Slowly Progressive, Low-Prevalence Rare 
Diseases With Substrate Deposition That 
Result From Single Enzyme Defects:  
Providing Evidence of Effectiveness for 
Replacement or Corrective Therapies 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
March 2020 
Rare Diseases 
 
   
Slowly Progressive, Low-Prevalence Rare 
Diseases With Substrate Deposition That 
Result From Single Enzyme Defects:  
Providing Evidence of Effectiveness for 
Replacement or Corrective Therapies  
Guidance for Industry 
 
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Center for Drug Evaluation and Research 
Food and Drug Administration 
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https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-
guidances 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER)  
 
March 2020 
Rare Diseases 
 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  DRUG DEVELOPMENT CONSIDERATIONS ........................................................... 2 
III.  TYPE AND QUANTITY OF EVIDENCE NECESSARY TO SUPPORT 
EFFECTIVENESS FOR REPLACEMENT OR CORRECTIVE THERAPIES ....... 4 
A.  Animal Toxicology/Pharmacology and Animal Models of Disease Activity   Major 
Considerations ................................................................................................................................ 4 
B.  First-in-Human Dosing and Dose Selection  Key Considerations ......................................... 6 
C.  Providing Evidence of Substrate Reduction ................................................................................ 7 
D.  Other Considerations ..................................................................................................................... 7 
REFERENCES .............................................................................................................................. 9 
 
 
  Contains Nonbinding Recommendations 
Slowly Progressive, Low-Prevalence Rare Diseases With Substrate 
Deposition That Result From Single Enzyme Defects:   
Providing Evidence of Effectiveness  
for Replacement or Corrective Therapies 
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 
 
This document provides guidance to sponsors on the evidence necessary to demonstrate the 
effectiveness of investigational new drugs2 or new drug uses intended for slowly progressive, 
low-prevalence rare diseases3 that are associated with substrate deposition and are caused by 
single enzyme defects.  This guidance applies only to those low-prevalence rare diseases with 
well-characterized pathophysiology, and in which changes in substrate deposition can be readily 
measured in relevant tissue or tissues. 
 
This guidance does not apply to products intended for low-prevalence rare diseases with rapidly 
progressive clinical coursessuch conditions can be evaluated by traditional approaches (i.e., 
using clinical endpoints such as survival, preservation of function, etc.)4or low-prevalence rare 
                                                 
1 This guidance has been prepared by the Office of New Drugs and the Office of the Center Director in the Center 
for Drug Evaluation and Research (CDER) in cooperation with the Center for Biologics Evaluation and Research 
(CBER) at the Food and Drug Administration. 
 
2 For the purposes of this guidance, all references to drugs or drug products include both human drugs and 
biological drug products regulated by CDER and CBER unless otherwise specified. 
 
3 For the purpose of this guidance, a disease of low prevalence may be defined as a condition affecting a very small 
population: for example, approximately a few thousand persons or fewer in the United States.  To be eligible for 
orphan drug designation, a product must be one for a disease or condition that:  (A) affects less than 200,000 
persons in the United States, or (B) affects more than 200,000 in the United States and for which there is no 
reasonable expectation that the cost of developing and making available in the United States a drug for such disease 
or condition will be recovered from sales in the United States of such drug (21 U.S.C. 360bb). 
 
4 Examples of rapidly progressive rare diseases include infantile-onset Pompe disease and infantile-onset lysosomal 
acid lipase disease. 
1 
  Contains Nonbinding Recommendations 
diseases with previously characterized endpoints predictive of clinical benefit (e.g., 
normalization of phenylalanine levels for phenylketonuria patients). 
 
FDA encourages sponsors to discuss with the relevant review divisions whether the approach 
outlined in this guidance applies to their specific drug development programs. 
 
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.  DRUG DEVELOPMENT CONSIDERATIONS  
 
There are many reasons that make demonstrating effectiveness extremely challenging for drugs 
intended to treat slowly progressive, low-prevalence rare diseases that result from defects in a 
single enzyme.  Following are some of those reasons: 
 
  Given the slow progression of the disease, demonstrating clinical stability or clinical 
improvement may require an extremely long time, even decades in some conditions. 
 
