Cytomegalovirus in 
Transplantation:  
Developing Drugs to 
Treat or Prevent Disease 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
May 2020 
Clinical/Antimicrobial 
 
   
Cytomegalovirus in 
Transplantation: 
Developing Drugs to 
Treat or Prevent Disease 
Guidance for Industry 
 
 
 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information 
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD  20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353; Email: druginfo@fda.hhs.gov 
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
May 2020 
Clinical/Antimicrobial
   
TABLE OF CONTENTS 
 
I.  INTRODUCTION..................................................................................................1 
II.  BACKGROUND ....................................................................................................2 
III.  DEVELOPMENT PROGRAM ............................................................................4 
A.  General Drug Development Considerations ................................................................... 4 
1. Early-Phase Development Considerations .......................................................................... 4 
a.  Pharmacology/toxicology development considerations ............................................... 4 
b.  Nonclinical virology development considerations ....................................................... 5 
c.  General considerations for phase 1 and phase 2 clinical development ......................... 9 
2. Drug Development Population .......................................................................................... 12 
3. Efficacy Considerations .................................................................................................... 12 
4. Safety Considerations ........................................................................................................ 13 
B.  Phase 3 Efficacy Trial Considerations ........................................................................... 13 
1. Trial Design ....................................................................................................................... 13 
a.  Preventing CMV disease ............................................................................................ 13 
b.  Treating CMV disease ................................................................................................ 15 
2. Trial Population ................................................................................................................ 16 
3. Entry Criteria .................................................................................................................... 17 
4. Randomization, Stratification, and Blinding ..................................................................... 18 
5. Pediatric Populations ........................................................................................................ 18 
6. Dose Selection ................................................................................................................... 19 
7. Use of Active Comparators ............................................................................................... 19 
8. Efficacy Endpoints ............................................................................................................. 19 
a.  CMV prophylaxis trials in SOT recipients ................................................................. 19 
b.  CMV prophylaxis trials in HSCT recipients .............................................................. 20 
c.  CMV preemptive therapy trials in SOT or HSCT recipients ...................................... 21 
d.  Treating CMV disease in SOT or HSCT recipients ................................................... 21 
9. Trial Procedures and Timing of Assessments ................................................................... 22 
10.  Endpoint Adjudication .................................................................................................. 22 
11.  Statistical Considerations ............................................................................................. 22 
a.  Analysis populations ................................................................................................... 22 
b.  Efficacy analyses ........................................................................................................ 23 
c.  Handling of missing data ............................................................................................ 23 
12.  Accelerated Approval Considerations .......................................................................... 23 
C.  Other Considerations ...................................................................................................... 24 
1. Clinical Virology Considerations ...................................................................................... 24 
2. Pharmacokinetic/Pharmacodynamic Considerations ....................................................... 25 
GLOSSARY OF ACRONYMS ......................................................................................26 
REFERENCES .................................................................................................................27 
APPENDIX .......................................................................................................................31 
 
  Contains Nonbinding Recommendations 
Cytomegalovirus in Transplantation:   
Developing Drugs to Treat or Prevent Disease 
Guidance for Industry1 
 
 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA staff responsible for this guidance as listed on the 
title page. 
 
 
 
 
I.  INTRODUCTION  
 
The purpose of this guidance is to assist sponsors in the clinical development of drugs to treat or 
prevent cytomegalovirus (CMV) disease in patients who have undergone solid organ 
transplantation (SOT) or hematopoietic stem cell transplantation (HSCT).2  Specifically, this 
guidance addresses the Food and Drug Administrations (FDAs) current thinking regarding the 
overall development program and clinical trial designs for the development of drugs and 
therapeutic biological products to support an indication for treating or preventing CMV disease 
in post-transplant populations.  This guidance is intended to facilitate continued discussions 
among the Division of Antiviral Products (DAVP), pharmaceutical sponsors, the academic 
community, and the public.3  This guidance does not address drug development for treating or 
preventing congenital CMV infection or CMV infection in patients other than those undergoing 
SOT or HSCT. 
 
This guidance also discusses the use of CMV viremia, measured as DNAemia (CMV 
deoxyribonucleic acid (DNA) in blood determined by polymerase chain reaction (PCR)), as a 
validated surrogate endpoint in clinical trials. 
 
This guidance does not contain discussion of the general issues of statistical analysis or clinical 
trial design.  Those topics are addressed in the ICH guidances for industry E9 Statistical 
                                                 
1 This guidance has been prepared by the Division of Antiviral Products in the Center for Drug Evaluation and 
Research at the Food and Drug Administration. 
  
2 For the purposes of this guidance, all references to drugs include both human drugs and therapeutic biological 
products unless otherwise specified. 
 
3 In addition to consulting guidances, sponsors are encouraged to contact the DAVP to discuss specific issues that 
arise during the development of anti-CMV drugs. 
 
1  Contains Nonbinding Recommendations 
Principles for Clinical Trials (September 1998) and E10 Choice of Control Group and Related 
Issues in Clinical Trials (May 2001), respectively.4 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
as recommendations, unless specific regulatory or statutory requirements are cited.  The use of 
the word should in Agency guidances means that something is suggested or recommended, but 
not required. 
 
 
II.  BACKGROUND 
 
CMV is a member of the beta-herpes virus group that causes infection worldwide with variable 
geographic distribution linked to socioeconomic status.  In the United States, CMV 
seroprevalence ranges from 40 percent to 80 percent (Cannon and Davis 2005; Bate et al. 2010).  
Primary infection occurs in CMV seronegative hosts and is usually acquired during the first 
decades of life.  In most cases, primary infection is benign and self-limited.  However, in patients 
with immature or compromised immune systems (e.g., transplant recipients, congenitally 
infected newborns, or patients with acquired immunodeficiency syndrome (AIDS)), primary 
CMV infection is often symptomatic and is associated with increased morbidity and mortality.  
As with all herpes viruses, CMV establishes lifelong latency after primary infection; thereafter, 
intermittent viral shedding and disease reactivation can occur, particularly in hosts with 
compromised immune systems (Ramanan and Razonable 2013). 
 
CMV is the single most frequent opportunistic pathogen in transplant recipients.  The incidence 
of CMV infection and disease in this population depends on a number of factors, such as 
transplant type, donor and recipient CMV serostatus, and the level of immunosuppression 
(Ramanan and Razonable 2013).  A transplant recipient is described by nomenclature that first 
describes the donors CMV serostatus followed by the recipients CMV serostatus.  For example, 
D+/R- refers to a seronegative recipient of a transplant from a seropositive donor.5 
 
In SOT recipients, observational studies have demonstrated an association between donor and 
recipient CMV serostatus and risk for CMV disease; D+/R- status is associated with a higher risk 
(with rates of 50 percent to 60 percent) for developing CMV disease than CMV seropositive 
recipients (D+/R+ or D-/R+), who have rates of 10 percent to 20 percent (Hartmann et al. 2006).  
The lowest rate of CMV infection (less than 5 percent) occurs in CMV seronegative SOT 
recipients who received a transplanted organ from a seronegative donor (D-/R-).  In HSCT 
recipients, CMV seropositive recipients (R+) are at the highest risk for developing CMV disease 
regardless of the donors CMV serostatus.  Without prophylaxis (treatment administered to all 
patients at risk for developing CMV disease), approximately 80 percent of CMV seropositive 
                                                 
4 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the FDA 
guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
 
5 CMV serostatus of donor (D) and recipient (R) is designated as D+ or D- and R+ or R-, respectively.  The term 
CMV seropositive refers to a donor or recipient with IgG antibodies to a previously acquired CMV infection and the 
term CMV seronegative denotes that anti-CMV IgG antibodies are absent. 
2  Contains Nonbinding Recommendations 
HSCT patients will experience CMV infection in the blood, and without preemptive therapy 
(treating patients with CMV replication in blood), approximately 30 percent of patients with 
CMV viremia will develop CMV disease (Ljungman et al. 2010). 
 
The clinical manifestations of CMV infection range from asymptomatic CMV viremia to tissue-
invasive (end-organ) CMV disease, including CMV syndrome, a subcategory of CMV disease 
described only in SOT recipients.  Any organ can be infected by CMV; however, CMV 
pneumonia is the most serious manifestation of CMV infection in HSCT recipients and has been 
associated with high mortality.  In contrast, CMV in SOT recipients has a predilection to 
replicate in the allograft.  CMV infection may also be associated with an increased risk of other 
opportunistic infections, graft failure, graft rejection, and mortality (Razonable et al. 2013). 
 
In general, because of the increased morbidity and mortality associated with CMV disease in 
transplant recipients, preventing CMV disease is generally recognized as a better strategy than 
treating established CMV disease.  Prophylactic therapy and preemptive therapy are the two 
major strategies used for prevention (Boeckh and Ljungman 2009; Tomblyn et al. 2009; 
Razonable et al. 2013; Kotton et al. 2018).  Both strategies have been shown to be effective for 
preventing CMV disease in SOT and HSCT recipients. 
 
