Designing a single-arm phase 2 clinical trial of mitapivat for adult patients with erythrocyte membranopathies (SATISFY): a framework for interventional trials in rare anaemias – pilot study protocol

Abstract

Introduction

Membranopathies encompass haemolytic disorders arising from genetic variants in erythrocyte membrane proteins, including hereditary spherocytosis and stomatocytosis. Congenital dyserythropoietic anaemia type II (CDA II) is associated with the SEC23B gene and can exhibit phenotypic similarities to membranopathies. Current treatment options for these conditions, apart from splenectomy, are primarily supportive. Mitapivat, a novel pyruvate kinase (PK) activator, has demonstrated efficacy in increasing haemoglobin levels and reducing haemolysis in patients with PK deficiency, thalassemia, sickle cell disease and a mouse model of hereditary spherocytosis.

Methods and analyses

Safety and efficacy of mitapivat sulfate in adult patients with erythrocyte membranopathies (SATISFY) is a prospective, multicentre, single-arm phase two trial involving approximately 25 adult patients (≥18 years) diagnosed with a membranopathy or CDA II. During the 8-week dose escalation period, subjects will receive an initial dose of 50 mg mitapivat two times per day and may increase to 100 mg two times per day at week 4 based on the safety and changes in haemoglobin levels. Patients tolerating mitapivat well may be eligible to continue in two consecutive 24-week fixed dose periods.

The primary objective of this study is to evaluate the safety of mitapivat, assessed through the occurrence of treatment-emergent adverse events. Secondary objectives include assessing the effects of mitapivat on haemoglobin levels, haemolysis, erythropoiesis, patient-reported outcome measures and spleen size.

SATISFY aims to assess the safety and efficacy of mitapivat in adult patients with red blood cell membranopathies and CDA II, with the aim of establishing proof-of-concept in patients living with these rare conditions.

Ethics and dissemination

NCT05935202/CTIS:2023-503271-24-01. Findings will be published in peer-reviewed journals.

Trial registration number

Clinicaltrials.gov, NCT05935202. CTIS:2023-503271-24-01. Registered 07-July-2023. Protocol number: 2.1. https://clinicaltrials.gov/study/NCT05935202.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• Prospective multicentre study design enhances the generalisability of results.

• Inclusion of both genetic and clinical criteria ensures accurate diagnosis.

• Utilisation of patient-reported outcome measures to capture patient-centric data.

• Collaboration with EuroBloodNet provides access to a specialised network and resources.

• Limited sample size and adult-only population may affect the statistical power and generalisability to paediatric cases.

Introduction

Red blood cell (RBC) membranopathies encompass a group of disorders characterised by the defects of structural membrane proteins or abnormalities in membrane ion channels and include disorders such as hereditary spherocytosis (HS) and hereditary stomatocytosis (HSt).

HS, the most frequent hereditary haemolytic disorder in people of northern European descent, is primarily caused by variants in genes encoding cytoskeleton proteins and proteins tethering the membrane cytoskeleton to the phospholipid bilayer.^{1 2}

Classically, patients present with Coombs-negative haemolytic anaemia, increased mean corpuscular haemoglobin (Hb) concentration (MCHC), palpable splenomegaly and a family history of haemolytic anaemia. Diagnostic tests for HS include the evaluation of RBC morphology, osmotic fragility test, flow cytometry-based eosin-5’-maleimide (EMA) binding, osmotic gradient ektacytometry and next-generation sequencing.^{3 4}

Dehydrated HSt (DHSt) or hereditary xerocytosis is a relatively rare haemolytic anaemia often associated with iron overload.⁵ DHSt is characterised by pathological efflux of potassium and water from the erythrocytes. Anaemia is often mild or even non-existent due to reduced levels of 2,3-diphosphoglycerate (2,3-DPG), which increases Hb oxygen affinity and consequently erythropoiesis.⁶ Etiologically, variants in the PIEZO1 or KCNN4 genes are found.⁷ Piezo1 is a mechanosensitive ion channel and gain-of-function variants lead to an increased influx of Ca²⁺, which in turn activates the Gardos channel (KCNN4). Activation of the Gardos channel by Piezo1 activation or by KCNN4 mutations leads to a leak of potassium out of the erythrocyte and dehydration. The prevalence of DHSt is not well characterised but has been estimated to be 1:8000 in the USA, based on general laboratory data (anaemia or other signs of haemolysis combined with a high MCHC).⁸ MCHC is increased in both HS and DHSt due to preserved Hb content in the setting of decreased cell volume due to either membrane loss (HS) or cell dehydration (DHSt).⁹ Overhydrated HSt (OHSt) is another haemolytic condition of RBC hydration. OHSt is commonly associated with mutations in RHAG,¹⁰ which results in RBC overhydration and haemolysis.

