
pediatric, and fetal imaging. Training and credentialing are recommended for each
159
subspecialty discipline (Bierig et al. 2005). However, such educational standards have not yet
160
been defined for nonclinical echocardiography. Therefore, it is difficult to determine whether
161
someone performing echocardiography in dogs is also competent to perform similar studies in
162
mice or rats. This raises the question as to whether there should be species-specific
163
echocardiography training. The specifics of training are not straightforward. In some cases, 12 | P a g e
Hausner and Chi, Regulatory Echocardiography 164
the technical and support staff both acquire and analyze the images. Training may be formal
165
or informal i.e. one staff member instructing another. There is some question as to whether the
166
informal methods of instruction show decay of technique over time or a lessening of rigor as
167
subsequent staff members become second and third generation educators (Donner et al. 2018).
168
At present, however, there is no consensus concerning education, credentialing, and
169
continuing education for the nonclinical sector. It has been proposed that clinical standards are
170
not necessarily appropriate for the nonclinical setting. For any specialty, best practices for a
171
given context are established, beginning with appropriate education, demonstration of
172
competence (credentialing) and maintenance of competence (continuing education).
173
4.
Image acquisition
174
4.1
Intra- and interobserver variability
175
Intra-observer variability is of critical importance when operators generate or analyze images
176
or both. That is, if the same operator generates the same image or measures the same
177
parameter several times, how much will the dimensions or parameters of the image vary?
178
When more than one observer is involved, interobserver variability must also be considered.
179
Normal physiologic variability also needs to be considered. For example, the change in heart
180
rate that may spontaneously occur throughout the day or the course of an examination.
181
Intra- and interobserver variability may change depending upon the parameter e.g.,
182
dimensions measured, functions evaluated, or imaging modality used, i.e., two-dimensional
183
versus M-mode versus three-dimensional (Grune et al. 2018). Angle-dependence of certain
184
echocardiography measurements is another example illustrating the importance of assessing
185
intra-observer variability. Variation in the angle of insonation affects reliability of such
13 | P a g e
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measurements, e.g., M-mode measurement of tricuspid annular plane systolic excursion
187
(TAPSE) (Visser, Sintov, and Oldach 2018). Another consideration for intra-observer
188
variability is the use of echocardiography in longitudinal studies. A longitudinal study has the
189
advantage of following change within a single animal during a period of drug exposure, and
190
potentially, following the same animal through a drug-free recovery period. This approach
191
may reduce animal numbers while at the same time generate informative reversibility data.
192
For this to be a realistic goal, there needs to be confidence in the repeatability of
193
echocardiography measurements and an understanding of variability inherent in the
194
technology (Gentile-Solomon and Abbott 2016).
195
There is no standardized evaluation or consensus on how best to determine or report intra- or
196
interobserver variability. Acceptable levels of intra-observer variability are unknown but
197
should be outside the margin of error. For example, if a 5% difference in ejection fraction is
198
proposed as a clinical endpoint, the intra-observer variability should be significantly below
199
5%. As reported in several publications, factors influencing intra-observer variability include
200
the type of measurement, software used, and the training and experience of the operator
201
(Gentile-Solomon and Abbott 2016; Santarelli et al. 2019; Visser, Sintov, and Oldach 2018;
202
Donner et al. 2018). A sampling of published intra-observer variability is shown in Table 2.
203
None of the reports identified within the FDA electronic files noted intra-observer variability
204
or indicated that it had been assessed.
205
206
207 14 | P a g e
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Table 2: Published Intra- and Interobserver Variability Species
Parameter
Intraobserver
Interobserver
variability
variability
Reference
Healthy dogs,
2D TAPSE
4.4CV, 1.9-7.6%
9.5CV, 7.1%
Visser et. al., J Vet Cardiol
multiple
MM TAPSE
2.3CV, 1.1-7.1%
8.4CV, 5.8%
(2018) 20:165-174
TMAD
4.44CV
4.12CV
Wolf et. al. J Vet Cardiol
5.2CV
(2018) 20:175-185
breeds Healthy dogs
GLS Dogs with and
GLS:
Within day
without
transmural +
3.5 CV vendor 1
8 vendor1
Santarelli et. al. Am J Vet
cardiac disease
endocardial
5.6
3.9 independent
Res (2019) 80:347-357
Late diastolic
14.2 vendor 1
14.2 vendor1
SR
13.1 independent
17.3 independent
ESV
2.3±6.0 (mean
3.8±6.0 (mean diff±SD)
Healthy C57BL/6J mice (M-
independent
Grune 2018
diff±SD) EDV
mode)
2.1±5.8(mean
6.2±6.2(mean diff±SD)
diff±SD)
Healthy dogs
ESV
18%
11%
(3D imaging)
EDV
12%
14%
LeBlanc 2017
209
TMAD= tissue motion annular displacement; GLS=global longitudinal strain; MM= m-mode; TAPSE= tricuspid
210
annular plane systolic excursion; SR= strain rate; STE= speckle tracking echocardiography; vendor1= Vendor 1-
211
dependent software; independent= vendor-independent software; ESV= end systolic volume; EDV= end diastolic
212
volume; CV = coefficient of variation
213
4.2
Guidelines
214
Ideally, a given echocardiography study should be conducted using uniform, pre-specified
215
methodology. This is especially important if multiple laboratories are conducting the 15 | P a g e
Hausner and Chi, Regulatory Echocardiography 216
examinations and data to be compiled into one report. . However, given the variability of
217
biological windows associated with individual anatomy, different body weights and
218
conformation, and the nature of the technology, the pre-specified methodology needs to be
219
flexible. Another way to help decrease variability is the use of pre-specified image guidelines
220
as set forth by the ASE. Other guidelines, such as those of the American College of
221
Veterinary Internal Medicine, may also be useful. A set of standardized criteria for acquisition
222
and measurement helps to decrease bias associated with improvement in technique or image
223
acquisition over time as operators gain experience or familiarity with effects within a study.