Currently, limited therapeutic options for treating or preventing CMV disease in transplant 
recipients are available.  As of May 2020, only five drugs have received FDA approval for 
systemic use for treating or preventing CMV disease:  letermovir, ganciclovir and its prodrug 
valganciclovir, foscarnet, and cidofovir.  Letermovir is approved for CMV prophylaxis in CMV 
seropositive recipients of an allogeneic HSCT; ganciclovir and valganciclovir are approved for 
preventing CMV disease in transplant recipients and for treating CMV retinitis in 
immunocompromised patients, including patients with AIDS.  Foscarnet and cidofovir have 
received FDA approval only for treating CMV retinitis in AIDS patients.  Moreover, ganciclovir, 
valganciclovir, foscarnet, and cidofovir are associated with significant toxicities.  These findings, 
coupled with the emergence of resistance to available drugs (Lurain and Chou 2010; Komatsu et 
al. 2014), strongly support the urgent need for new therapeutic agents that are effective and less 
toxic. 
 
During the past 15 years, all phase 3 trials designed to support marketing applications for CMV 
drugs were prophylaxis trials in SOT and/or HSCT recipients.  The primary endpoint used in 
these prophylaxis trials in SOT recipients was the incidence of CMV disease, including both 
symptomatic CMV infection (also called CMV syndrome) and/or tissue-invasive CMV disease 
(e.g., CMV colitis, hepatitis, or pneumonia).  CMV syndrome is defined better in SOT recipients 
than it is in HSCT recipients, mainly because the symptoms associated with CMV syndrome can 
have several other causes in the setting of HSCT, including other viral infections.  Until recently, 
the primary endpoint used in prophylaxis trials in HSCT patients was the incidence of tissue-
invasive CMV disease. 
 
However, the results of recent trials revealed that in the current era of preemptive therapy for 
CMV viremia based on optimized PCR assays, the incidence of tissue-invasive CMV disease in 
HSCT recipients at 6 months post-transplantation was less than 5 percent (Marty et al. 2011).  
These results call into question whether trials with tissue-invasive CMV disease as an endpoint 
3  Contains Nonbinding Recommendations 
in HSCT patients are feasible, considering the sample sizes needed for such trials given the low 
frequency of CMV disease in these patients.  The accumulated clinical literature supports the 
premise that CMV viremia predicts development of CMV disease in transplant recipients (Gor et 
al. 1998; Emery et al. 1999; Emery et al. 2000; Jang et al. 2012; Natori et al. 2018) and that 
CMV viremia predicts mortality (Green et al. 2016).  Prophylaxis or preemptive therapy for 
CMV viremia prevents CMV disease (Green et al. 2016), the suppression of viremia is 
associated with clinical resolution of CMV disease (Asberg et al. 2007), and CMV prophylaxis 
in HSCT recipients is associated with decreased mortality (Marty et al. 2017). 
 
These observations have prompted the FDA to consider CMV viremia (DNAemia) as a validated 
surrogate endpoint to be used as a part of a composite endpoint to support traditional approval.  
Therefore, traditional approval for new drug applications (NDAs) for CMV prophylaxis trials in 
HSCT recipients can be based on a composite endpoint defined as either the occurrence of 
tissue-invasive CMV disease or the initiation of anti-CMV preemptive therapy based on 
clinically significant CMV DNAemia.  The use of CMV DNAemia as a part of a composite 
endpoint for other indications (e.g., treatment) is also discussed in this guidance. 
 
 
III.  DEVELOPMENT PROGRAM 
 
 
A.  General Drug Development Considerations 
 
1.  Early-Phase Development Considerations 
 
General considerations pertinent to nonclinical development and early clinical development are 
outlined in this section.  Sponsors considering developing drugs for treating or preventing CMV 
disease are encouraged to communicate with the FDA through the pre-investigational new drug 
application (pre-IND) consultation program.6,7  Pre-IND consultation with the FDA is optional.  
It may be particularly helpful for sponsors with limited experience in the IND process or for 
sponsors who want to obtain FDA recommendations for developing drugs with unique 
considerations based on mechanistic action, novel treatment approaches, or the use of novel 
biomarkers. 
 
a.  Pharmacology/toxicology development considerations 
 
Pharmacology/toxicology development for CMV antiviral drugs should follow existing guidance 
for drug development.  For detailed recommendations regarding pharmacology/toxicology 
development for single antiviral drugs and for two or more new investigational drugs to be used 
in combination, sponsors should consult the following ICH guidances on nonclinical safety 
studies:  For small molecules, see the ICH guidance for industry M3(R2) Nonclinical Safety 
                                                 
6 https://www.fda.gov/drugs/pre-ind-consultation-program/division-anti-viral-products-davp-pre-ind-letter-
instruction. 
 
7 https://www.fda.gov/drugs/pre-ind-consultation-program/getting-started-division-antiviral-products-pre-ind-
process. 
4  Contains Nonbinding Recommendations 
Studies for the Conduct of Human Clinical Trials and Marketing Authorization for 
Pharmaceuticals (January 2010); for biological products and other biotechnology-derived 
pharmaceuticals, see the ICH guidance for industry S6 Preclinical Safety Evaluation of 
Biotechnology-Derived Pharmaceuticals (July 1997). 
 
We recommend carcinogenicity studies if the expected treatment duration, including intermittent 
use, is 6 months or longer (e.g., prophylaxis indications).8  Carcinogenicity studies can be 
submitted with an initial marketing application (i.e., NDA or biologics license application) or as 
required postmarketing studies. 
 
For drugs to be used in combination, ICH M3(R2) includes a discussion of nonclinical safety 
studies appropriate in a combination drug development setting involving two early-stage 
entities.9  ICH M3(R2) defines early-stage entities as compounds with limited clinical experience 
(i.e., phase 2 studies or earlier). In general, nonclinical combination studies of an early-stage 
entity plus an approved therapy are not recommended.10 Therefore, unless data from nonclinical 
studies of an early-stage entity suggest a potential for serious synergistic toxicity with an 
approved therapeutic drug, combination toxicology studies are not recommended. 
 
b.  Nonclinical virology development considerations 
 
Nonclinical virology studies can facilitate initial dose selection, enable the design of a clinical 
proof-of-concept study, and support an antiviral drug claim.  The sponsor should conduct studies 
to support initial human trials before submitting an IND.  Nonclinical virology studies for 
treating or preventing CMV should follow existing guidance for drug development.11  Additional 
recommendations for nonclinical and clinical virology assessments specific to developing drugs 
for treating or preventing CMV disease are summarized throughout this guidance. 
 
  Mechanism of action 
 
The sponsor should investigate the mechanism by which a drug exhibits anti-CMV activity by 
using cell culture and biochemical, structural, and/or genetic studies to evaluate the effect of the 
drug on relevant stages of the virus life cycle and to identify the CMV target protein or proteins 
for direct-acting antiviral drugs.  Mechanism-of-action investigations should include appropriate 
controls for assessing the specificity of anti-CMV activity, which may include assessments of 
activity against other CMV proteins, relevant host proteins, other viruses, and/or cells infected 
with investigational drug-resistant CMV variants.  Biochemical or subcellular quantitative assays 
                                                 
8 See the ICH guidance for industry S1A The Need for Long-Term Rodent Carcinogenicity Studies of 
Pharmaceuticals (March 1996). 
 
9 See ICH M3(R2), section XVII., Combination Drug Toxicity Testing. 
 
10 See the guidances for industry Chronic Hepatitis C Virus Infection: Developing Direct-Acting Antiviral Drugs for 
Treatment (November 2017) and Human Immunodeficiency Virus-1 Infection: Developing Antiretroviral Drugs for 
Treatment (November 2015). 
 