Congenital dyserythropoietic anaemia type II (CDA II) is the most common form of CDA at 0.71 cases/million,¹¹ with more than 100 sequence variants described.¹² CDA is characterised by increased peripheral RBC destruction¹¹ resulting in anaemia, jaundice and splenomegaly and often leading to liver iron overload and gallstones.¹³ Genetically, it is caused by variants affecting the secretory COPII coat component SEC23B.¹⁴ Diagnostically CDA II can be mistaken for HS as results from EMA binding test and osmotic gradient ektacytometry¹⁵ can be similar to HS as a consequence of membrane abnormalities including reduced glycosylation of band 3.^{3 16} Haemolysis in CDA II has been attributed to the hypoglycosylation of band 3, resulting in clustering of band 3 on the RBC surface membrane.¹⁷

A common feature of the hereditary anaemias mentioned above is impaired RBC deformability, as measured by osmotic gradient ektacytometry. For decades, ATP has been deemed essential for keeping RBCs viable through the maintenance of membrane flexibility, integrity and intracellular ion concentrations.^{18 19} Increasing ATP generation therefore is an obvious pharmaceutical target in these hereditary anaemias.²⁰

Mitapivat, also known as mitapivat sulphate or AG-348, is an allosteric activator of pyruvate kinase (PK), which plays a crucial role in maintaining energy homeostasis in RBCs. In ex vivo experiments conducted on RBCs, mitapivat was also shown to increase ATP levels and decrease 2,3-DPG levels in PK-deficient cells and wild-type control cells.²¹ Genetic variants of PK are a major cause of hereditary non-spherocytic haemolytic anaemia. Based on two pivotal phase 3 studies,^{22 23} mitapivat is approved for treatment of adults with genetic PK deficiency. In other haemolytic anaemias, patients typically have wild-type PK enzyme responsive to activation by mitapivat. This may explain the notable response rate observed in the recent phase 2 trials of mitapivat in other haemoglobinopathies.^{24 25}

In a band 4.2 /- mouse model of HS, mitapivat ameliorated anaemia, reduced haemolysis and decreased iron overload.²⁰ Considering that patients with DHSt exhibit decreased PK activity,²⁶ the therapeutic activation by mitapivat presents a rational approach for this condition as well.

In this study, we hypothesise that mitapivat will be safe, increase Hb and ameliorate haemolysis in patients with RBC membranopathies or CDAII, and that this may lead to reduced fatigue and dyspnoea and improvements in physical activity and health-related quality of life (HRQoL).

Methods and analyses

Study design

SATISFY (ClinicalTrials.gov, NCT05935202) is a prospective, single-armed, exploratory multicentre pilot study designed to study the safety and efficacy of mitapivat in RBC membranopathies and CDAII. Patients will be recruited from centres of expertise in the European Union (EU) starting with Denmark and the Netherlands, and outside the EU in Canada. For this purpose, two sibling studies will be conducted: one in the EU and one in Canada. The study report will be a combined analysis of all patients (EU+Canada). This protocol is for the EU portion of the study.

Subjects who have taken at least one dose of mitapivat will return for a safety follow-up visit 4 weeks after their last dose.

Maximal study duration per subject is a screening period (≤50 days) +56 weeks on the study drug (figure 1). A follow-up extension study (fixed dose extension period) may be considered for responders who safely tolerate mitapivat, or the current study may be amended as such, allowing responders to remain on their optimal dose of mitapivat. Other mechanisms to provide study drug to responders may also be considered as the study ends.

Figure 1. Safety and efficacy of mitapivat sulfate in adult patients with erythrocyte membranopathies (SATISFY) study flow chart. The study is divided into four phases: screening (50 days), dose escalation (4 weeks), fixed dose period 1 (24 weeks) and fixed dose period 2 (24 weeks). BID, two times per day. W, week.