224
4.3
Sedation, anesthesia, conscious state
225
Several investigators have proposed consideration of the conscious state of the animal to
226
improve reliability of echo studies (Lindsey et al. 2018). If medication is to be used to
227
facilitate the examination, sedative or anesthetic agents must be consistent and have minimal
228
or known effects on the parameters of interest. Pre-specification of time after induction of
229
anesthesia for the conduct of the study and heart rate range to be used for analysis should be
230
established. Moreover, inclusion and exclusion criteria should be defined ahead of time.
231
4.4
Landmarks for measurements
232
Other criteria to be pre-specified include the point of image capture in the cardiac cycle, and
233
acceptable image clarity (Lindsey et al. 2018). Dickson et al. (2016) examined aortic valve
234
diameter and area at three points in diastole in 163 dogs and 40 cats, both healthy and those
235
with heart disease. Measurements were obtained at early diastole, during the P-wave, and at
236
end diastole. The investigators analyzed within-animal and within-timepoint variability for
237
aortic valve linear dimensions and area measurements. Observed aortic valve measurements 16 | P a g e
Hausner and Chi, Regulatory Echocardiography 238
changed throughout diastole. These changes were clinically relevant for dogs, but deemed
239
unlikely to be so for cats. In dogs, the median within-subject measurement variability was 5%
240
for liner dimensions and 8% for area measurements. In cats, the variability was 4.5% for
241
linear dimensions and 10.4% for area measurements (Dickson et al. 2016). The same
242
phenomenon of variability of aortic dimensions has been reported in humans due to the
243
deformation and stretch accompanying pulsatility, a characteristic that can be applied across
244
species (Blanke et al. 2012). These examples illustrate the need for a precise description of
245
timepoints during the cardiac cycle when images are analyzed to reduce within-subject
246
variability. Linking a time of analysis to a mechanical or electrocardiogram event would
247
provide objective criteria for an operator.
248 249
5.
Image analysis: mathematics
250
The analysis process is susceptible to certain biases. To minimize operator-dependent bias,
251
use of a coding system conducted by personnel independent of both acquisition and analysis is
252
prudent. By not having knowledge of a given animal’s treatment group, a more objective
253
evaluation is possible. A study report or protocol should specify what information is and is not
254
accessible to the operator.
255
5.1
Software
256
Different ultrasound machines have different software, usually with default formulas.
257
However, many parameters have multiple options for formulas with the final choice decided
258
by the operator. Each formula has different sets of assumptions about cardiac shape and
259
function and most are derived from use in human clinics. It is not clear which of the formulas
260
is most appropriate for use in various laboratory animal species. Some investigators choose to 17 | P a g e
Hausner and Chi, Regulatory Echocardiography 261
export data generated from an echocardiography study to a spreadsheet for analysis with
262
customized formulas and software, preferably validated for the species of interest. However,
263
these software contribute different degrees of variability depending upon the measurement
264
under examination (Santarelli et al. 2019).
265
Studies conducted at multiple sites with different machines containing different software or
266
different versions of the same software may yield inconsistent data. For example, updates to
267
the software may be a confounding factor because of the potential to affect reference ranges
268
and other parameters.
269
5.2
Interpretation
270
Interpretation should not rely on a single parameter but should be integrative and scientifically
271
supported by known pharmacology and existing safety information. Use of a single parameter
272
for interpretation may be unduly influenced by variability in anatomy, physiology, and
273
operator factors (Konstam and Abboud 2017). Also, historical data for the testing facility may
274
be important for identifying the range of normal variations in cardiac parameters. While shifts
275
in group means may be important, animals perceived as outliers or spurious may actually be
276
important signals, identified by knowledge of historical data ranges.
277
Another factor to complicate interpretation of a study is growth of an animal. Duration of a
278
study, depending on the species, may encompass significant growth and development. In a
279
study with a reversibility component, this may be particularly challenging. However, in
280
addition to a control comparator, one possibility is to pre-specify a late selection of animals
281
demonstrating changes of potential interest to be a subset of animals included in the drug-free
282
recovery examinations. 18 | P a g e
Hausner and Chi, Regulatory Echocardiography 283
The analysis of nonclinical echocardiography studies is sometimes used to determine
284
optimum parameters to include in clinical safety monitoring. Statistical significance is often
285
set at p=0.05. Any given ultrasound machine can generate data for numerous parameters. The
286
more parameters that are recorded, the more likely that some will be marked as statistically
287
significant by random chance rather than due to true biological effect. Therefore, the more
288
parameters measured, the more opportunities for spurious results. Ideally, one or more
289
parameters should be prespecified for evaluation based on information known about the
290
product and the hypothesized effect. Alternatively, a descriptive exploratory study may be a
291
valuable first step, followed by a focused, hypothesis-driven study. Both of these alternatives
292
help avoid spurious findings.
293
294
6.
Future Directions
295
Echocardiography has the potential to provide valuable pharmacodynamic and safety
296
assessment information, which can be translatable to clinical monitoring. The addition of
297
echocardiography to nonclinical animal studies is also consistent with the principles of the
298
3Rs: to reduce, refine, and replace animal studies. This technology is a refinement of animal
299
studies and may help to reduce numbers of animals used. However, the utility of
300
echocardiography in nonclinical studies is contingent upon a demonstration of the reliability
301
of the information generated. To that end, we suggest the assembly of an expert multi-
302
disciplinary working group to include human echocardiographers, cardiologists, veterinary
303
cardiologists, safety pharmacologists, toxicologists, and regulators. The purpose of this group
19 | P a g e
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would be to identify practical standards and quality control measures for nonclinical
305
echocardiography studies. This would encompass:
306
•
training, certification, and continuing education of a sonographer
307
•
best practices for a nonclinical study
308
•
validation, or at least examination, of formulas used for different parameters in different species
309 310
•
recommendations for sedation and anesthesia, and
311
•
collection of historical control data and establishment of species-specific normative
312
data for echocardiography parameters.