11 See the guidance for industry Antiviral Product Development  Conducting and Submitting Virology Studies to 
the Agency (June 2006). 
5  Contains Nonbinding Recommendations 
supporting the mechanism of action should report the inhibitory concentration values (IC  and 
50
IC ). 
90
 
  Antiviral activity data from cell culture studies 
 
The sponsor should characterize antiviral activity of an investigational drug in cell culture to 
identify a target plasma concentration for evaluation in CMV-infected patients.  The sponsor 
should assess the antiviral activity of investigational drugs by using CMV laboratory isolates as 
well as several (more than 20) geographically and temporally distinct isolates, the majority of 
which should be U.S. isolates.  The 50 percent and 90 percent effective concentration values 
(EC  and EC ) should be determined.  These studies should include different CMV types (i.e., 
50 90
the four gB (UL55) genotypes (gB1 through gB4) and the two gH (UL75) genotypes (gH1 and 
gH2)).  Additional analyses with worldwide isolates are encouraged.  If differences in 
susceptibility are observed for different clinical isolates, the sponsor should conduct additional 
genotypic and phenotypic characterizations to identify genetic polymorphisms that may affect 
CMV susceptibility to the investigational drug.  The sponsor should also assess sequestration of 
the drug by serum proteins and determine a serum-adjusted EC  value.  We recommend 
50
evaluating the drugs antiviral activity at different concentrations of human serum and 
extrapolating the EC  value in the presence of 100 percent human serum. 
50
 
  Combination antiviral activity relationships 
 
If future combination therapy is anticipated, early in development the sponsor should 
characterize combination antiviral activity relationships between the investigational drug and 
approved drugs for CMV. The sponsor should identify any combinations for which the 
investigational drug is antagonistic by using cell culture assays.  The sponsor should also assess 
each component of a drug that contains multiple novel agents (e.g., combinations of monoclonal 
antibodies) individually for antagonism with approved drugs.  For all combination antiviral 
activity assessments, the sponsor should provide combination index values when the two agents 
are combined at their individual EC  values, and studies should include controls for 
50
cytotoxicity.  The sponsor should also assess combination antiviral activity relationships for 
nucleos(t)ide and deoxynucleos(t)ide CMV investigational drugs with approved nucleos(t)ide 
and deoxynucleos(t)ide antiviral drugs targeting other viruses (e.g., hepatitis B virus (HBV), 
hepatitis C virus (HCV), and human immunodeficiency virus (HIV-1)), as appropriate, before 
testing combinations of the agents in co-infected patients. 
 
  Cytotoxicity and mitochondrial toxicity 
 
The sponsor should quantify directly the cytotoxic effects of the drug, including those that target 
the host, for the cells used to assess CMV antiviral activity.  A 50 percent cytotoxic 
concentration (CC ) should be determined, and the therapeutic index (CC  value/EC  value) 
50 50 50
should be calculated.  The sponsor should assess cytotoxicity by using various human cell lines 
and primary cells cultured under proliferating conditions for several cell divisions and 
nonproliferating conditions. 
 
6  Contains Nonbinding Recommendations 
The sponsor should assess mitochondrial toxicity in both glucose- and galactose-containing 
media (Marroquin et al. 2007).  In addition, for nucleoside analogs, the sponsor should evaluate 
the inhibition of mitochondrial ribonucleic acid polymerase as well as the potential inhibition of 
host DNA and/or RNA synthesis (Arnold et al. 2012).  Positive controls for mitochondrial 
toxicity studies should be relevant to the class of the investigational drug whenever possible. 
 
The sponsor should conduct these biochemical and cell-based assessments for potential cellular 
and mitochondrial toxicity as a complement to in vivo toxicology assessments and not in lieu of 
in vivo studies. Results from these studies should be interpreted in the context of the in vivo 
toxicology, nonclinical, and clinical pharmacokinetic data to help assess clinical risk. Antibodies 
specifically targeting viral proteins are unlikely to be cytotoxic and FDA typically would not 
recommend these assessments. 
 
  Considerations for antisense RNA and siRNA candidates 
 
Knockdown of viral protein expression via antisense RNA and siRNA has shown promise for the 
development of antiviral drugs.  Drugs of this nature, which bind to a nucleic acid target, present 
potential mismatch issues that could lead to species-specific toxicities not detected in classical 
toxicity studies.  Therefore, in addition to the cytotoxicity and toxicology assessments, we 
recommend that the following bioinformatic studies be conducted for drugs that target a nucleic 
acid: 
 
  Potential off-target matches should be identified in the human transcriptome, regardless 
of tissue expression.  For each of these, available information on mouse knockouts and 
human genetic diseases should be described.  A plan for monitoring for significant off-
target effects should be included in clinical trial protocols. 
 
  The conservation among the candidate off-target human genes should be determined with 
their respective mouse genes that are three or fewer mismatched bases different from the 
drug to determine if these sites are sufficiently conserved in the mouse such that toxicities 
related to off-target matches would be present in mice. 
 
  Potential off-target matches should be identified in the human mitochondrial 
transcriptome (see https://omictools.com/the-mitochondrial-genome-browser-tool or 
http://www.mtdb.igp.uu.se/, as well as other public sources for mitochondrial genome 
information). 
 
  The variation within the off-target matches should be determined in the transcriptomes of 
different populations in the United States to assess whether different populations would 
be more susceptible than others to off-target effects. 
 
  The effect of different mismatches with respect to off-target effects should be determined 
(i.e., comparing purine to purine versus other mismatches). 
 
7  Contains Nonbinding Recommendations 
  Antiviral activity in animal models12 
 
Demonstrating CMV antiviral activity in an animal model is not required.  However, if such 
studies are conducted and provided as part of nonclinical development, reported data should 
include the CMV strain designation, gB (UL55) and gH (UL75), genotypes (as well as any other 
CMV genes used for characterization, if known (Puchhammer-Stockl and Gorzer 2006)), the 
EC  value of the challenge virus, time course plots of viral load data for each animal, and an 
50
assessment of resistance development. 
 
  Resistance and cross-resistance 
 
The ability of CMV to develop resistance when subjected to drug pressure should be examined 
in appropriate cell culture models selecting and characterizing genotypically and phenotypically 
several independent resistant isolates.  The sponsor should determine and validate amino acid 
substitutions associated with the development of resistance to the investigational drug by 
introducing the changes into the CMV genome (e.g., using bacterial artificial chromosome 
technology) and determining the fold-shift in susceptibility relative to the parental strain using 
appropriate cell culture and/or biochemical assays.  Results from these studies should be used to: 
(1) characterize the genetic barrier to resistance (e.g., number of mutations required to confer 
reduced susceptibility, number of passages); (2) predict whether the genetic barrier for resistance 
may vary as a function of concentration of the investigational drug; (3) reveal potential resistance 
pathways and the potential for cross-resistance with other anti-CMV drugs; (4) assess the 
potential effect of polymorphisms at amino acid positions associated with resistance using 
available sequence databases; (5) provide preliminary information on assays that may be used in 
clinical studies; and (6) support the drugs hypothesized mechanism of action.  Resistant viruses 
selected in cell culture can provide important controls for assessing clinical isolates 
phenotypically. 
 
Resistance studies should include evaluation of the potential for cross-resistance, both to 
approved drugs and to drugs in development (when possible), particularly focusing on those in 
the same drug class and other classes with the same viral target.  The sponsor should determine 
the antiviral activity of approved drugs against viruses resistant to the investigational drug and 
the antiviral activity of the investigational drug against viruses resistant to approved drugs.  The 
resistance and cross-resistance studies may be important to support studies in patients who have 
developed resistance to approved treatments. 
 
Some deoxynucleoside analogs for treating CMV infection have also been found to have 
antiviral activity against HIV-1 and can select for resistant variants (Tachedjian et al. 1995; 
Lisco et al. 2008; McMahon et al. 2008).  Sponsors of such drugs should determine the cell 
culture antiviral activity of the active moiety against HIV-1 because these may be used in HIV-1-
infected patients.  If the drug demonstrates antiviral activity, the sponsor should determine the 
development of resistance to the investigational drug genotypically and phenotypically by 
                                                 
12 We support the principles of the 3Rs (reduce/refine/replace) for animal use in testing.  FDA encourages sponsors 
to consult with review divisions when considering a nonanimal testing method believed to be suitable, adequate, 
validated, and feasible.  FDA will consider if the alternative method could be assessed for equivalency to an animal 
test method. 
8  Contains Nonbinding Recommendations 
selecting resistant HIV-1 variants.  Resistance studies should include evaluation of cross-
resistance to approved nucleos(t)ide reverse transcriptase inhibitors for HIV-1. 
 
  Targeting host factors 
 
For drugs targeting host factors, the sponsor should assess polymorphisms in the human 
population to determine whether the drug will be more or less effective in different populations.  
If a nonclinical assay to assess the drug effect is available (e.g., assays that are used to evaluate 
the binding activity of drugs targeting a specific receptor), the sponsor should evaluate the target 
host protein/factor from each of the key racial groups in the United States to determine whether 
race may be a factor in efficacy.  Additionally, the sponsor should collect samples during clinical 
trials to determine the genotype of subjects who respond less favorably to treatment.  We 
recommend that drugs targeting host functions be evaluated in animal models to demonstrate 
activity and assess for the potential for toxicities in infected animals. 
 