Informed consent will be obtained from all participants prior to any study procedure. The study is conducted in accordance with the Declaration of Helsinki, the Medical Research Involving Human Subjects Act, and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) harmonised guideline integrated addendum to ICH E6(R1): Guideline for Good Clinical Practice ICH E6(R2) ICH Consensus Guideline.

Patient and public involvement statement

Patients and the public were not directly involved in the design of this study.

EuroBloodNet Association

The study is the first clinical study in the context of the European Reference Network EuroBloodNet (https://eurobloodnet.eu) and will be sponsored by its closely associated non-profit, EuroBloodNet Association.

Safety monitoring and assessments

A Safety Assessment Committee (SAC) will oversee participant safety. The SAC will review data periodically, with a focus on an interim safety analysis at the end of the week-8 visits, evaluating standard safety thresholds against the risk profile in the investigator’s brochure. An independent Data Monitoring Committee is not required given the study’s non-critical disease focus, small patient population, absence of blinding or randomisation, adult-only participants, short duration with a preplanned 8-week interim analysis and well-documented safety profile of mitapivat in other haemolytic anaemias.22,24

Objectives and endpoints

The study aims to assess the safety of mitapivat and establish proof of concept regarding its efficacy in patients with RBC membranopathies or CDAII. If safety and proof of concept are demonstrated, the findings may inform the design of larger clinical trials, including paediatric studies. Safety is the primary endpoint and will be evaluated throughout the study. Secondary and exploratory endpoints will be assessed during the fixed dose periods, which include measures related to Hb response, haemolysis, erythropoiesis, patient-reported symptoms and spleen size.

The overall objective of this study is to assess the safety of mitapivat and provide proof of concept regarding its efficacy in patients with RBC membranopathies or CDAII. Successful demonstration of safety and proof of concept in this study may lead to the design of larger clinical trials involving both adults and paediatric populations. The dose escalation period, spanning 8 weeks, aims to determine the maximal tolerated dose of mitapivat for use in the fixed dose periods (figure 1).

Throughout the study, the primary endpoint of safety will be evaluated, while secondary and exploratory endpoints will be assessed during the fixed dose periods 1 and 2, each lasting 24 weeks. Fixed dose period 1 will evaluate the initial response, while fixed dose period 2 will assess the longer-term response. Data analysis will be conducted separately for each period. A comprehensive list of study objectives and endpoints is provided in table 1. Schedule of assessments and outcome assessments are detailed in the study protocol (online supplemental material). Outcome assessments are summarised in the following sections.

Table 1. Study objectives and endpoints.

Primary objective	Primary endpoint
To evaluate the safety of mitapivat.	Type, incidence, severity and relationship of mitapivat to AE and SAE.
Secondary objectives	Secondary endpoints
To evaluate the effect of mitapivat on changes in haemoglobin (Hb).	Hb response (HR) defined as a ≥10 g/L (1 g/dL) increase in Hb concentration from baseline (mean of screening and D0) that is sustained at two or more points during fixed dose period 1.
To evaluate the long-term effect of mitapivat on Hb.	Change from baseline in Hb levels to the average of scheduled visits in fixed dose period 2.
To evaluate the effect of mitapivat on additional measures of Hb response.	Average change in mean Hb concentration during fixed dose periods 1 and 2 compared with baseline (mean of screening and D0).
	Proportion of subjects who achieve Hb concentration in the sex-associated normal range that is sustained at two or more points in the fixed dose periods 1 and 2.
To evaluate the effect of mitapivat on markers of haemolysis.	Change from baseline in lactate dehydrogenase (LDH), bilirubin and haptoglobin at the end of fixed dose periods 1 and 2.
To evaluate the effect of mitapivat on markers of erythropoiesis.	Change from baseline in reticulocytes erythropoietin, erythroferrone and sTfR at the end of fixed dose periods 1 and 2.
To evaluate the effect of mitapivat on symptoms and impacts.	Change from baseline in fixed dose periods 1 and 2: Health related Quality of Life (HRQoL) (SF-36 V.1) Pyruvate Kinase Deficiency Impact Assessment (PKDIA)
To evaluate the effect of mitapivat on spleen size in non-splenectomised patients.	Change from baseline in spleen volume in fixed dose periods 1 and 2.
Exploratory objectives	Explorative endpoints
To explore PK activity and thermostability on purified red cells and red cells of different age/density in relation to red cell ATP, 2,3-DPG and other metabolites, and the effect of mitapivat on the relation between these parameters.	Maximum change in p50, 2,3-DPG and ATP levels, PK-R and PK-M2 isozyme distribution levels (Western Blot, MSD) in whole blood and red cells of different age. Change in metabolism in whole blood and red cells of different age, as evaluated by untargeted metabolomics.
To explore the effect of mitapivat on membrane flexibility on purified red cells and red cells of different age/density.	Change in total red cell membrane flexibility and membrane health of whole blood and red cells of different age, as evaluated by osmotic gradient ektacytometry and membrane stability test (Lorrca, RR Mechatronics). Change in membrane surface area of whole blood and red cells of different age as evaluated by EMA-binding test.
To explore the effect of mitapivat on iron metabolism.	Change in hepcidin, ferritin and TSAT.
	Change in hepatic and cardiac iron concentration evaluated by R2 MRI.
To explore the effect of mitapivat on RBC autoantibodies.	Improvement in flow cytometric DAT for autoantibodies.
To explore the effect of mitapivat on changes in Hb in the disease entities.	Primary and secondary endpoint in: HS HSt CDAII
To explore the effect of mitapivat on RBC lifespan.	Increase in HbA1c standardised to fructosamine.
To evaluate the effect of mitapivat on splenic biomarkers in splenectomised patients.	Change from baseline in pitted RBC count.