313
314
7. Conclusion
315
Defining best practices for nonclinical echocardiography will be a profound and challenging
316
undertaking. However, it is a necessary step to establish echocardiography as an informative
317
tool in drug development.
318
Acknowledgements
319
We thank Shetarra Walker, MD, FACC, Norman Stockbridge MD, PhD, and Karen Davis-
320
Bruno PhD for their critical reading of this manuscript and valuable assistance.
321
322
323
Funding 20 | P a g e
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This research did not receive any specific grant from funding agencies in the public,
325
commercial, or not-for-profit sectors.
326
327
Disclaimer
328
This article reflects the views of the authors and should not be construed to represent FDA
329
views or policies.
330
21 | P a g e
References Bierig, SM., D. Ehler, ML. Knoll, and AD . Waggoner. 2005. "Minimum standards for the cardiac sonographer: a position paper." In American Society of Echocardiography. Blanke, P., M. Russe, J. Leipsic, J. Reinohl, U. Ebersberger, P. Suranyi, M. Siepe, G. Pache, M. Langer, and U. J. Schoepf. 2012. 'Conformational pulsatile changes of the aortic annulus: impact on prosthesis sizing by computed tomography for transcatheter aortic valve replacement', JACC Cardiovasc Interv, 5: 984-94. Dickson, D., D. Caivano, M. Patteson, and M. Rishniw. 2016. 'The times they are a-changin': Twodimensional aortic valve measurements differ throughout diastole', J Vet Cardiol, 18: 15-25. Donner, D. G., H. Kiriazis, X. J. Du, T. H. Marwick, and J. R. McMullen. 2018. 'Improving the quality of preclinical research echocardiography: observations, training, and guidelines for measurement', Am J Physiol Heart Circ Physiol, 315: H58-H70. Gentile-Solomon, J. M., and J. A. Abbott. 2016. 'Conventional echocardiographic assessment of the canine right heart: reference intervals and repeatability', J Vet Cardiol, 18: 234-47. Grune, J., A. Blumrich, S. Brix, S. Jeuthe, C. Drescher, T. Grune, A. Foryst-Ludwig, D. Messroghli, W. M. Kuebler, C. Ott, and U. Kintscher. 2018. 'Evaluation of a commercial multi-dimensional echocardiography technique for ventricular volumetry in small animals', Cardiovasc Ultrasound, 16: 10. Konstam, M. A., and F. M. Abboud. 2017. 'Ejection Fraction: Misunderstood and Overrated (Changing the Paradigm in Categorizing Heart Failure)', Circulation, 135: 717-19. Lindsey, M. L., Z. Kassiri, J. A. I. Virag, L. E. de Castro Bras, and M. Scherrer-Crosbie. 2018. 'Guidelines for measuring cardiac physiology in mice', Am J Physiol Heart Circ Physiol, 314: H733-H52. Santarelli, G., M. Baron Toaldo, J. Bouvard, T. M. Glaus, and J. Fernandez Del Palacio. 2019. 'Variability among strain variables derived from two-dimensional speckle tracking echocardiography in dogs by use of various software', Am J Vet Res, 80: 347-57. Visser, L. C., D. J. Sintov, and M. S. Oldach. 2018. 'Evaluation of tricuspid annular plane systolic excursion measured by two-dimensional echocardiography in healthy dogs: repeatability, reference intervals, and comparison with M-mode assessment', J Vet Cardiol, 20: 165-74.
Highlights •
Nonclinical echocardiography potentially provides translational information
•
Generation and analysis of images are operator-dependent
•
A Multidisciplinary Expert Working Group may help to define best practices
Keywords: echocardiography, nonclinical, reproducibility, regulatory, cardiovascular
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Elizabeth A. Hausner -S
Digitally signed by Elizabeth A. Hausner -S DN: c=US, o=U.S. Government, ou=HHS, ou=FDA, ou=People, 0.9.2342.19200300.100.1.1=1300147069, cn=Elizabeth A. Hausner -S Date: 2019.11.18 07:43:49 -05'00'
Xuan Chi -S
Digitally signed by Xuan Chi -S DN: c=US, o=U.S. Government, ou=HHS, ou=FDA, ou=People, cn=Xuan Chi -S, 0.9.2342.19200300.100.1.1=0014066742 Date: 2019.11.18 08:55:04 -05'00'
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
S0273-2300(20)30041-6
DOI:
https://doi.org/10.1016/j.yrtph.2020.104615
Reference:
YRTPH 104615
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 18 November 2019 Revised Date:
7 February 2020
Accepted Date: 10 February 2020
Please cite this article as: Hausner, E.A., Chi, X., Echocardiography in nonclinical studies: Where are we?, Regulatory Toxicology and Pharmacology (2020), doi: https://doi.org/10.1016/j.yrtph.2020.104615. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Inc.