  Developing new disease-specific instruments and endpoints to assess clinical response 
(e.g., patient-reported outcomes, observer-reported outcomes, new biomarkers) may not 
be feasible because of the rarity of the disease, geographical distribution of patients, or 
slow progression of disease manifestations. 
 
  Information on the natural history of the disease may be insufficient to inform the 
selection of a historical comparator or to inform clinical endpoint selection in future 
clinical trials. 
 
  In rare circumstances, conducting clinical trials may be impossible because of the 
extremely low number of patients with a specific disease or with a clinical manifestation 
of interest for a given disease. 
 
  When more than one potential therapy is investigated concomitantly, the pool of potential 
patients is further reduced. 
 
Sponsors should take the following into consideration when developing a rational approach to 
drug development: 
 
  A genetic defect affecting a single enzyme can result in either the absence of, or a low 
level of, enzyme activity, with subsequent accumulation of substrates that may be toxic to 
various tissues.  Residual enzyme activity often inversely correlates with substrate 
accumulation. 
 
2 
  Contains Nonbinding Recommendations 
  An increase in enzyme activity resulting from the administration of an exogenous enzyme 
product, by reducing the amount of substrate accumulated and/or by slowing substrate 
accumulation, may alter the rate of disease progression or, over time, shift the disease 
phenotype to a milder one. 
 
  The amount of enzyme activity necessary to prevent or reduce abnormal substrate 
accumulation can vary considerably among tissues. 
 
  Replacement enzymes may penetrate different tissues and subcellular compartments to a 
different extent, which may result in differences in response to treatment in various 
tissues. 
 
  Evidence of activity requires not only evidence that the drug reaches the target organ and 
subcellular compartment of interest but also a demonstration that the drug reduces 
substrate accumulation. 
 
  Some biomarkers (other than substrates) are very closely linked to the underlying 
pathophysiology of the disease (e.g., they can be directly linked to a missing metabolite 
on a critical metabolic pathway).  Sponsors can use changes in such biomarker levels 
during drug development for dose selection or patient selection, and these changes can 
serve as an early demonstration of drug activity, but they are not a replacement for 
demonstration of reduction in substrate deposition in the tissues of interest in clinical 
trials. 
 
Sponsors can apply several strategies for treating slowly progressive, low-prevalence rare 
diseases that result from defects in a single enzyme, including the following: 
 
  Administering a fully functional exogenous enzyme that reaches the organ or organs of 
interest.  This is commonly referred to as enzyme replacement therapy. 
 
  Ameliorating the enzyme defect by using a pharmacologic chaperone that binds to the 
mutant enzyme, inducing proper folding, ensuring correct intracellular trafficking, and 
preventing premature enzyme degradation. 
 
  Reducing the rate of synthesis of substrates. 
 
  Diverting an accumulating substrate to an alternative metabolic pathway. 
 
  Introducing the wild type gene into somatic cells using viral vectors. 
 
 
3 
  Contains Nonbinding Recommendations 
III.  TYPE AND QUANTITY OF EVIDENCE NECESSARY TO SUPPORT 
EFFECTIVENESS FOR REPLACEMENT OR CORRECTIVE THERAPIES 
 
As discussed in section II., for certain slowly progressive, low-prevalence rare diseases, sponsors 
can pursue various treatment strategies to halt or slow the abnormal accumulation of substrate in 
tissues.  When the pathophysiology of a disease is well understood and the mechanism of action 
of the drug/biologic is well characterized, specific drug-induced substrate reduction in relevant 
tissue or tissues can have a reasonable likelihood of predicting clinical effectiveness.  In such a 
case, a clear demonstration in clinical trial or trials that an exogenously administered enzyme or 
drug results in substrate reduction (i.e., it reaches the tissue of interest ) can serve as the basis for 
accelerated approval. 
 
For drugs granted accelerated approval, postmarketing confirmatory trials are required to verify 
and describe clinical benefit by evaluating one or more clinical endpoints.5  In some instances, 
further evaluating (e.g., longer duration of treatment and progressive reduction or resolution of 
substrate deposition) the same histological endpoint that was used to support accelerated 
approval in the same or similar population could provide persuasive evidence of clinical benefit 
and could support full approval. 
 