AEadverse eventATPAdenosine TriphosphateCDAIIcongenital dyserythropoietic anaemia type IIDATdirect antiglobulin test2,3-DPG2,3-diphosphoglycerateEMAeosin-5'-maleimide binding testHbHemoglobinHShereditary spherocytosisHSthereditary stomatocytosisPKpyruvate kinaseRBCred blood cellsSAEserious adverse eventSTfRsoluble transferrin receptorTSATtransferrin saturation

Primary study parameters

Safety analysis

The overall safety profile of the study drug will be assessed in terms of the following safety and tolerability endpoints: incidence of treatment-emergent adverse events (TEAEs), clinical laboratory values, ECGs (standard 12-lead), physical examination findings and dual-energy X-ray absorptiometry (DEXA) scans.

Secondary study parameters

Hb response

The key secondary endpoint is Hb response, defined as a ≥1.0 g/dL increase in Hb concentration sustained at two scheduled visits in the fixed dose period compared with baseline. The proportion of subjects who achieved an Hb response (response rate) will be summarised for each disease entity (HS, HSt and CDAII). Hb concentrations assessed within 8 weeks after an RBC transfusion will be excluded from the analysis of the primary endpoint. Subjects who do not have at least two on-treatment Hb concentration assessments in the fixed dose period will be considered non-responders.

Average change in mean Hb

The average changes from baseline in Hb concentrations at scheduled visits will be analysed and compared with baseline. Furthermore, individual patient’s changes from baseline at the end of the fixed dose periods will be summarised. Mean Hb change by disease entity (eg, HS, HSt and CDAII) will be described.

Hb concentration in the normal range

The number and percentage of subjects who achieve Hb levels in the normal range sustained at two or more scheduled assessments in the fixed dose period will be provided, with descriptions by disease entity (HS, HSt and CDAII).

Markers of haemolysis and erythropoiesis

Changes from baseline at each visit over time in markers of haemolysis, haematopoietic activity and indicators of clinical activities will be summarised.

HRQoL assessments

The number and proportion of subjects in each response level will be summarised by study visit. Changes from baseline at scheduled visits in the fixed dose period will be summarised.

Spleen size

Changes from baseline in the length and volume of the spleen will be summarised for each scheduled scan. The change will be described in terms of absolute size change and percentage.

Dosing rationale

Dosing was selected based on results from patients with thalassemia, where a mitapivat 100 mg two times per day was safe and effective.²⁴ Higher doses were also safe, but not associated with increased response in PK deficiency.²⁷

Study population

The study will include patients with RBC membranopathies or CDAII. Approximately 16 patients from the EU study sites and nine patients from the Canadian study will be enrolled. Both male and female subjects are eligible to participate. Prior splenectomy is allowed, except within the last 6 months of screening. If a subject withdraws from the study before the first evaluation after the first dosing, they may be replaced.