Echocardiography in Nonclinical Studies: Where are we? Elizabeth A. Hausner* and Xuan Chi Running title: Nonclinical Echocardiography Division of Cardiovascular and Renal Products, Center for Drug Evaluation and Research (CDER), United States Food and Drug Administration (FDA), USA *
Corresponding author
Elizabeth A. Hausner 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 [email protected] Telephone: 301-796-1084 Fax: 301-796-9841 Word count Abstract: 178 Text: 3172 References: 10 references, 330 words
Hausner and Chi, Regulatory Echocardiography
2|Page
Hausner and Chi, Regulatory Echocardiography
Echocardiography in Nonclinical Studies: Where are we? Elizabeth A. Hausner and Xuan Chi
Keywords: echocardiography, nonclinical, reproducibility, regulatory, cardiovascular
3|Page
1
Abstract
2
Echocardiography is a powerful, noninvasive tool used both in clinical and nonclinical
3
settings, including in drug development. When used appropriately, it can provide valuable
4
translational information about pharmacodynamics, safety pharmacology, or toxicology,
5
helping to define no-observed-adverse-effect levels and providing guidance for clinical
6
monitoring and dose selection. Echocardiography is advantageous in conducting longitudinal
7
studies and reducing the number of animals used in safety assessments. To this end, there has
8
been no clear enunciation of what constitutes appropriate use of this imaging technology in a
9
nonclinical drug development setting. In this review, we describe the use of echocardiography
10
in nonclinical studies in regulatory submissions to the US Food and Drug Administration
11
Center for Drug Evaluation and Research. In addition, we discuss three main areas: the
12
operator, image acquisition, and image analysis, where variability may affect the reliability of
13
information generated in an echocardiography study. As a path forward, our recommendation
14
is for a multi-disciplinary expert working group to establish guidelines for education and
15
credentialing of nonclinical echocardiographers as well as quality assurance standards for
16
nonclinical echocardiography labs.
17
Keywords: echocardiography, translational, cardiovascular, safety, toxicology
18
19
20
21
Hausner and Chi, Regulatory Echocardiography 22 23
1.
Introduction
24
Echocardiography has evolved into a powerful noninvasive tool for both clinical medicine and
25
research. Nonclinical echocardiography is an area in need of input from the pharmaceutical
26
industry, contract research organizations, and regulatory agencies in a collaborative manner to
27
develop a standardized approach to image attainment, data collection, and to the extent
28
possible, interpretation of results. In this manuscript, we discuss the parameters we consider
29
important for translational use of echocardiography in nonclinical drug development studies,
30
based on our experience with nonclinical echocardiography data. In regulatory submissions,
31
most nonclinical echocardiography data are generated to demonstrate either an efficacy or
32
safety endpoint, including cardiac contractility, cardiac wall function, and blood flow and
33
valve performance.
34
The advantages nonclinical echocardiography offers include dynamic, longitudinal monitoring
35
of both functional and morphological changes, ease of use, and relatively low cost. Image
36
acquisition, measurement or data collection, and interpretation are operator-dependent with a
37
potential for significant intra- and inter-observer variability. This variability along with
38
relatively low spatial resolution as compared to histopathology contributes to potentially
39
relatively low sensitivity of echocardiography to detect functional and morphological cardiac
40
changes in nonclinical studies. The degree of change that can reliably be detected may depend
41
upon the species, homogeneity of the subjects, sonographic equipment and analytical skill as
42
well as the parameter examined. An important question to be considered during our review is
43
- what standards should be applied to echocardiography data to optimize reproducibility, data
44
rigor, and translational value? Moreover, what is the framework of training, design and
45
conduct of studies, analysis, and reporting, that defines an informative, reliable study? The 6|Page
Hausner and Chi, Regulatory Echocardiography 46
“design of studies” includes components such as how many prestudy exams,logistics of exams
47
including timing relative to pharmacokinetics parameters, how often and at what time points
48
should echocardiography data be collected. Design of studies, is highly dependent on the
49
overall goals for the specific study and is beyond the scope of this manuscript.
50 51
2.
How has echocardiography been used in nonclinical studies to date?
52
We searched the US Food and Drug Administration (FDA) Center for Drug Evaluation and
53
Research (CDER) electronic document archival database, which houses all CDER electronic
54
reviews and correspondence for drugs and biologics in clinical development since 2001. Our
55
search consisted of several steps. We identified nonclinical reviews that reported or discussed
56
the use of echocardiography. These reviews were retrieved and separated into those that
57
assessed nonclinical echocardiography and those that referenced clinical use. For reviews of
58
nonclinical echocardiography, we retrieved original study reports to better understand the
59
utility of echocardiography in regulatory nonclinical studies. With the implementation of the
60
Standard for Exchange of Nonclinical Data (SEND) and data warehouse tools, future
61
echocardiography data in safety pharmacology or toxicology studies may be modeled in
62
SEND. These standardized echocardiography data will be more amenable to cross-study
63
analyses.
64
2.1
Results of our search
65
We sorted Investigational New Drug (IND) applications involving nonclinical
66
echocardiography studies by CDER review divisions. The submissions were distributed across
67
twelve review divisions with the Division of Cardiovascular and Renal Products (DCRP) and
68
the Division of Metabolic and Endocrinology Products (DMEP) receiving the largest number 7|Page
Hausner and Chi, Regulatory Echocardiography 69
of submissions (Table 1). In addition, we categorized studies by the following types: support of
70
pharmacodynamic evaluation, cardiovascular safety pharmacology, and general toxicology.