  Developing monoclonal antibodies 
 
The development of monoclonal antibodies (mAbs) for treating or preventing CMV disease 
should follow the same recommendations described above.  In addition, the sponsor should 
assess the conservation (identity) at each amino acid position for the mAb binding site in 
available CMV sequence data for each CMV type/subtype as well as the dependence of binding 
upon the target proteins conformation.  The sponsor should also identify the amino acid residues 
that may affect susceptibility for any isolates showing reduced susceptibility in cell culture 
studies.  Sponsors developing monoclonal antibodies should evaluate any potential detrimental 
effects of the antibody, such as antibody-dependent enhancement of infection (Manley et al. 
2011), and Fc-mediated, complement-dependent cytotoxicity. 
 
c.  General considerations for phase 1 and phase 2 clinical development 
 
In general, the sponsor should conduct phase 1 trials to assess pharmacokinetics and safety of the 
investigational drug and, when possible, antiviral activity.  Phase 2 trials should characterize 
doses of the investigational drug with regard to both antiviral activity and safety for further study 
in phase 3 trials.  Specific trial design issues for CMV drug development depend on the intended 
indication and the intended patient population (SOT or HSCT recipients). 
 
The following recommendations and examples provide information for potential phase 1 and 
phase 2 trial designs for CMV antiviral drugs. 
 
  Phase 1a/first-in-human studies 
 
For the first-in-human studies, we recommend studies in healthy adult subjects to assess safety, 
pharmacokinetics, and the ability to achieve target concentrations based on cell culture antiviral 
activity studies.  The sponsor can also conduct single-dose and short-duration multiple-dose 
pharmacokinetic studies in subjects at risk for CMV disease (e.g., immunocompromised hosts), 
particularly if nonclinical data indicate that a drug may be genotoxic or otherwise unacceptable 
for studies in healthy volunteers. 
9  Contains Nonbinding Recommendations 
 
  Phase 2 proof-of-concept trials  
 
For other antiviral drugs (e.g., drugs for treating HIV-1, HBV, or HCV infection), proof of 
concept for antiviral activity generally is demonstrated via short-term administration of the 
investigational drug to chronically infected patients with measurable levels of circulating virus.  
A reduction from baseline in plasma viral load over days or weeks is assessed to establish initial 
antiviral activity and to evaluate exposure-response relationships.  For anti-CMV drugs, proof-
of-concept trials may be somewhat more challenging because transplant recipients with CMV 
DNAemia are typically started immediately on antiviral treatment and generally would not be 
considered candidates for delaying approved treatments to participate in short-term monotherapy 
trials of investigational drugs without proven activity in humans. 
 
Phase 2 trial design options to demonstrate proof of concept could include evaluation of 
reductions in CMV DNAemia or CMV replication in other compartments in patients with 
measurable virus with or without overt disease.  Selection of patients and concomitant treatment 
are key considerations to avoid situations in which patients would not receive adequate standard 
of care (SOC).  Examples of such designs include: 
 
  Randomized, placebo-controlled, dose-ranging trial in which the investigational drug or 
placebo is added to SOC treatment (e.g., ganciclovir) or, in some cases, could be directly 
compared with SOC treatment in patients being treated for CMV viremia.  The treatment 
period would be short (2 to 3 weeks), with a switch to SOC for the remaining duration of 
therapy.  Antiviral activity is assessed as the degree of reduction in plasma CMV 
DNAemia from baseline after 2 to 3 weeks of treatment or proportion of patients with 
CMV DNAemia below the lower limit of quantitation (LLOQ) (target not detected or 
target detected) at a specified time point or rate of reduction of CMV DNA.  A similar 
proof-of-concept trial could also be conducted in patients with CMV DNAemia that is 
resistant to SOC therapy. 
 
  Assessment of antiviral activity in renal transplant recipients at low risk for progression 
to tissue-invasive CMV disease (e.g., D-/R+) with CMV viruria or low-level CMV 
viremia in a placebo-controlled trial with a switch to rescue therapy for progressive 
viremia above a prespecified threshold may be feasible in some settings. 
 
  Randomized, placebo-controlled, dose-ranging trial to measure reductions in CMV 
shedding in semen or in urine in asymptomatic patients with underlying immune 
suppression, such as HIV-1 infection, who generally would not be treated for 
asymptomatic CMV infection. 
 
Before adding the investigational drug to other approved therapies, the sponsor should assess the 
potential for drug-drug interactions, as drug interaction studies may be recommended if there is a 
likelihood of a pharmacokinetic interaction. 
 
Ideally, doses selected for early phase 2 trials should provide plasma and/or tissue drug 
exposures that exceed by severalfold the protein binding-adjusted, cell culture EC  value of the 
50
10  Contains Nonbinding Recommendations 
drug.  When evaluating doses, the sponsor should also take into account any safety margins 
previously identified in both animal toxicology studies and studies conducted in healthy 
volunteers. 
 
The sponsor can use results from proof-of-concept antiviral activity trials to guide dose selection 
for subsequent phase 2b or phase 3 trials in which anti-CMV therapy is studied for longer 
durations. 
 
  Phase 2b trials 
 
The same trial designs discussed for phase 3 (section III. B., Phase 3 Efficacy Trial 
Considerations) could be used for phase 2b trials; however, phase 2b trials generally should 
include more doses and fewer subjects per arm compared with phase 3 trials.  The primary goal 
in phase 2b trials should be to determine doses and durations based on safety and efficacy 
considerations for further evaluation in phase 3 trials.  Further dose discrimination for efficacy 
and safety can be evaluated in phase 3 trials with greater statistical power to detect smaller 
differences. 
 
Trial randomization should be stratified according to baseline characteristics predicted to have a 
significant effect on treatment outcome (e.g., donor and recipient CMV serostatus).  Initial trials 
should include frequent CMV virologic monitoring and individual and trial stopping rules for 
poor virologic outcomes (e.g., virologic breakthrough or relapse or progression to CMV disease).  
Protocols should include opportunities for patients with virologic failure or clinical progression 
to receive appropriate therapeutic rescue regimens.  The sponsor should collect and include in 
final study reports and/or other appropriate regulatory submissions efficacy outcome data from 
all subjects, including those who received a therapeutic rescue regimen or regimens, as these data 
could be informative for future clinical trials.  As safer and more tolerable and efficacious drugs 
become available, we anticipate that the risk-benefit considerations for patient populations will 
evolve. 
 
Specific information recommended to support phase 3 trials includes: 
 
  Single- and multiple-dose pharmacokinetics and safety in healthy subjects or other 
populations, as appropriate 
 
  Antiviral (anti-CMV) activity data from phase 2 clinical trials 
 
  Human safety data in approximately 100 patients for the highest dose that will be 
evaluated further in phase 3 trials 
 
  Data from clinical trials or other sources supporting the proposed doses and duration of 
study drug chosen for further study 
 
  Drug-drug interaction data if in vitro and/or in vivo study results suggest a potential for a 
drug interaction with other drugs likely to be used concomitantly in phase 3 trials 
 
11  Contains Nonbinding Recommendations 
For an end-of-phase 2 meeting, efficacy and safety data from each of the regimens under study in 
phase 2 trials should be available to select drug regimens and patient populations for study in 
phase 3. 
 
2.  Drug Development Population 
 
The drug development population for efficacy trials should be transplant recipients at risk for 
CMV disease, including: 
 
  HSCT recipients 
  SOT recipients, including kidney, liver, heart, lung, pancreas, and other SOT recipients 
 
FDA may recommend supportive data to define safety and pharmacokinetics before trials are 
conducted in specific subgroups.  This may include data from hepatic or renal impairment trials 
and drug-drug interaction trials (e.g., drug-drug interaction trials with immunosuppressants used 
post-transplantation). 
 
Trials should include adequate U.S. subject representation to ensure the applicability of trial 
results to the U.S. population.  An adequate representation of sexes, races, ages, and virus types 
is also recommended during drug development.  Sponsors should share their pretrial initiation 
work with the FDA to ensure the sites selected have a sufficient number of subjects from these 
populations (e.g., women, Black/African Americans, Hispanic/Latinos, Asian Americans) to 
enroll in phase 2 and phase 3 clinical trials.  Extending trial site enrollment caps to allow for 
enrollment of underrepresented populations can also help to increase trial diversity. 
 
3.  Efficacy Considerations 
 
Sponsors can submit a marketing application to gain approval of a drug for a single indication 
(prophylaxis or treatment) in one or more populations or can submit a marketing application for 
multiple indications.  Generally, applications include at least two adequate and well-controlled 
trials.13  However, two trials may not be needed for every indication and population.  Generally, 
FDA would consider trials for different indications (prophylaxis or treatment) and in different 
populations (HSCT or SOT recipients) supportive of each other.  Sponsors should consult the 
guidance for industry Providing Clinical Evidence of Effectiveness for Human Drug and 
Biological Products (May 1998) regarding circumstances in which one phase 3 clinical trial may 
be supportive of approval. 
 