Study procedures

Eligibility

Patients with RBC membranopathy or CDAII confirmed genetically by the American College of Medical Genetics (ACMG) class 3 variant of uncertain significance (VUS), 4 or 5 variant are eligible for the study. Average of at least two Hb concentration measurements during the screening period must be <13.0 g/dL for males and 11.0 g/dL for females. Patients with average Hb >10.0 g/dL for males and females must meet at least one of the following additional criteria: splenomegaly, fatigue attributed to haemolysis or paraclinical haemolysis. Adequate organ function is required. Patients cannot be included if they have a diagnosis of PK deficiency or received RBC transfusions (≥5 units in the last 12 months or any within the last 3 months).

Additional key inclusion and exclusion criteria are listed in figure 2 and box 1.

Figure 2. Graphical representation of key inclusion and exclusion criteria for the SATISFY study. ACMG, American College of Medical Genetics; CDAII, congenital dyserythropoietic anaemia type II; Hb, haemoglobin; PK, pyruvate kinase.

Box 1. Inclusion and exclusion criteria.

Inclusion criteria

• Male or female with RBC membranopathy or CDAII. Diagnosis must be supported genetically by the ACMG class 3 (VUS), 4 or 5 variant.

• Age ≥18 years at the first screening.

• Average haemoglobin (Hb) concentration (average of at least 2 Hb measurements separated by a minimum of 7 days during the screening period) must be <13.0 g/dL for males and 11.0 g/dL for females. Patients with average Hb >10.0 g/dL for males and females must meet at least one of the following additional criteria:

  • Splenomegaly (length ≥12.5 cm).

  • Fatigue attributed to haemolysis.

  • Active haemolysis as evaluated by one or more of the following: haptoglobin, bilirubin, LDH and reticulocytes.

• Subjects must start or continue taking at least the equivalent of daily 0.8 mg oral folic acid for the duration of the study.

• Have adequate organ function, as defined by:

  • Serum aspartate aminotransferase (AST) ≤2.5 × ULN (unless the increased AST is assessed by the investigator as due to haemolysis and/or hepatic iron deposition) and alanine aminotransferase (ALT) ≤2.5 × ULN.

  • Normal or elevated levels of serum bilirubin. In subjects with serum bilirubin>ULN, the elevation must be attributed to haemolysis with or without Gilbert’s syndrome and must not be associated with choledocholithiasis, cholecystitis, biliary obstruction or hepatocellular disease Elevated bilirubin attributed to haemolysis with or without Gilbert’s syndrome is not exclusionary.

  • Estimated glomerular filtration rate ≥45 mL/min/1.73 m² by Chronic Kidney Disease Epidemiology Collaboration creatinine.

• Be willing and able to give written informed consent and comply with all study procedures for the duration of the study.

• For women of reproductive potential, have a negative urine or serum pregnancy test during the Screening Period (Day 50–Day 1). Women of reproductive potential are defined as sexually mature women who have not undergone a hysterectomy, bilateral oophorectomy or tubal occlusion; or who have not been naturally postmenopausal (ie, who have not menstruated at all for at least the preceding 12 months prior to signing informed consent), or has a known diagnosis of hypogonadotropic hypogonadism.

• For women of reproductive potential as well as men with partners who are women of reproductive potential, be abstinent as part of their usual lifestyle, or agree to use two forms of contraception, one of which must be considered highly effective, from the time of giving informed consent, during the study, and for a highly effective form of contraception is defined as combined (oestrogen and progestin containing) hormonal contraceptives (oral, intravaginal or transdermal) associated with inhibition of ovulation; progestin-only hormonal contraceptives (oral, injectable or implantable) associated with inhibition of ovulation; intrauterine device; intrauterine hormone releasing system; bilateral tube occlusion or vasectomized partner. The second form of contraception can include an acceptable barrier method, which includes male or female condoms with or without spermicide and a cervical cap, diaphragm or sponge with spermicide. Women of reproductive potential using hormonal contraception as a highly effective form of contraception must also use an acceptable barrier method while enrolled in the study and for at least 28 days after their last dose of the study drug.

Exclusion criteria

• Known history of pyruvate kinase (PK) deficiency (decreased PK activity or two pathogenic PKLR alleles). PK activity and PKLR testing is not required.