71
72
Table 1: Distribution of Nonclinical Echocardiography Studies Across CDER Review
73
Divisions and by the purpose of studies (From January, 2001through June, 2019 ) Type of Study CDER Review
# of INDs with Nonclinical
Division
Echo Reviews
Pharmacology
DCRP
24
Safety Toxicology
(Pharmacodynamics)
Pharmacology
22 (rat; dog; goat)
1 (dog)
2 (rat, dog) 15 (mice, rat, dog,
18 DMEP
2 (mouse; rat)
DAP
4
OHOP
3
DAIP
2
DBRUP
2
1 (NHP)
monkey) 4 (dog, monkey)
1 (dog and monkey)
2 (monkey) 2 (dog and monkey) 1 (dog)
DPARP
1 (NHP) 1 (juvenile NHP)
2 1 (dog) DTOP
2
DNP
2
DGIEP
1
DDDP
1
DAAAP
1
8|Page
1 (dog)
1 (rat)
1 (rat) 1 (DNP)
1 (rat) 1 (juvenile rat) 1 (dog)
Hausner and Chi, Regulatory Echocardiography 74
NHP = non-human primate; DGIEP = Division of Gastroenterology and Inborn Errors Products; DDDP =
75
Division of Dermatology and Dental Products; DBRUP: = Division of Bone, Reproductive and Urologic
76
Products; DPARP = Division of Pulmonary, Allergy, and Rheumatology Products; DAAAP = Division of
77
Anesthesia, Analgesia, and Addiction Products; DNP = Division of Neurology Products; DCRP = Division of
78
Cardiovascular and Renal Products; DMEP = Division of Metabolism and Endocrinology Products; OHOP =
79
Office of Hematology and Oncology Products; DAP = Division of Antiviral Products; DAIP = Division of Anti-
80
Infective Products; DTOP = Division of Transplant and Ophthalmology Products
81
82
Nonclinical echocardiography studies submitted to DCRP were primarily in vivo
83
pharmacology studies to show efficacy in animal models for drugs in development for cardiac
84
indications. In contrast, nonclinical echocardiography studies submitted to other divisions
85
were primarily included as safety studies to evaluate potential cardiac toxicities (cardiac
86
contractility or morphological changes). In some submissions, nonclinical echocardiography
87
data provided critical proof-of-concept information such as a dose-response relationship for
88
contractility or safety and tolerability data. These data helped determine first-in-human doses.
89
Since similar investigations and measurements can be done in laboratory animals and humans,
90
nonclinical echocardiography data can help advise the clinical monitoring plan. In our review
91
of submissions containing nonclinical echocardiography data, we observed a lack of details of
92
how these studies were conducted, thereby limiting data interpretability. As echocardiography
93
is a highly operator- and instrument-dependent modality, technical details are crucial for
94
regulatory confidence.
95
The nonclinical studies using echocardiography were primarily conducted in rodents and dogs
96
for pharmacology, and dogs and nonhuman primates (NHPs) for safety pharmacology and
9|Page
Hausner and Chi, Regulatory Echocardiography 97
toxicology, besides being conducted in pigs, goats, and sheep. Two of the studies summarized
98
in Table 1 were conducted in juvenile animals (rat and NHP).
99
2.2
Questions raised by the studies identified
100
Our search indicates that the reporting of nonclinical echocardiography for regulatory
101
purposes occurs infrequently. Variability in the frequency of nonclinical echocardiography
102
study reports submitted to specific review divisions is most likely due to the nature of the
103
therapeutic areas and the likelihood of cardiovascular involvement. Why certain divisions
104
receive echocardiography data primarily for pharmacologic support while the others receive
105
for safety assessment is beyond the scope of this manuscript.
106
Information about the technique and study design supplied in the sponsor’s reports is variable.
107
Some reports provide detailed information regarding the frequency at which studies were
108
conducted, angles of insonation, and formulas used for analysis. Other reports are
109
minimalistic and simply report the use of the technology and present summary results. This is
110
an understandable consequence of the use of a relatively new tool for which industry
111
standards and regulatory guidances have not yet been established. Currently, no regulatory
112
authority has published standards regarding the use of echocardiography in nonclinical
113
studies.
114
There are several general concerns for any new technology added to nonclinical safety
115
assessment. First, there is a learning curve for those implementing the technology and for
116
those reviewing the data. When positive controls are included in these studies, the tendency is
117
to use comparators with profound effects. Although detection of these obvious effects may be
118
somewhat reassuring, it is not indicative of more subtle effects being reliably identified. 10 | P a g e
Hausner and Chi, Regulatory Echocardiography 119
Second, echocardiography, a new tool in early drug development, possibly raises the bar for a
120
new drug. That is, a new drug is subjected to scrutiny not used in the past and this creates
121
more stringent expectations for a new entity. It is important to establish species-specific
122
normative data based on historical controls so that the significance of changes can be
123
interpreted. Moreover, it is critical to correlate the clinical experience from approved drugs
124
with findings from nonclinical echocardiography studies to understand what constitutes
125
acceptable background effects. Participation of veterinary cardiologists in safety assessment
126
studies may help bridge the clinical applicability of nonclinical studies and safety monitoring
127
in human clinical trials.
128
2.3
Sources of variability
129
The information available to CDER reviewers for any given nonclinical study is what is
130
provided by the sponsor, and the content and quality varies across different submissions. To
131
help determine the reliability of the echo information provided, reviewers need to know the
132
acquisition and analysis methods used. This includes information regarding who generated
133
and acquired the images, and how the analysis was conducted.
134 135
3.
The observer : who is generating the data?
136
According to the Good Laboratory Practices (GLP) (21CFR58), it is at the discretion of a
137
study director to decide if an individual involved in a GLP study has the appropriate
138
combination of education, training, and experience to perform the assigned functions. The
139
GLP regulations also direct a testing facility to maintain a current summary of the training,
140
experience, and job description for each person engaged in the conduct of a nonclinical
11 | P a g e
Hausner and Chi, Regulatory Echocardiography 141
laboratory investigation. This information is required to be available for audit during an FDA
142
inspection.