                                                 
13 See section 505(d) of the Federal Food, Drug & Cosmetic Act (FD&C Act)(21 U.S.C. 355(d)). 
 
12  Contains Nonbinding Recommendations 
Because CMV disease in transplant recipients is considered serious and life-threatening, and 
because only limited therapeutic options are available to treat or prevent CMV disease, 
investigational drugs for CMV may be eligible for fast track, priority review, or breakthrough 
therapy designation.14 
 
4.  Safety Considerations  
 
The FDA recommends that sponsors engage in early discussions with the DAVP on trial designs 
as well as on the proposed size of the safety database that depends on the patient population and 
proposed indication.  Because CMV disease is serious and life-threatening in 
immunocompromised patients, a safety database of 300 to 500 patients who received the 
proposed dose and duration of the drug generally should be sufficient to assess risk-benefit for an 
initial marketing application.  FDA may recommend a safety database with fewer patients for 
investigational drugs that demonstrate substantial improvement in efficacy and safety compared 
with currently available therapeutic options.  On occasion, specific findings from nonclinical or 
clinical development may lead FDA to recommend a larger safety database to adequately 
evaluate potential drug toxicity.  If significant safety signals emerge during drug development, 
we may recommend that the safety database be increased or that specific safety studies be 
conducted.  For marketing applications containing trials evaluating treatment of CMV disease in 
patients who have failed or developed resistance to approved treatments, a safety database of 
approximately 300 patients may be recommended. 
 
Ideally, safety data from controlled and comparative trials are recommended to assess the safety 
of an investigational drug.  We recommend that sponsors provide controlled and comparative 
safety data to an approved and clinically accepted SOC treatment (or placebo, if appropriate).  In 
some situations, we may recommend uncontrolled or historically controlled data as supportive 
data for marketing applications. 
 
B.  Phase 3 Efficacy Trial Considerations 
 
1.  Trial Design 
 
Phase 3 trial design depends on the proposed indication or indications and the intended 
population or populations for use.  Following are examples of trial designs that could be 
considered for evaluating CMV antiviral therapy in transplant recipients. 
 
a.  Preventing CMV disease 
 
Preventing CMV disease in transplant recipients includes both prophylaxis (administering an 
anti-CMV drug to at-risk subjects with no evidence of CMV DNAemia or CMV disease) and 
preemptive therapy (preventing CMV disease by treating subjects with CMV DNAemia).  The 
                                                 
14 See sections 506(a) and (b) of the FD&C Act (21 U.S.C. 356(a) and (b)) and the current Prescription Drug User 
Fee Act goals letter, which is available at 
https://www.fda.gov/downloads/forindustry/userfees/prescriptiondruguserfee/ucm511438.pdf.  For more 
information, see the guidance for industry Expedited Programs for Serious Conditions  Drugs and Biologics (May 
2014). 
13  Contains Nonbinding Recommendations 
following sections discuss examples of trial designs for CMV prophylaxis or preemptive therapy 
in SOT or HSCT populations. 
 
  CMV prophylaxis trials in SOT recipients 
 
Examples of clinical trial designs that can be considered for evaluating CMV prophylaxis in SOT 
recipients include the following: 
 
  Noninferiority Trials.  In a randomized, double-blinded, active-controlled trial, high-risk 
(D+/R-) SOT recipients would be randomized to receive the SOC regimen or the 
investigational drug for at least 100 days (200 days for kidney transplant recipients) post-
transplantation.  The primary endpoint would be the proportion of subjects who develop 
CMV disease (CMV syndrome or tissue-invasive CMV disease).  The duration of follow-
up depends on the duration of prophylaxis, type of organ transplant, and other factors, 
such as expected timing of immune recovery post-transplantation.  In general, subjects 
should be followed for an adequate time to ensure that they are not at increased risk for 
late-onset CMV disease. 
 
The size of the noninferiority margin would depend on the specific patient population 
being studied as well as other factors.  Sponsors should discuss with the DAVP their 
justification for the proposed noninferiority margin, the proposed trial design, the data 
analysis plan, and plans for long-term follow-up postmarketing.  See the Appendix for 
additional considerations regarding clinical trials to evaluate CMV prophylaxis in liver 
transplant recipients. 
 
  Superiority Trials.  In a randomized, double-blinded superiority trial, the SOC would be 
used as comparator.  Alternatively, in an add-on superiority trial, transplant recipients 
would be randomized to receive the investigational drug plus SOC or SOC alone.  The 
primary endpoint would be the incidence of CMV disease. 
 
  CMV prophylaxis trials in HSCT recipients 
 
Examples of clinical trial designs that can be considered for evaluating CMV prophylaxis in 
HSCT recipients include the following: 
 
  Noninferiority Trials.  In a randomized, double-blinded, active-controlled trial, high-risk 
(CMV seropositive) HSCT recipients would be randomized to receive the SOC regimen 
or the investigational drug for at least 100 days post-transplantation.  The primary 
endpoint would be a composite endpoint defined as the occurrence of either tissue-
invasive CMV disease or the initiation of anti-CMV preemptive therapy based on 
clinically significant CMV DNAemia.  The endpoint would likely be driven by the 
incidence of CMV DNAemia.  The FDA considers CMV viremia (DNAemia) a validated 
surrogate endpoint whose use in efficacy studies would enable the Agency to grant 
traditional approval for NDAs for prophylaxis trials in HSCT recipients. 
 
14  Contains Nonbinding Recommendations 
  Superiority Trials.  A superiority trial of the investigational drug using a blinded 
comparison against the SOC may be appropriate in CMV seropositive HSCT recipients.  
Enrolled transplant recipients would be randomized to receive SOC or the investigational 
drug for at least 100 days post-transplantation or until a time when most patients are 
expected to achieve immune recovery.15  Alternatively, in an add-on superiority trial, 
transplant recipients would be randomized to receive the investigational drug plus SOC 
or SOC alone. The primary endpoint would be a composite endpoint, as defined above. 
 
  Preemptive therapy in SOT or HSCT recipients 
 
Preemptive therapy (antiviral therapy initiated when CMV DNAemia is detected at a level above 
a predetermined threshold without evidence of tissue-invasive CMV disease or CMV syndrome) 
depends on frequent and regular monitoring for CMV DNAemia.  The goal of preemptive 
therapy is to prevent tissue-invasive CMV disease.  In the past, establishing universal 
quantitative viral thresholds for initiation of preemptive therapy has been difficult because of 
differences in assay performance and sample type (whole blood versus plasma) but may now be 
more feasible with the publication of the World Health Organization standard for CMV DNA 
quantification (Fryer et al. 2010) and with the availability of approved assays.16 
 
Some examples of preemptive therapy trial designs that could be used in these populations 
include: 
 
  Superiority Trials.  Superiority trials of the investigational drug versus intravenous 
ganciclovir or oral valganciclovir, or add-on superiority trials in which subjects are 
randomized to the investigational drug or placebo added to an SOC background therapy 
(e.g., intravenous ganciclovir or oral valganciclovir), may be feasible.  In superiority 
trials for this indication, efficacy could be assessed using the clinical endpoint of the 
absence of CMV disease (tissue-invasive CMV disease or CMV syndrome in SOT 
recipients or tissue-invasive CMV disease in HSCT recipients) or by using a composite 
endpoint (undetectability of CMV DNAemia and absence of CMV disease at a specific 
time point). 
 
Other trial designs could include duration of treatment or dose-ranging superiority trials 
in which shorter and longer durations of treatment or higher versus lower doses are 
compared.  Superiority of the longer duration or of the higher dose would demonstrate 
efficacy of the investigational drug. 
 
b.  Treating CMV disease 
 
The following section discusses considerations for clinical trial design for treating CMV disease 
in SOT or HSCT recipients, including treating CMV infections resistant or refractory to current 
SOC therapy. 
 
                                                 
15 Other treatment durations may be proposed based on scientific rationale. 
 
16 https://www.fda.gov/medical-devices/vitro-diagnostics/nucleic-acid-based-tests. 
15  Contains Nonbinding Recommendations 
  Treating CMV disease in SOT and HSCT recipients 
 
In the SOT setting, CMV disease refers to either tissue-invasive CMV disease or CMV 
syndrome, as defined in section III. B. 8., Efficacy Endpoints.  In HSCT recipients, CMV disease 
refers only to tissue-invasive CMV disease. 
 
Options for trial designs for CMV disease treatment trials in either SOT or HSCT recipients 
include: 
 
  Superiority Trials.  Trials to demonstrate superiority to SOC therapy, or add-on 
superiority trials in which subjects are randomized to the investigational drug or placebo 
added to an SOC therapy (e.g., intravenous ganciclovir or oral valganciclovir), are 
feasible and appropriate.  The primary endpoints would include both resolution or 
improvement of clinical signs and symptoms of CMV disease and CMV DNAemia below 
LLOQ (target not detected or target detected). 
 
  Noninferiority Trials.  No antiviral drugs have been approved for treating CMV disease 
in SOT or HSCT recipients.  Therefore, noninferiority trials are not feasible for this 
indication unless the treatment effect for the SOC anti-CMV therapy over placebo can be 
determined for treating CMV disease in these populations to support a noninferiority 
margin. 
 