The following sections describe what FDA considers substantial evidence of effectiveness to 
support accelerated approval for an investigational new drug replacement or corrective therapy 
or new drug use intended to treat a slowly progressive, low-prevalence rare disease with 
substrate deposition that is caused by a single enzyme defect. 
 
In the absence of a way to directly characterize the clinical response to the drug of interest (i.e., 
how a patient feels, functions, or survives), the nonclinical and, particularly, the clinical 
pharmacology components of the drug development program become the main source of data 
that 1) support a safe dose that can be used to initiate human studies and 2) inform dose 
exploration, which is essential to final dose selection for clinical trials. 
 
The following sections emphasize how sponsors can use nonclinical and clinical pharmacology 
information, along with additional sources of information (e.g., in vitro data), to inform dose 
selection for clinical trials meant to lead to marketing approval. 
 
A.  Animal Toxicology/Pharmacology and Animal Models of Disease Activity   
Major Considerations 
 
This section highlights some aspects of the nonclinical program that could inform drug 
development in slowly progressive, low-prevalence rare diseases. 
 
                                                 
5 Section 506(c)(2)(A) of the Federal Food, Drug, and Cosmetic Act.  See also 21 CFR part 314, subpart H and 21 
CFR part 601, subpart E. 
 
4 
  Contains Nonbinding Recommendations 
  Evaluation of the toxicological profile in animals is important for all drug development 
programs.6 
 
  Disease-specific animal models are desirable for drug development in rare diseases.  
Conserving metabolic pathways and essential intermediary components between animal 
species and humans (e.g., ligands, cognate receptors, critical enzyme domains) can 
generate a wealth of relevant pharmacokinetic/pharmacodynamic and proof-of-concept 
information (e.g., animal disease improvement, survival) that can guide testing of 
investigational drug products in humans. 
 
  Some animal models of single-gene human storage disorders display phenotypes that 
mimic to a large extent the clinical manifestations and overall course of the human 
disease (e.g., tripeptidyl peptidase null dachshund dog model for tripeptidyl peptidase 
deficiency) and offer opportunities for evaluating the effect of human enzymes in 
situations in which there is significant structural and functional conservation of the 
missing enzyme across species.  Animal models can provide opportunities for 
histological studies and demonstrate penetrance of a specific drug in the tissue of interest, 
including reaching specific subcellular compartments (e.g., lysosomes).  Moreover, such 
animal models can provide evidence of enzyme activity by demonstrating the reduction 
or disappearance of disease-specific substrates. 
 
  Although not all animal models mimic the human phenotype, FDA encourages sponsors 
to develop relevant models, given the potential benefit for future drug development. 
 
  Demonstration of benefit in animal models for a specific drug product may support 
initiation of clinical studies in pediatric patients by meeting 21 CFR subpart D 
requirements for prospect of direct benefit.7 
 
                                                 
6 See the draft guidance for industry Investigational Enzyme Replacement Therapy Products:  Nonclinical 
Assessment (October 2019).  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.  See also the ICH guidances for industry M3(R2) Nonclinical Safety 
Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (January 2010), 
M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for 
Pharmaceuticals  Questions and Answers (February 2013), and S6(R1) Preclinical Safety Evaluation of 
Biotechnology-Derived Pharmaceuticals (May 2012).  For recommendations on the substance and scope of 
nonclinical information needed to support clinical trials for cell therapy and gene therapy products, see the guidance 
for industry Preclinical Assessment of Investigational Cellular and Gene Therapy Products (November 2013).  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/RegulatoryInformation/Guidances/default.htm.  For complex biological 
products (e.g., gene therapy), alternative approaches may be needed for animal studies as well as for demonstration 
of effectiveness.  FDA encourages sponsors to discuss their proposals with the appropriate CBER product office.  
FDA also 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 as a potential replacement to an animal test method. 
 
7 21 CFR 50.52. 
 