143
The appropriate combination of education, training, and experience for a nonclinical
144
echocardiography operator is not well defined from a regulatory standpoint. The regulations
145
do not specify credentials for various specialties involved in a nonclinical study, e.g., clinical
146
pathology or histopathology, but there are credentialing bodies for veterinary experts for these
147
and other areas. Several scientific societies have established criteria for knowledge, skills, and
148
ability in other areas of nonclinical studies (e.g. diplomates of toxicology, safety
149
pharmacology). Demonstrating competence and how best to maintain this competence may be
150
dependent on the kind of studies conducted and the species of animals studied. Improvements
151
in technology, updates to individual imaging systems, and changes in the studies (e.g.,
152
pharmacology versus safety pharmacology) all necessitate continuing education.
153
The American Society of Echocardiography (ASE) has produced several documents
154
pertaining to the initial and continuing education of a sonographer. The ASE identifies three
155
elements for demonstrating competence in a sonographer: credentialing and formal education,
156
technical competence, and continuing education. The type of training described is intended to
157
address unique imaging considerations for human subspecialty disciplines including adult,
158
pediatric, and fetal imaging. Training and credentialing are recommended for each
159
subspecialty discipline (Bierig et al. 2005). However, such educational standards have not yet
160
been defined for nonclinical echocardiography. Therefore, it is difficult to determine whether
161
someone performing echocardiography in dogs is also competent to perform similar studies in
162
mice or rats. This raises the question as to whether there should be species-specific
163
echocardiography training. The specifics of training are not straightforward. In some cases, 12 | P a g e
Hausner and Chi, Regulatory Echocardiography 164
the technical and support staff both acquire and analyze the images. Training may be formal
165
or informal i.e. one staff member instructing another. There is some question as to whether the
166
informal methods of instruction show decay of technique over time or a lessening of rigor as
167
subsequent staff members become second and third generation educators (Donner et al. 2018).
168
At present, however, there is no consensus concerning education, credentialing, and
169
continuing education for the nonclinical sector. It has been proposed that clinical standards are
170
not necessarily appropriate for the nonclinical setting. For any specialty, best practices for a
171
given context are established, beginning with appropriate education, demonstration of
172
competence (credentialing) and maintenance of competence (continuing education).
173
4.
Image acquisition
174
4.1
Intra- and interobserver variability
175
Intra-observer variability is of critical importance when operators generate or analyze images
176
or both. That is, if the same operator generates the same image or measures the same
177
parameter several times, how much will the dimensions or parameters of the image vary?
178
When more than one observer is involved, interobserver variability must also be considered.
179
Normal physiologic variability also needs to be considered. For example, the change in heart
180
rate that may spontaneously occur throughout the day or the course of an examination.
181
Intra- and interobserver variability may change depending upon the parameter e.g.,
182
dimensions measured, functions evaluated, or imaging modality used, i.e., two-dimensional
183
versus M-mode versus three-dimensional (Grune et al. 2018). Angle-dependence of certain
184
echocardiography measurements is another example illustrating the importance of assessing
185
intra-observer variability. Variation in the angle of insonation affects reliability of such
13 | P a g e
Hausner and Chi, Regulatory Echocardiography 186
measurements, e.g., M-mode measurement of tricuspid annular plane systolic excursion
187
(TAPSE) (Visser, Sintov, and Oldach 2018). Another consideration for intra-observer
188
variability is the use of echocardiography in longitudinal studies. A longitudinal study has the
189
advantage of following change within a single animal during a period of drug exposure, and
190
potentially, following the same animal through a drug-free recovery period. This approach
191
may reduce animal numbers while at the same time generate informative reversibility data.
192
For this to be a realistic goal, there needs to be confidence in the repeatability of
193
echocardiography measurements and an understanding of variability inherent in the
194
technology (Gentile-Solomon and Abbott 2016).
195
There is no standardized evaluation or consensus on how best to determine or report intra- or
196
interobserver variability. Acceptable levels of intra-observer variability are unknown but
197
should be outside the margin of error. For example, if a 5% difference in ejection fraction is
198
proposed as a clinical endpoint, the intra-observer variability should be significantly below
199
5%. As reported in several publications, factors influencing intra-observer variability include
200
the type of measurement, software used, and the training and experience of the operator
201
(Gentile-Solomon and Abbott 2016; Santarelli et al. 2019; Visser, Sintov, and Oldach 2018;
202
Donner et al. 2018). A sampling of published intra-observer variability is shown in Table 2.
203
None of the reports identified within the FDA electronic files noted intra-observer variability
204
or indicated that it had been assessed.
205
206
207 14 | P a g e
Hausner and Chi, Regulatory Echocardiography 208
Table 2: Published Intra- and Interobserver Variability Species
Parameter
Intraobserver
Interobserver
variability
variability
Reference
Healthy dogs,
2D TAPSE
4.4CV, 1.9-7.6%
9.5CV, 7.1%
Visser et. al., J Vet Cardiol
multiple
MM TAPSE
2.3CV, 1.1-7.1%
8.4CV, 5.8%
(2018) 20:165-174
TMAD
4.44CV
4.12CV
Wolf et. al. J Vet Cardiol
5.2CV
(2018) 20:175-185
breeds Healthy dogs
GLS Dogs with and
GLS:
Within day
without
transmural +
3.5 CV vendor 1
8 vendor1
Santarelli et. al. Am J Vet
cardiac disease
endocardial
5.6
3.9 independent
Res (2019) 80:347-357
Late diastolic
14.2 vendor 1
14.2 vendor1
SR
13.1 independent
17.3 independent
ESV
2.3±6.0 (mean
3.8±6.0 (mean diff±SD)
Healthy C57BL/6J mice (M-
independent
Grune 2018
diff±SD) EDV
mode)
2.1±5.8(mean
6.2±6.2(mean diff±SD)
diff±SD)
Healthy dogs
ESV
18%
11%
(3D imaging)
EDV
12%
14%
LeBlanc 2017
209
TMAD= tissue motion annular displacement; GLS=global longitudinal strain; MM= m-mode; TAPSE= tricuspid
210
annular plane systolic excursion; SR= strain rate; STE= speckle tracking echocardiography; vendor1= Vendor 1-
211
dependent software; independent= vendor-independent software; ESV= end systolic volume; EDV= end diastolic
212
volume; CV = coefficient of variation
213
4.2
Guidelines
214
Ideally, a given echocardiography study should be conducted using uniform, pre-specified
215
methodology. This is especially important if multiple laboratories are conducting the 15 | P a g e
Hausner and Chi, Regulatory Echocardiography 216
examinations and data to be compiled into one report. . However, given the variability of
217
biological windows associated with individual anatomy, different body weights and
218
conformation, and the nature of the technology, the pre-specified methodology needs to be
219
flexible. Another way to help decrease variability is the use of pre-specified image guidelines
220
as set forth by the ASE. Other guidelines, such as those of the American College of
221
Veterinary Internal Medicine, may also be useful. A set of standardized criteria for acquisition
222
and measurement helps to decrease bias associated with improvement in technique or image
223
acquisition over time as operators gain experience or familiarity with effects within a study.