• Receiving regularly scheduled blood (RBC) transfusion therapy (also termed chronic, prophylactic or preventive transfusion), defined as more than five transfusion episodes in the 12-month period up to the first day of study treatment, and/or have received a transfusion within the past 3 months prior to the first day of study treatment.

• Have a significant medical condition that confers an unacceptable risk to participating in the study, and/or that could confound interpretation of the study data. Such significant medical conditions include, but are not limited to:

  • Poorly controlled hypertension (defined as systolic blood pressure >150 mm Hg or diastolic blood pressure >90 mm Hg) refractory to medical management.

  • Any history of congestive heart failure; myocardial infarction or unstable angina pectoris; haemorrhagic, embolic or thrombotic stroke; or recent (<6 months prior to signing informed consent) deep venous thrombosis or pulmonary or arterial embolism.

  • Cardiac dysrhythmias judged as clinically significant by the investigator.

  • Clinically symptomatic cholelithiasis or cholecystitis. Prior cholecystectomy is not exclusionary. Subjects with symptomatic cholelithiasis or cholecystitis may be rescreened once the disorder has been treated and clinical symptoms have resolved.

  • History of drug-induced cholestatic hepatitis.

  • Severe iron overload as evaluated by the investigator. This includes cardiac (eg, clinically significant impaired left ventricular ejection fraction) or hepatic (eg, fibrosis and cirrhosis) dysfunction.

  • Have a diagnosis of any other congenital or acquired blood disorder or any other haemolytic process, except mild allo-immunisation, as a consequence of transfusion therapy.

  • Positive test for HBsAg or HCVAb with signs of active hepatitis B or C virus infection. Subjects with hepatitis C may be rescreened after receiving appropriate hepatitis C treatment.

  • Positive test for HIV-1 or HIV-2 antibodies.

  • Active infection requiring the use of parenteral antimicrobial agents or grade ≥3 in severity (per NCI CTCAE) within 2 months prior to the first dose of study treatment.

  • Diabetes mellitus judged to be under poor control by the investigator or requiring >3 antidiabetic agents, including insulin (all insulins are considered one agent); use of insulin per se is not exclusionary.

  • History of any primary malignancy, with the exception of curatively treated non-melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; or other primary tumour treated with curative intent, no known active disease present, and no treatment administered during the last 3 years.

  • Unstable extramedullary haematopoiesis that could pose a risk of imminent neurological compromise.

  • Severe hepatic issues such as liver fibrosis (F3 or worse), significant cirrhosis or non-alcoholic fatty liver disease (non-alcoholic steatohepatitis).

  • Current or recent history of psychiatric disorder that, in the opinion of the investigator, could compromise the ability of the subject to cooperate with study visits and procedures.

• Are currently enrolled in another therapeutic clinical trial involving ongoing therapy with any investigational or marketed product or placebo. Participation in registry studies is allowed.

• Have exposure to any investigational drug, device or procedure within five half-lives or 3 months (whichever is longer) to the first dose of study treatment.

• Have had any prior treatment with a PK activator.

• Have a prior bone marrow or stem cell transplant.

• Are currently pregnant or breastfeeding or planning to become pregnant during the course of the study.

• Have a history of major surgery within 6 months prior to signing informed consent. Note that procedures such as laparoscopic gallbladder surgery are not considered major in this context.

• Are receiving medications that are strong inhibitors of CYP3A4 that have not been stopped for ≥5 days or a timeframe equivalent to five half-lives (whichever is longer); or strong inducers of CYP3A4 that have not been stopped for ≥4 weeks or a timeframe equivalent to five half-lives (whichever is longer), prior to the first dose of study treatment.

• Are currently receiving haematopoietic stimulating agents (eg, erythropoietins, granulocyte colony stimulating factors and thrombopoietins) that have not been stopped for a duration of at least 28 days prior to the first dose of study treatment.

• Known allergy to mitapivat or its excipients (microcrystalline cellulose, croscarmellose sodium, sodium stearyl fumarate and mannitol) or history of acute allergic reaction to drugs characterised by acute haemolytic anaemia, drug-induced liver injury, anaphylaxis, rash of erythema multiforme type or Stevens-Johnson syndrome, cholestatic hepatitis or other serious clinical manifestations.