5 
  Contains Nonbinding Recommendations 
B.  First-in-Human Dosing and Dose Selection  Key Considerations 
 
In selecting specific doses for slowly progressive, low-prevalence rare diseases that are caused 
by a defect in a single enzyme, sponsors should consider the following: 
 
  Because efficient patient utilization remains a critical component of any rare disease 
clinical program, sponsors should use any available sources of information (e.g., 
publications, experience with similar compounds, experience in related patient 
populations) during dose selection. 
 
  Testing enzyme replacement therapies in healthy subjects may not be appropriate because 
of the potential risk of inducing an immune response to the investigational drug product 
and cross-reactivity of the elicited antibodies with the endogenous protein and the risk of 
inducing a deficient state in such subjects. 
 
  Using nonhuman data obtained in animal models of disease and in vitro data may be, in 
some cases, the only way to estimate a starting human dose that sponsors hypothesize 
will provide clinical benefit.8  Sponsors can obtain additional dosing information from 
predictive models based on current understanding of in vitro enzyme kinetics (including 
characterizing the enzyme kinetics in relevant cell lines) and allometric scaling. 
 
  Animal toxicology data may inform a safe starting human dose.9 
 
  Sponsors may be able to use effective dose finding in an informative animal model of 
human disease with the knowledge of blood levels and tissue levels to identify an initial 
estimate of a human equivalent dose with appropriate extrapolation (e.g., allometric 
scaling).  Such data can also provide initial estimates of dose-response relationships. 
 
  The selection of the dose and regimen for clinical trials may be optimized based on 
available clinical and nonclinical observations or mechanistic/model-based approaches 
that consider dose- or concentration-response relationships, factors affecting 
pharmacokinetics (e.g., body weight, organ function), and disease characteristics (e.g., 
baseline deficit of the enzyme/enzyme function, severity). 
 
  In clinical trials, sponsors should consider evaluating two or more dose levels.  Although 
a parallel design is considered the best approach for dose exploration, this approach may 
not be feasible for some diseases or patient populations.  Dose evaluation within subjects 
may provide an alternative approach.  For within-subject dose exploration, sponsors 
should take into consideration carryover effects from the previous dose, and the treatment 
duration should be sufficiently long to allow adequate evaluation of the response at each 
dose level. 
                                                 
8 See the guidance for industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for 
Therapeutics in Adult Healthy Volunteers (July 2005). 
 
9 See ICH M3(R2), ICH M3(R2)Questions and Answers, and the guidance for industry Estimating the Maximum 
Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. 
6 
  Contains Nonbinding Recommendations 
 
  Sponsors should consult with the Agency as early as possible to discuss important issues 
such as model-based strategies, dose selection, study design, and endpoint analyses. 
 
 
C.  Providing Evidence of Substrate Reduction 
 
Sponsors should discuss with the Agency any plan to generate evidence of substrate reduction in 
clinical trials.  Such evidence should be generated in tissues in which changes in substrate 
deposition can be readily measured, and the relevance of changes in these tissues to the overall 
disease process must be well understood and clearly justified.  Sponsors should also address how 
the treatment effect size relates to the variability in the test measure.  To this end, sponsors 
should consider the following: 
 
  If substrate levels are heterogeneous in the tissue of interest, efforts to produce a 
representative measurement of the substrate in the tissue may improve the ability to 
detect a treatment effect.  For example, sponsors may obtain multiple specimens from the 
subject to produce an average result at a given time point as a way to address 
heterogeneity in the tissue. 
 
  Sponsors should perform complete analytical validation for all assays used to measure the 
substrate levels.  This validation should include acceptance criteria for analytical 
performance characteristics.  FDA recommends centralized testing of substrate level 
endpoints.  If local assays are necessary for the purposes of conducting the trial (e.g., for 
adaptive dosing), sponsors should also obtain specimens for centralized testing.  If 
centralized testing is not feasible, sponsors should perform cross-validation of the assays 
conducted at local laboratories. 
 
  Preanalytical sample handling can significantly influence assay performance.  Sponsors 
should establish standard operating procedures for the collection, storage, and shipment 
of biospecimens that should be followed at each trial site with deviations recorded.  
Preanalytic reagents and instrumentation should also be validated. 
 