224
4.3
Sedation, anesthesia, conscious state
225
Several investigators have proposed consideration of the conscious state of the animal to
226
improve reliability of echo studies (Lindsey et al. 2018). If medication is to be used to
227
facilitate the examination, sedative or anesthetic agents must be consistent and have minimal
228
or known effects on the parameters of interest. Pre-specification of time after induction of
229
anesthesia for the conduct of the study and heart rate range to be used for analysis should be
230
established. Moreover, inclusion and exclusion criteria should be defined ahead of time.
231
4.4
Landmarks for measurements
232
Other criteria to be pre-specified include the point of image capture in the cardiac cycle, and
233
acceptable image clarity (Lindsey et al. 2018). Dickson et al. (2016) examined aortic valve
234
diameter and area at three points in diastole in 163 dogs and 40 cats, both healthy and those
235
with heart disease. Measurements were obtained at early diastole, during the P-wave, and at
236
end diastole. The investigators analyzed within-animal and within-timepoint variability for
237
aortic valve linear dimensions and area measurements. Observed aortic valve measurements 16 | P a g e
Hausner and Chi, Regulatory Echocardiography 238
changed throughout diastole. These changes were clinically relevant for dogs, but deemed
239
unlikely to be so for cats. In dogs, the median within-subject measurement variability was 5%
240
for liner dimensions and 8% for area measurements. In cats, the variability was 4.5% for
241
linear dimensions and 10.4% for area measurements (Dickson et al. 2016). The same
242
phenomenon of variability of aortic dimensions has been reported in humans due to the
243
deformation and stretch accompanying pulsatility, a characteristic that can be applied across
244
species (Blanke et al. 2012). These examples illustrate the need for a precise description of
245
timepoints during the cardiac cycle when images are analyzed to reduce within-subject
246
variability. Linking a time of analysis to a mechanical or electrocardiogram event would
247
provide objective criteria for an operator.
248 249
5.
Image analysis: mathematics
250
The analysis process is susceptible to certain biases. To minimize operator-dependent bias,
251
use of a coding system conducted by personnel independent of both acquisition and analysis is
252
prudent. By not having knowledge of a given animal’s treatment group, a more objective
253
evaluation is possible. A study report or protocol should specify what information is and is not
254
accessible to the operator.
255
5.1
Software
256
Different ultrasound machines have different software, usually with default formulas.
257
However, many parameters have multiple options for formulas with the final choice decided
258
by the operator. Each formula has different sets of assumptions about cardiac shape and
259
function and most are derived from use in human clinics. It is not clear which of the formulas
260
is most appropriate for use in various laboratory animal species. Some investigators choose to 17 | P a g e
Hausner and Chi, Regulatory Echocardiography 261
export data generated from an echocardiography study to a spreadsheet for analysis with
262
customized formulas and software, preferably validated for the species of interest. However,
263
these software contribute different degrees of variability depending upon the measurement
264
under examination (Santarelli et al. 2019).
265
Studies conducted at multiple sites with different machines containing different software or
266
different versions of the same software may yield inconsistent data. For example, updates to
267
the software may be a confounding factor because of the potential to affect reference ranges
268
and other parameters.
269
5.2
Interpretation
270
Interpretation should not rely on a single parameter but should be integrative and scientifically
271
supported by known pharmacology and existing safety information. Use of a single parameter
272
for interpretation may be unduly influenced by variability in anatomy, physiology, and
273
operator factors (Konstam and Abboud 2017). Also, historical data for the testing facility may
274
be important for identifying the range of normal variations in cardiac parameters. While shifts
275
in group means may be important, animals perceived as outliers or spurious may actually be
276
important signals, identified by knowledge of historical data ranges.
277
Another factor to complicate interpretation of a study is growth of an animal. Duration of a
278
study, depending on the species, may encompass significant growth and development. In a
279
study with a reversibility component, this may be particularly challenging. However, in
280
addition to a control comparator, one possibility is to pre-specify a late selection of animals
281
demonstrating changes of potential interest to be a subset of animals included in the drug-free
282
recovery examinations. 18 | P a g e
Hausner and Chi, Regulatory Echocardiography 283
The analysis of nonclinical echocardiography studies is sometimes used to determine
284
optimum parameters to include in clinical safety monitoring. Statistical significance is often
285
set at p=0.05. Any given ultrasound machine can generate data for numerous parameters. The
286
more parameters that are recorded, the more likely that some will be marked as statistically
287
significant by random chance rather than due to true biological effect. Therefore, the more
288
parameters measured, the more opportunities for spurious results. Ideally, one or more
289
parameters should be prespecified for evaluation based on information known about the
290
product and the hypothesized effect. Alternatively, a descriptive exploratory study may be a
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valuable first step, followed by a focused, hypothesis-driven study. Both of these alternatives
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help avoid spurious findings.