  Treating CMV infections resistant or refractory to CMV antiviral drugs in transplant 
recipients 
 
Trials for treating CMV infections resistant or refractory to treatment with available drugs (e.g., 
ganciclovir/valganciclovir, foscarnet) could include treating CMV disease or CMV viremia. 
Definitions of resistant and refractory CMV infection for use in trials in this population should 
be discussed with and agreed upon by DAVP.  For trials that include both groups of patients 
(resistant and refractory to treatment), the sponsor should demonstrate statistical significance in 
the overall population.  Efficacy in the subgroups of patients who are resistant or refractory to 
CMV antiviral drugs should be consistent with the overall treatment effect. 
 
Trial design options for these populations would include a superiority trial comparing the 
investigational drug versus SOC therapy or an add-on superiority trial comparing the 
investigational drug plus SOC versus SOC treatment alone (if the two drugs did not demonstrate 
antagonism in combination antiviral activity assessments). 
 
2.  Trial Population 
 
As mentioned, this guidance focuses on treating or preventing CMV disease in SOT and HSCT 
recipients.  Some of the specific issues with regard to trial population for these indications are 
discussed below: 
 
16  Contains Nonbinding Recommendations 
  CMV Prophylaxis in SOT Recipients.  For trials evaluating an investigational drug for 
CMV prophylaxis in SOT recipients, patients should be high risk based on CMV 
serostatus (D+/R-). 
 
  CMV Prophylaxis in HSCT Recipients.  Trials of the investigational drug versus SOC 
should be conducted in CMV seropositive (R+) HSCT recipients, who are at the highest 
risk for CMV infection and disease. 
 
  Preemptive Therapy in SOT or HSCT Recipients.  Preemptive therapy can be studied in 
any transplant recipient who has evidence of CMV DNAemia at levels above a 
prespecified threshold. 
 
  Treating CMV Disease.  Any SOT or HSCT recipient with CMV disease, regardless of 
CMV serostatus of donor and recipient, could be included in treatment trials.  In trials 
evaluating treatment in SOT recipients, a sufficient number of subjects with tissue-
invasive CMV disease should be enrolled (and not just those with CMV syndrome) to 
support an indication for treating CMV disease. 
 
  Treating CMV Infections Resistant or Refractory to CMV Antiviral Drugs in Transplant 
Recipients.  Any SOT or HSCT recipient with CMV infection or disease resistant or 
refractory to available CMV antiviral drugs could be included in these trials. 
 
3.  Entry Criteria 
 
Following are specific considerations for trial entry criteria for CMV treatment or prevention 
trials: 
 
  Prophylaxis Trials in SOT or HSCT Recipients.  To be enrolled in a CMV prophylaxis 
trial, the transplant recipient should have no detectable CMV infection post-
transplantation, as documented by CMV DNA testing with PCR in plasma (less than 
LLOQ, target not detected), within 5 days before initiation of therapy. 
 
  Preemptive Therapy Trials in SOT or HSCT Recipients.  In clinical practice, virologic 
thresholds for initiating preemptive therapy in HSCT recipients have been based on 
preestablished risks for CMV disease (Boeckh and Ljungman 2009).  For clinical trials, 
optimal virologic thresholds for initiating preemptive therapy have not been established.  
Proposed virologic thresholds for initiating preemptive therapy for CMV viremia in 
clinical trials should be discussed with and agreed upon by the DAVP. 
 
  Treatment Trials in SOT or HSCT Recipients with CMV Disease.  To be enrolled in a 
CMV treatment trial, transplant recipients should have virological evidence of CMV 
replication with signs and symptoms of CMV syndrome or tissue-invasive CMV disease 
(SOT recipients) or with clinical evidence of tissue-invasive CMV disease (HSCT 
recipients). 
 
17  Contains Nonbinding Recommendations 
  Treatment Trials in Patients with CMV Infections Resistant or Refractory to CMV 
Antiviral Drugs.  CMV isolates at baseline should have evidence of resistance to CMV 
antiviral drugs by genotypic analysis.  Patients with CMV disease refractory to treatment 
can be included, but the inclusion criteria for subjects refractory to therapy should be 
rigorously defined in the protocol. 
 
4.  Randomization, Stratification, and Blinding 
 
Sponsors should conduct randomized, double-blinded trials whenever feasible.  For add-on 
superiority trials of an investigational drug added to SOC therapy compared with SOC therapy 
alone, subjects randomized to the latter should receive a matching placebo. 
 
Sponsors designing trials in which blinding may be difficult or infeasible should discuss their 
proposals with the DAVP in advance to review potential modifications that might facilitate 
blinding and to discuss the potential effect of open-label therapy on interpretation of results. 
 
Sponsors should consider stratifying subjects by important baseline risk factors for CMV 
infection/disease in HSCT recipients, such as CMV serostatus of donor and recipient and other 
factors associated with risk of CMV disease.  For SOT recipients, sponsors should consider 
stratifying by CMV serostatus of donor and recipient and the type of transplant (e.g., kidney, 
liver, lung). 
 
In trials that include both SOT and HSCT recipients, sponsors should consider stratifying by type 
of transplant (SOT or HSCT). 
 
5.  Pediatric Populations 
 
Sponsors are encouraged to begin discussions about their pediatric formulation and clinical 
development plan early in development because pediatric clinical trials are a required part of the 
overall drug development program.17  Under the Pediatric Research Equity Act, sponsors must 
submit an initial pediatric study plan to the FDA no later than 60 days after the end-of-phase 2 
meeting.18 
 
FDA recommends that inclusion of pediatric patients in clinical trials generally can be initiated 
after sufficient safety, pharmacokinetic, and efficacy data are available from adults.  If clinical 
trials in adults have demonstrated no significant safety concerns that would preclude study in 
children, the FDA encourages evaluating adolescents using the adult dose and formulation 
(Momper et al. 2013).  However, sponsors should discuss with the DAVP the initial pediatric 
pharmacokinetic data and results of available modeling and simulation before selecting doses for 
                                                 
17 See section 505B(e) of the FD&C Act (21 U.S.C. 355c(e)). 
 
18 See section 505B(e)(2)(A) of the FD&C Act (21 U.S.C. 355c(e)(2)(A)) and the draft guidance for industry 
Pediatric Study Plans:  Content of and Process for Submitting Initial Pediatric Study Plans and Amended Initial 
Pediatric Study Plans (March 2016).  When final, this guidance will represent the FDAs current thinking on this 
topic. For the most recent version of a guidance, check the FDA guidance web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
 
 
18  Contains Nonbinding Recommendations 
pediatric treatment trials.  Depending on results of the adult clinical trials, and on whether 
efficacy in adults can be extrapolated to pediatric patients (i.e., if the course of disease and the 
effect of the drug are sufficiently similar in adults and pediatric patients), either comparative or 
single-arm trials may be recommended in pediatric subjects.19  Sponsors pediatric study plans 
should include information to support pediatric extrapolation if extrapolation is planned. 
 
6.  Dose Selection 
 
To guide optimal dose selection and treatment durations in phase 3 trials, sponsors should 
consider safety and efficacy results from previous trials and exposure-response relationships for 
safety and efficacy.  For treatment trials, we recommend that sponsors develop a mechanistic 
model of the kinetics of viral load reduction that can assist with the optimization of dose 
selection and treatment duration to reduce the risk of selecting for resistant viruses caused by 
subtherapeutic exposures.  Such a model should include a mechanistically appropriate targeted 
drug effect, components to describe virologic breakthroughs and virologic responses, and 
relevant covariates for describing differences in response.  When applicable, these mechanistic 
modeling approaches can use viral kinetic model structures and corresponding disease 
progression-parameter values from the literature. 
 
Sponsors can select a range of doses and treatment durations for phase 3 trials if there are 
uncertainties about the optimal regimen or if the model indicated a different dose or treatment 
duration to be better for certain subpopulations, such as patients having CMV with baseline 
ganciclovir resistance.  Sponsors can also consider an adaptive design for the dose selection. 
 
7.  Use of Active Comparators  
 
In general, the active comparator in a noninferiority trial should be an FDA-approved drug that is 
considered the SOC for the specific indication and population being studied.  Sponsors should 
justify proposed noninferiority margins and discuss them with the DAVP.  See the guidance for 
industry Non-Inferiority Clinical Trials to Establish Effectiveness (November 2016) for 
additional information on determining noninferiority margins. 
 
8.  Efficacy Endpoints 
 
FDA recommends the definitions for CMV infection and disease for use in clinical trials that are 
advocated by Ljungman and colleagues (Ljungman et al. 2017). 
 
a.  CMV prophylaxis trials in SOT recipients 
 
FDA recommends that the primary endpoint for trials of CMV prophylaxis in SOT recipients be 
a clinical endpoint of CMV disease and includes both CMV syndrome and tissue-invasive CMV 
disease measured at 6 or 12 months post-transplantation depending on duration of prophylaxis.  
                                                 
19 For additional information on pediatric extrapolation, see the draft guidance for industry General Clinical 
Pharmacology Considerations for Pediatric Studies for Drugs and Biological Products (December 2014).  When 
final, this guidance will represent the FDAs current thinking on this topic. 
19  Contains Nonbinding Recommendations 
The diagnosis of CMV syndrome and tissue-invasive CMV disease should be confirmed by an 
independent, blinded, clinical adjudication committee. 
 