D.  Other Considerations 
 
The following considerations are intended to inform the assessments of efficacy or safety in 
clinical trials: 
 
  Because most rare diseases are pediatric diseases or have onset of manifestations in 
childhood, pediatric studies will be a critical part of drug development.  However, 
treatment in pediatric patients cannot proceed without addressing ethical considerations 
for conducting investigations in vulnerable populations.  Unless the risks of an 
investigational drug are no more than a minor increase over minimal risk (21 CFR 50.53), 
the administration of an investigational drug in children must offer a prospect of direct 
clinical benefit to individually enrolled patients, the risk must be justified by the 
anticipated benefit, and the anticipated risk-benefit profile must be at least as favorable as 
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  Contains Nonbinding Recommendations 
that presented by accepted alternative treatments (21 CFR 50.52).  Additionally, sponsors 
must make adequate provisions to obtain the permission of the parents and the assent of 
the child as per 21 CFR 50.55.10 
 
  Sponsors should perform genetic testing and provide genotypes for the defect or defects 
of interest in all clinical trial subjects. 
 
  For therapeutic protein products, sponsors should evaluate immunogenicity in all trial 
subjects using an analytically validated assay.  Also, sponsors should determine cross-
reactive immunologic material status in all clinical trial subjects as part of the 
immunogenicity assessment. For this, FDA recommends that sponsors collect and store 
blood (or other relevant tissue) samples at baseline before initiating treatment.  Refer to 
the appropriate guidances regarding assessment of immunogenicity.11 
 
  Sponsors should consult with FDA regarding additional clinical outcome data that could 
be systematically collected to assess clinical benefits in individual subjects. 
 
                                                 
10 21 CFR 50.52. 
 
11 See the guidance for industry Immunogenicity Assessment for Therapeutic Protein Products (August 2014) and 
the draft guidance for industry Assay Development and Validation for Immunogenicity Testing of Therapeutic 
Protein Products (April 2016).  When final, this guidance will represent the FDAs current thinking on this topic. 
8 
  Contains Nonbinding Recommendations 
1  REFERENCES 
2   
3  Literature 
4   
5  Fan J-Q, 2003, A Contradictory Treatment for Lysosomal Storage Disorders: Inhibitors Enhance 
6  Mutant Enzyme Activity, Trends Pharmacol Sci, 24(7):355360. 
7   
8  Germain DP, Giugliani R, Hughes DA, Mehta A, Nicholls K, Barisoni L, Jennette CJ, Bragat A, 
9  Castelli J, Sitaraman S, Lockhart DJ, and Boudes PF, 2012, Safety and Pharmacodynamic 
10  Effects of a Pharmacological Chaperone on -Galactosidase A Activity and 
11  Globotriaosylceramide Clearance in Fabry Disease:  Report from Two Phase 2 Clinical Studies, 
12  Orphanet J Rare Dis, 7:91101. 
13   
14  Platt FM and Butters TD, 1998, New Therapeutic Prospects for the Glycosphingolipid 
15  Lysosomal Storage Diseases, Biochem Pharmacol, 56(4):421430. 
16   
17   
18  Guidances for industry 
19   
20  Draft guidance for industry Rare Diseases:  Common Issues in Drug Development (February 
21  2019)1 
22   
23  Guidance for industry Expedited Programs for Serious Conditions  Drugs and Biologics (May 
24  2014) 
25   
26  Guidance for industry Providing Clinical Evidence of Effectiveness for Human Drug and 
27  Biological Products (May 1998) 
28   
29   
30  Prescribing information 
31   
32  Cholbam (cholic acid) available at 
33  https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/205750s000lbl.pdf. 
34   
35  Fabrazyme (agalsidase beta) available at 
36  https://www.accessdata.fda.gov/drugsatfda_docs/label/2010/103979s5135lbl.pdf. 
37   
38  Kanuma (sebelipase alfa) available at 
39  https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/125561s000lbl.pdf. 
40   
41  Myozyme (alglucosidase alfa) available at 
42  https://www.accessdata.fda.gov/drugsatfda_docs/label/2014/125141s219lbl.pdf. 
43   
                                                 
1 When final, this guidance will represent the FDAs current thinking on this topic. 
9