• For men and women of reproductive potential: unwillingness to be abstinent or use double anticonception during the trial period.

Adherence assessments

To enhance the validity of data, medication adherence will be recorded using a dosing diary and pill counting. Unused tablets will be counted and recorded on the appropriate electronic case report form.

Monitoring and quality assurance

Data monitoring will be performed by a certified clinical research associate of the sponsor. Details will be provided in the monitoring plan.

Data management

The study site’s electronic medical record system will be used as the source document for medical data. All study data will be entered into a secured REDCap (Research Electronic Data Capture) database, which allows for audit trail. A data management plan will be generated to describe data collection, handling, storage and back-up, analysis, archiving and sharing.

Dose reduction and interruption of study treatment

Subjects should not abruptly discontinue mitapivat treatment, except in medical emergencies under the supervision of the investigator. If a subject exhibits an excessive Hb response exceeding the local sex-based upper limit of normal, a dose decrease to the next lower level should be considered without tapering.

Study procedures

The study procedures and assessments are detailed in the Appendix II: Schedule of Assessments in the study protocol (online supplemental material). The key assessments are presented below.

Laboratory assessments

Clinical laboratory measures including complete blood count and haemolysis markers will be measured at all visits.

Patient-reported outcomes measures

SATISFY is a part of the European Rare Disease Research Coordination and Support Action consortium (ERICA; http://erica-rd.eu) work package 3 on patient-centred research for rare diseases, which promotes the implementation of standardised patient-reported outcome measures (PROMs).

The following PROMs will be evaluated during screening and throughout the study: the medical outcome study 36 short form (SF-36) V.1.0 and PK Deficiency Impact Assessment (PKDIA).

The PKDIA is a 12-item PROM of the common impacts of PK deficiency on the activities of daily living.²⁸ Subjects rate how their disease has impacted aspects of daily living in the past 7 days, including impacts on relationships, perceived appearance, work performance and leisure, and social, mental and physical activities. PKDIA was validated for PK deficiency in the ACTIVATE study (A Study to Evaluate Efficacy and Safety of AG-348 in Not Regularly Transfused Adult Participants With Pyruvate Kinase Deficiency).²⁹ Given the phenotypic overlap between pyruvate kinase deficiency (PKD) and the haemolytic anaemia included in this study, PKDIA is likely to capture the patient HRQoL well in this setting.

The SF-36 is a generic measure of HRQoL,²⁸ validated across various populations and domains of HRQoL.³⁰

RBC assessment

Specialised RBC assessment will involve several tests and measurements at weeks 0 and 32. These include flow cytometric direct antiglobulin test for autoantibodies,³¹ RBC pit counts to assess spleen function and RBC health,³² assessment of membrane flexibility, osmotic resistance and band 3 content using osmotic gradient ektacytometry³³ supplemented by EMA binding test,³⁴ cell membrane stability testing,³⁵ measurement of PK activity normalised to hexokinase activity,³⁶ PK stability test,³⁷ measurement of Hb oxygen affinity using Hemox-analyzer (TCS, New Hope, Pennsylvania) and/or automated venous blood gas analyser (Radiometer, Copenhagen, Denmark). Measurements will be performed on the total RBC population as well as after separation according to RBC age by density centrifugation. Membrane and cytosolic proteins will be measured by mass spectrometry³⁸ on RBCs. Metabolomic analyses will be carried out as previously described.³⁹

Markers of Erythropoiesis and iron metabolism

We will evaluate the effect of mitapivat on a range of known and candidate markers of erythropoiesis and iron metabolism at weeks 0, 8, 32, 56 and end of study visits. These include erythroferrone,⁴⁰ hepcidin,⁴¹ erythropoietin and soluble transferrin receptor.

Scans

MRI of the heart and liver will be conducted to assess the effect of iron overload and to measure the size of the spleen at weeks 0, 32 and 56. DEXA scans are performed as a safety measure at weeks 0, 32 and 56 to assess bone density, although prior studies did not find the loss of bone function during mitapivat treatment.^{22 23}

Sample size justification and statistical considerations

The study is primarily descriptive and does not involve formal hypothesis testing. The sample size in each dose arm will be determined based on feasibility.