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6.
Future Directions
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Echocardiography has the potential to provide valuable pharmacodynamic and safety
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assessment information, which can be translatable to clinical monitoring. The addition of
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echocardiography to nonclinical animal studies is also consistent with the principles of the
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3Rs: to reduce, refine, and replace animal studies. This technology is a refinement of animal
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studies and may help to reduce numbers of animals used. However, the utility of
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echocardiography in nonclinical studies is contingent upon a demonstration of the reliability
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of the information generated. To that end, we suggest the assembly of an expert multi-
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disciplinary working group to include human echocardiographers, cardiologists, veterinary
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cardiologists, safety pharmacologists, toxicologists, and regulators. The purpose of this group
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would be to identify practical standards and quality control measures for nonclinical
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echocardiography studies. This would encompass:
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training, certification, and continuing education of a sonographer
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best practices for a nonclinical study
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validation, or at least examination, of formulas used for different parameters in different species
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recommendations for sedation and anesthesia, and
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collection of historical control data and establishment of species-specific normative
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data for echocardiography parameters.
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7. Conclusion
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Defining best practices for nonclinical echocardiography will be a profound and challenging
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undertaking. However, it is a necessary step to establish echocardiography as an informative
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tool in drug development.
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Acknowledgements
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We thank Shetarra Walker, MD, FACC, Norman Stockbridge MD, PhD, and Karen Davis-
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Bruno PhD for their critical reading of this manuscript and valuable assistance.
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Hausner and Chi, Regulatory Echocardiography 324
This research did not receive any specific grant from funding agencies in the public,
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commercial, or not-for-profit sectors.
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Disclaimer
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This article reflects the views of the authors and should not be construed to represent FDA
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views or policies.
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References Bierig, SM., D. Ehler, ML. Knoll, and AD . Waggoner. 2005. "Minimum standards for the cardiac sonographer: a position paper." In American Society of Echocardiography. Blanke, P., M. Russe, J. Leipsic, J. Reinohl, U. Ebersberger, P. Suranyi, M. Siepe, G. Pache, M. Langer, and U. J. Schoepf. 2012. 'Conformational pulsatile changes of the aortic annulus: impact on prosthesis sizing by computed tomography for transcatheter aortic valve replacement', JACC Cardiovasc Interv, 5: 984-94. Dickson, D., D. Caivano, M. Patteson, and M. Rishniw. 2016. 'The times they are a-changin': Twodimensional aortic valve measurements differ throughout diastole', J Vet Cardiol, 18: 15-25. Donner, D. G., H. Kiriazis, X. J. Du, T. H. Marwick, and J. R. McMullen. 2018. 'Improving the quality of preclinical research echocardiography: observations, training, and guidelines for measurement', Am J Physiol Heart Circ Physiol, 315: H58-H70. Gentile-Solomon, J. M., and J. A. Abbott. 2016. 'Conventional echocardiographic assessment of the canine right heart: reference intervals and repeatability', J Vet Cardiol, 18: 234-47. Grune, J., A. Blumrich, S. Brix, S. Jeuthe, C. Drescher, T. Grune, A. Foryst-Ludwig, D. Messroghli, W. M. Kuebler, C. Ott, and U. Kintscher. 2018. 'Evaluation of a commercial multi-dimensional echocardiography technique for ventricular volumetry in small animals', Cardiovasc Ultrasound, 16: 10. Konstam, M. A., and F. M. Abboud. 2017. 'Ejection Fraction: Misunderstood and Overrated (Changing the Paradigm in Categorizing Heart Failure)', Circulation, 135: 717-19. Lindsey, M. L., Z. Kassiri, J. A. I. Virag, L. E. de Castro Bras, and M. Scherrer-Crosbie. 2018. 'Guidelines for measuring cardiac physiology in mice', Am J Physiol Heart Circ Physiol, 314: H733-H52. Santarelli, G., M. Baron Toaldo, J. Bouvard, T. M. Glaus, and J. Fernandez Del Palacio. 2019. 'Variability among strain variables derived from two-dimensional speckle tracking echocardiography in dogs by use of various software', Am J Vet Res, 80: 347-57. Visser, L. C., D. J. Sintov, and M. S. Oldach. 2018. 'Evaluation of tricuspid annular plane systolic excursion measured by two-dimensional echocardiography in healthy dogs: repeatability, reference intervals, and comparison with M-mode assessment', J Vet Cardiol, 20: 165-74.
Highlights •
Nonclinical echocardiography potentially provides translational information
•
Generation and analysis of images are operator-dependent
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A Multidisciplinary Expert Working Group may help to define best practices
Keywords: echocardiography, nonclinical, reproducibility, regulatory, cardiovascular
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Elizabeth A. Hausner -S
Digitally signed by Elizabeth A. Hausner -S DN: c=US, o=U.S. Government, ou=HHS, ou=FDA, ou=People, 0.9.2342.19200300.100.1.1=1300147069, cn=Elizabeth A. Hausner -S Date: 2019.11.18 07:43:49 -05'00'
Xuan Chi -S
Digitally signed by Xuan Chi -S DN: c=US, o=U.S. Government, ou=HHS, ou=FDA, ou=People, cn=Xuan Chi -S, 0.9.2342.19200300.100.1.1=0014066742 Date: 2019.11.18 08:55:04 -05'00'
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.