FDA recommends that the secondary endpoints in CMV prophylaxis trials for SOT recipients 
could include but are not limited to the following.  However, only a limited number of such 
endpoints should be considered for testing using appropriate statistical methods for multiplicity: 
 
  The proportion of subjects with CMV disease at time points other than the time point 
used for the primary endpoint 
 
  The time to development of CMV disease 
 
  The proportion of subjects with investigator-treated CMV disease  
 
  The initiation of other anti-CMV therapy 
 
  The proportion of subjects with CMV DNAemia at different time points 
 
  The time to development of CMV DNAemia 
 
  Survival at different time points 
 
  The proportion of subjects experiencing biopsy-proven acute rejection 
 
  The proportion of subjects with graft loss 
 
  The proportion of subjects with opportunistic infections 
 
  The proportion of subjects developing genotypic changes associated with CMV resistance 
to the investigational drug 
 
b.  CMV prophylaxis trials in HSCT recipients 
 
FDA recommends that the primary endpoint for a phase 3 prophylaxis trial in HSCT recipients 
be a composite endpoint defined as the occurrence of CMV disease or the initiation of 
preemptive therapy based on clinically significant CMV DNAemia (as measured by a central 
virology laboratory) within 6 months post-transplantation. 
 
Viral load thresholds for initiating preemptive therapy should be based on the risks for CMV 
disease (Boeckh and Ljungman 2009).  FDAs recommendations regarding virologic thresholds 
for initiating preemptive therapy will depend on the assay and specimen (whole blood versus 
plasma), the risk of CMV infection/disease in the population under study, and individual patient 
risk factors.  Sponsors should agree upon virologic thresholds with the DAVP before initiating 
trials. 
 
FDA recommends that the secondary endpoints in CMV prophylaxis trials for HSCT recipients 
could include but are not limited to: 
20  Contains Nonbinding Recommendations 
 
  The proportion of subjects with tissue-invasive CMV disease 
 
  The proportion of subjects with CMV DNAemia 
 
  The time to onset of CMV infection (DNAemia)/tissue-invasive CMV disease through 6 
months or 12 months post-transplantation 
 
  Survival at 6 and 12 months post-transplantation 
 
  The proportion of subjects with opportunistic infections other than CMV infection 
 
  The proportion of subjects developing resistance to the investigational drug 
 
c.  CMV preemptive therapy trials in SOT or HSCT recipients 
 
FDA recommends that the primary endpoint for phase 3 trials of preemptive therapy in either 
SOT or HSCT patients be a composite endpoint defined as the proportion of subjects with CMV 
DNA less than LLOQ (target not detected or target detected) and absence of CMV disease at a 
prespecified time point after treatment initiation. 
 
d.  Treating CMV disease in SOT or HSCT recipients 
 
FDA recommends that the primary endpoint in a phase 3 trial in either SOT or HSCT recipients 
with tissue-invasive CMV disease (for SOT or HSCT) or CMV syndrome (for SOT) be the 
proportion of responders at a prespecified time point after treatment is initiated.  Response 
should include the following elements: 
 
  Substantial improvement/resolution of signs and symptoms of tissue-invasive CMV 
disease or CMV syndrome 
 
  CMV DNAemia less than LLOQ (target not detected or target detected) in two 
consecutive tests taken at least 5 to 7 days apart 
 
  No new occurrence of CMV disease at other sites 
 
  No evidence for relapse (CMV disease or DNAemia) within a prespecified time frame 
after stopping therapy 
 
Specific details regarding the primary endpoint should be discussed with and agreed upon by the 
DAVP. 
 
FDA recommends that the secondary endpoints could include but are not limited to: 
 
  The time to CMV DNA less than LLOQ (target not detected or target detected) 
 
21  Contains Nonbinding Recommendations 
  The time to resolution of signs and symptoms of tissue-invasive CMV disease or CMV 
syndrome 
 
  Patient survival 
 
  The development of opportunistic infections, graft rejection or failure 
 
  The development of antiviral resistance 
 
9.  Trial Procedures and Timing of Assessments 
 
For trials of investigational drugs for treating or preventing CMV disease in the post-transplant 
setting, rescue therapy for developing CMV disease or CMV viremia should be included in the 
protocol.  Quantitative CMV DNA should be measured frequently during clinical trials.  
Treatment of CMV disease should continue at least until CMV DNAemia is less than LLOQ 
(target not detected or target detected) for at least two consecutive measurements performed at a 
prespecified interval, and treatment duration should be recorded.  Sponsors should consider 
longer treatment based on the kinetics of viral load reduction because several logs of CMV may 
be present when an assay reports less than LLOQ (target detected).  In prophylaxis trials, CMV 
DNA should be monitored routinely during the trial and subjects should be monitored for 
development of signs and symptoms of CMV disease.  In treatment trials (including preemptive 
therapy), frequent monitoring of CMV DNA should continue after discontinuation of therapy to 
detect relapse of CMV viremia during the risk period. 
 
10.  Endpoint Adjudication 
 
Determination of tissue-invasive CMV disease and CMV syndrome endpoints should be 
adjudicated by an independent endpoint-assessment committee conducting a blinded review of 
clinical source data (Ljungman et al. 2017). 
 
11.  Statistical Considerations 
 
In general, sponsors should submit a detailed statistical analysis plan stating the trial hypotheses 
and analysis methods before initiating the trial.  Statistical analysis methods and issues are 
discussed in detail in the guidances for industry Providing Clinical Evidence of Effectiveness for 
Human Drug and Biological Products and Non-Inferiority Clinical Trials to Establish 
Effectiveness and the article Statistical Considerations on Subgroup Analysis in Clinical Trials 
(Alosh et al. 2015). 
 
a.  Analysis populations 
 
Generally, sponsors should include in the primary efficacy analysis all subjects who are 
randomized and receive at least one dose of assigned therapy during the trial.  However, if a 
substantial proportion of randomized subjects do not receive treatment in either or both arms, 
then FDA may recommend additional analyses. 
 
22  Contains Nonbinding Recommendations 
b.  Efficacy analyses 
 
The primary efficacy analyses in prophylaxis trials in SOT recipients should compare the 
incidence of CMV disease within 6 or 12 months post-transplantation across treatment arms. 
 
The primary efficacy analyses in prophylaxis trials in HSCT recipients should compare the 
incidence of tissue-invasive CMV disease and clinically significant CMV DNAemia requiring 
initiation of preemptive therapy within 6 months post-transplantation across treatment arms. 
 
The primary efficacy analyses in preemptive therapy trials should compare the proportion of 
SOT recipients or HSCT recipients with CMV DNAemia below LLOQ (target not detected or 
target detected) in the absence of CMV disease at a prespecified time point across treatment 
arms. 
 
For subgroups, the analysis of the primary efficacy endpoint should be performed for important 
demographic and baseline characteristics (e.g., geographic region (United States, non-United 
States), sex, race, age group, high- versus low-risk group, donor CMV serostatus (D+ or D-), 
recipient CMV serostatus (R- or R+)).  The purpose of these analyses is to explore the 
consistency of the primary efficacy endpoint result across these subgroups. 
 
c.  Handling of missing data 
 
Sponsors should make every attempt to limit loss of subjects from the trial.  We recommend that 
sponsors collect detailed data on reasons for trial discontinuation (e.g., opportunity to enter 
another trial offering a promising new treatment, death or events leading to death, disease 
progression, adverse events, loss to follow-up, withdrawal of consent, noncompliance, 
pregnancy, protocol violations, not discontinued or not known to be discontinued but data were 
missing at the final visit).  For subjects who discontinue treatment early, sponsors should 
determine if these subjects switched treatments or added additional therapy. 
 
Analyses excluding subjects with missing data or other post-treatment outcomes can be biased 
because subjects who do not complete the trial may differ substantially in both measured and 
unmeasured ways from subjects who remain in the trial.  The method of how missing data will 
be handled should be prespecified in the protocol or the statistical analysis plan.  FDA may 
recommend sensitivity analyses to demonstrate that the primary analysis results are robust to the 
assumptions regarding missing data. 
 
12.  Accelerated Approval Considerations 
 
As explained above, FDA considers CMV viremia (DNAemia) a validated surrogate endpoint 
for use as part of a composite endpoint that includes a clinical component to support traditional 
approval; therefore, accelerated approval regulations generally are not applicable for CMV 
treatment and prevention indications. 
 