Considering the variability in Hb concentration among anaemic patients, approximately 10% of subjects are expected to achieve an Hb response due to natural variability alone.⁴² Mitapivat has not been studied in patients with HS, but in patients with thalassemia, a 70% response rate was observed.²⁴ Based on the effectiveness of mitapivat in a mouse model of HS,²⁰ a 50% response rate therefore is a realistic estimate. To detect a 50% response rate, enrolling 6 subjects in each arm will provide 80% power at a 2-sided significance level of 0.05, assuming that 10% of individuals would achieve a response due to natural variability alone (without treatment).

The expected study population includes 16 patients from the SATISFY trial in the EU and 9 patients from the SATISFY trial in Canada. Approximately 80% (n=20) of the study population will consist of patients with membranopathies, with around 60% (n=15) having HS. Genetic characterisation of HS patients in Denmark showed that 69% of genetically confirmed HS patients had autosomal dominant pathogenic variants,³³ corresponding to approximately 10/15 HS patients with a similar genotype. Additionally, 40% had SPTB variants, corresponding to 6/15 patients with the same autosomal dominant genotype. Therefore, enrolling a total of 15 HS patients will ensure sufficient power to evaluate the efficacy of mitapivat on Hb levels in typical forms of HS. The effects on other membranopathies and CDAII will be investigated on an exploratory basis, as well as HS patients with SPTA1 variants due to their genetic complexity.

Data analysis will be performed from the start to the end of each study period (dose escalation, fixed dose period 1 and fixed dose period 2). The primary evaluation point for safety and efficacy endpoints is the end of the fixed dose period 1. Categorical variables will be summarised using frequency distributions, and percentages will include missing observations as a separate category. Safety analysis will include the incidence of TEAEs, clinical laboratory values, ECGs, physical examination findings and DEXA scans. Summaries will be presented by organ class and preferred term using frequency counts and percentages.

The key secondary endpoint is the Hb response, defined as a ≥1.0 g/dL increase in Hb concentration sustained at two scheduled visits during the fixed dose period compared with baseline. The proportion of subjects achieving an Hb response will be summarised for each disease entity (HS, HSt and CDAII). Changes from baseline in Hb concentrations at scheduled visits and individual patient changes at the end of the fixed dose periods will also be summarised. Mean Hb change by disease entity will be described. Additional endpoints are summarised in box 1.

Ethics and dissemination

This trial (version 1.0) was registered on Clinicaltrials.gov on 07-July-2023 (NCT05935202; https://clinicaltrials.gov/study/NCT05935202). It is approved centrally in the EU in the Clinical Trials Information System (CTIS) (2023-503271-24-01) and encompasses regulatory authorities in the member states, specifically the Danish Research Ethics Committees and Medical Ethics Review Committee NedMec, the Netherlands. Patients provide informed consent to participate. Positive and negative findings will be reported in an international scientific journal.

Discussion

Current treatment of RBC membranopathies is primarily supportive, and although splenectomy can improve Hb level in many cases, it carries lifelong risks of thrombosis and infection.⁴³ For patients with HSt, splenectomy poses an unacceptable risk of severe thrombosis leaving them with limited therapeutic options.⁴⁴

Mitapivat has demonstrated beneficial effects in haemoglobinopathies and is proven to be safe and effective in PK deficiency.22,24 Based on its efficacy in a mouse model of HS,²⁰ our objective is to demonstrate a therapeutic potential in patients with RBC membranopathies and CDAII. Anaemia may be less pronounced in these patients since many have compensated haemolysis but nonetheless suffer from the complications of chronic haemolysis, which highlights the importance of capturing the burden of disease and therapeutic effect by the PROMs of the study. Ongoing paediatric trials of mitapivat in thalassemia and PKD will provide valuable safety data on mitapivat in children, potentially paving the way for a phase 3 study that offers an alternative to splenectomy for anaemic children with HS or CDAII.

This study represents the first utilisation of the EuroBloodNet and ERICA framework to conduct a clinical trial in rare anaemias. Leveraging the new CTIS, we have registered the trial simultaneously across the EU as it will be conducted in Denmark and the Netherlands, facilitating the expansion of other trials for rare diseases to all 27 member states. The sibling SATISFY study in Canada serves as a model for global expansion, which can be applied in future studies. This framework has the potential to greatly benefit future intervention trials in rare diseases with limited patient populations and where effective treatment options are lacking.