23  Contains Nonbinding Recommendations 
C.  Other Considerations 
 
1.  Clinical Virology Considerations 
 
Sponsors should use an FDA-approved assay to quantify CMV DNA in plasma.  We recommend 
that CMV DNA in whole blood also be quantified for short-term monotherapy studies because 
this may improve sensitivity to detect antiviral activity.  Additionally, plasma CMV DNA has 
been shown to be highly fragmented, so care should be taken when interpreting the CMV DNA 
levels (Boom et al. 2002).  Virology analyses should be conducted at a central virology 
laboratory. 
 
Proof-of-concept and efficacy trials should assess the development of CMV genotypic resistance 
to the investigational drug.  In prophylaxis trials, resistance testing should be performed for 
subjects who have detectable CMV DNA at any time point or confirmed diagnosis of CMV 
disease, regardless of viral load.  Observations of particular interest that should be reported 
include multiple occurrences of substitutions from the reference sequence or sequences at highly 
conserved amino acid residues, substitutions at positions identified in cell culture selection 
studies and treatment trials, and multiple occurrences of unusual substitutions at polymorphic 
residues. 
 
In treatment trials, sponsors should perform resistance testing for subjects who demonstrate 
virologic breakthrough (defined as a greater than or equal to 1 log  increase in CMV DNA 
10
above nadir, or detectable CMV DNA, while on treatment, after an initial drop to undetectable), 
an incomplete antiviral response (e.g., detectable CMV DNA at end of treatment or slower rate 
of decline than the average response), a decline to a plateau viral load decay phase, or virologic 
relapse after treatment cessation.  Sponsors should include a proposal of the subjects to be 
evaluated for resistance in their resistance analysis plans.  Any amino acid changes, including 
mixtures, in the coding sequence of the targeted genome region present in on-treatment or 
follow-up samples, but not in the baseline sample, should be reported as having developed 
during therapy.  In addition, sponsors should analyze baseline samples to identify CMV genetic 
polymorphisms that are associated with differential antiviral activity with the new investigational 
drug. 
 
Sponsors should consider genotyping regions outside the direct CMV genome target depending 
on the characteristics of the antiviral drug and interactions of the target with other viral proteins 
or whole genome sequencing, if viral loads are adequate.  In cases when resistance is suspected 
based on viral DNA kinetics but genotypic evidence of resistance is not detected, sponsors 
should also consider performing additional genotypic analyses using a method sufficiently 
sensitive to detect minority variants (e.g., next-generation sequencing).  Ganciclovir/ 
valganciclovir resistance-associated substitutions have been detected in specific compartments 
exclusively and not in blood.  Therefore, sponsors should also consider genotyping samples 
collected from specific compartments. 
 
Viral resistance-associated substitutions and baseline polymorphisms affecting response 
observed in clinical trials but not identified and characterized in nonclinical virology experiments 
should be evaluated phenotypically by introducing the changes into the CMV genome and 
24  Contains Nonbinding Recommendations 
determining the conferred fold-shift in susceptibility to the drug using appropriate cell culture 
and/or biochemical assays.  In addition, sponsors should perform phenotypic analyses using 
baseline and on-treatment clinical isolates from a subset of trial subjects representative of the 
CMV genetic diversity and virologic responses observed in clinical trials.  Phenotypic assays 
should include wild-type reference virus and resistant virus (initially from cell culture selection 
studies) controls. 
 
For quantification of CMV DNA, we recommend that sponsors use an FDA-approved PCR assay 
or assays using a central laboratory.  Sponsors should collect results from local laboratory tests, 
identifying the assay or assays used.  If investigational assays are used, performance 
characteristics with geographically and temporally distinct isolates should be provided.  Sponsors 
should report values that are less than LLOQ as less than LLOQ, target not detected or less 
than LLOQ, target detected, as appropriate. 
 
The FDA performs independent assessments of virologic and resistance data.  Before submitting 
virology datasets, sponsors should consult with the DAVP to obtain information on the most 
recent format and, in the case of next-generation sequence analysis, the procedure for submitting 
FASTQ files. 
 
2.  Pharmacokinetic/Pharmacodynamic Considerations 
 
Sponsors should assess pharmacokinetics and the relationship between systemic drug exposure 
and virologic or clinical endpoints and safety.  Virologic or clinical endpoints to be used for 
analyses depend on the proposed indication and trial designs. 
 
Sponsors can use a combination of intensive and sparse blood sampling throughout development 
to characterize the pharmacokinetics of the investigational drug.  An intensive sampling schedule 
is recommended in early-phase trials.  In longer-term/late phase trials, however, an intensive 
blood sampling schedule might not be feasible or may be feasible only in a subset of subjects or 
over a limited period of time.  Sponsors should obtain sparse pharmacokinetic samples from as 
many subjects in longer-term/late phase duration trials as possible and can combine the 
pharmacokinetic data from these trials with intensive pharmacokinetic data from earlier trials for 
analysis. 
 
Sponsors can use pharmacokinetics and the relationship between systemic drug exposure and 
virologic or clinical responses in early-phase trials (i.e., proof-of-concept studies) to aid the 
design of phase 2b or phase 3 trials (e.g., dose selection and treatment duration).  When 
sufficient efficacy and pharmacokinetic data are available, sponsors can use a simplified analysis 
relating proportion of subjects with treatment failure and appropriate exposure variable (e.g., 
trough concentration or area under the plasma drug concentration versus time curve) to support 
evidence of effectiveness of different dosage regimens.  Sponsors should also perform analyses 
of the exposure-safety relationships using similar approaches to assist in evaluating the balance 
between effectiveness and safety toxicity of different dosage regimens. 
25  Contains Nonbinding Recommendations 
GLOSSARY OF ACRONYMS 
 
AIDS    acquired immunodeficiency syndrome 
CC    cytotoxic concentration 
CMV    cytomegalovirus 
DAVP   the Division of Antiviral Products 
DNA    deoxyribonucleic acid 
EC    effective concentration 
FDA    Food and Drug Administration 
HBV     hepatitis B virus 
HCV     hepatitis C virus 
HIV    human immunodeficiency virus 
HSCT   hematopoietic stem cell transplantation 
IND    investigational new drug application 
LLOQ   lower limit of quantitation 
mAb    monoclonal antibody 
NDA    new drug application 
PCR    polymerase chain reaction 
pre-IND  pre-investigational new drug application 
SOC    standard of care 
SOT    solid organ transplantation 
 
26  Contains Nonbinding Recommendations 
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30  Contains Nonbinding Recommendations 
1  APPENDIX 
2   
3  CLINICAL TRIAL DESIGN CONSIDERATIONS FOR CMV PROPHYLAXIS 
4  IN LIVER TRANSPLANT RECIPIENTS 
5   
6  At this time, FDA does not recommend a noninferiority trial with valganciclovir as comparator 
7  to evaluate efficacy in liver transplant recipients as the sole population in the trial because the 
8  efficacy of valganciclovir in this population has not been adequately demonstrated.  In a 
9  randomized controlled trial in solid organ transplant recipients submitted for marketing 
10  authorization, valganciclovir was noninferior to oral ganciclovir in the overall trial population for 
11  preventing cytomegalovirus (CMV) disease (CMV syndrome and tissue-invasive CMV disease) 
12  post-transplantation.20  However, among liver transplant recipients, who made up the largest 
13  subgroup (approximately 50 percent of patients enrolled), almost five times more tissue-invasive 
14  CMV disease (as determined by an adjudication committee) was reported with valganciclovir 
15  than with oral ganciclovir as prophylaxis (valganciclovir package insert). 
16   
17  These findings remain unexplained, and currently no antiviral drugs other than oral ganciclovir 
18  have been approved in the United States for CMV prophylaxis in liver transplant recipients.  
19  However, because valganciclovir generally is considered the standard of care in this population 
20  (Levitsky et al. 2008; Kotton et al. 2018) and because oral ganciclovir currently is not available 
21  in the United States, FDA may recommend that valganciclovir be used as a comparator in a 
22  superiority trial.  Additionally, FDA may recommend that a noninferiority trial including 
23  recipients of different types of organ transplants (e.g., liver, heart, kidney, kidney-pancreas) 
24  using valganciclovir as comparator may be appropriate to demonstrate efficacy in liver transplant 
25  recipients if noninferiority is demonstrated for the overall trial population and the rate of CMV 
26  disease is similar between the liver transplant recipients and the other subpopulations for both the 
27  new treatment and the valganciclovir comparator.  However, if the rate of tissue-invasive CMV 
28  disease is higher for liver transplant recipients than for other organ transplant recipients in the 
29  valganciclovir comparator arm, then noninferiority could not be concluded for liver transplant 
30  recipients. 
31   
                                                 
20 In a placebo-controlled trial, oral ganciclovir was shown to decrease the incidence of CMV disease in liver 
transplant recipients during the first 6 months post-transplantation (ganciclovir capsules package insert).  However, 
oral ganciclovir is currently not available in the United States. 
31