Haemoglobin vesicles as artificial red blood cells developed for use as a transfusion alternative: an open-label, single-centre phase Ib study protocol in Japan

Abstract

Introduction

Haemoglobin vesicles (HbVs) (product name, NMU-HbVs [Nara Medical University-Haemoglobin Vesicles]), which contain purified human haemoglobin encapsulated within liposomes, have been developed as a potential alternative to blood transfusions in emergency situations. A previous phase I study examined doses up to 100 mL in 11 healthy volunteers. Here, we describe the protocol for a phase Ib study, wherein we will evaluate the safety and pharmacokinetics of NMU-HbV in healthy Japanese adults.

Methods and analysis

This single-centre, open-label, dose-escalation study will enrol 16 healthy volunteers divided into four cohorts. Planned doses are 100 mL for cohorts 1 and 2, 200 mL for cohort 3 and 400 mL for cohort 4, with infusion rates gradually increasing to a maximum of 5.0 mL/min. The primary endpoint will be safety, which will be assessed as the incidence of adverse events within 14 days and significant clinical changes within 72 hours after administration. Safety evaluations will include subjective symptoms, vital signs, electrocardiograms and laboratory test results compared with the baseline. The secondary endpoint will be pharmacokinetics, which will be assessed as changes in NMU-HbV concentration immediately after infusion until day 4 to determine the maximum blood concentration, time to reach the maximum blood concentration, area under the blood concentration-time curve and elimination half-life. This study will provide data on the safety and pharmacokinetic profiles of NMU-HbV at doses up to 400 mL. The findings are expected to support the further development of NMU-HbV as a viable alternative to emergency transfusions.

Ethics and dissemination

The study protocol was approved by the Institutional Review Board of Nara Medical University on 10 December 2024. Dissemination plans include publishing in peer-reviewed scientific journals and presentation at international conferences.

Trial registration number

Japan Registry of Clinical Trials (jRCT2051240249). Registered on 27 January 2025 (https://jrct.mhlw.go.jp/en-latest-detail/jRCT2051240249).

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• This will be a prospective, single-centre, phase Ib dose-escalation trial, allowing a structured assessment of safety across incremental dose levels of NMU-HbV.

• Standardised premedication and monitoring protocols (vital signs, ECG, laboratory tests) will be applied uniformly, enhancing internal validity and reproducibility.

• Pharmacokinetic analyses will be performed using validated non-compartmental methods, enabling reliable estimation of maximum blood concentration, time to reach the maximum blood concentration, area under the blood concentration-time curve and half-life.

• The small sample size (n=16) and restriction to healthy Japanese volunteers may limit generalisability to patient populations and other ethnic groups.

• The open-label, single-centre design without a comparator group may restrict the ability to detect subtle or delayed adverse events and may introduce observer bias.

Introduction

In settings such as prehospital care, remote islands, emergencies, disasters and surgery, it is sometimes not possible to quickly secure blood for transfusion for patients in shock from massive bleeding; the only alternative is transfusion with fluids that lack oxygen-carrying capacity.¹ Therefore, even if circulatory volume is restored, tissue oxygen deficiency cannot be quickly corrected, often leading to mortality or a substantially reduced quality of life. An oxygen carrier that could replace blood for transfusion would make it possible to avoid critical situations, save lives and improve quality of life. In other words, such a development would revolutionise emergency and disaster medicine, as well as the initial treatment of critical bleeding in remote areas. There is much hope for the realisation of such oxygen carriers in obstetrics, emergency medical care and blood companies.²

Haemoglobin vesicles (HbVs) have been developed over many years by researchers at Waseda University, Nara Medical University, Asahikawa Medical University and other institutions, with support from the government. They contain high concentrations of human haemoglobin molecules purified from human red blood cells encapsulated within liposome particles.³ The liposomes are structurally similar to several preparations already used in clinical settings. However, while many existing liposome preparations exert their efficacy by releasing the encapsulated drug from the liposome inside the body, in this preparation, importantly, the haemoglobin molecules remain encapsulated inside the liposome, which allows the NMU-HbV to act safely in the body as an oxygen carrier in place of red blood cells. Many animal experiments have shown that NMU-HbV circulates in blood vessels while still encapsulating human haemoglobin molecules, transports oxygen to tissues, and is eventually captured by the reticuloendothelial system and decomposed and excreted, similar to red blood cells.4,10 In other words, when sufficient red blood cells are present, it is difficult to distinguish the biological function (oxygen transport function) of the vesicles from that of the red blood cells and evaluate the two separately.

The primary use of NMU-HbV for emergency administration to patients with haemorrhagic shock, providing a haemoglobin dose equivalent to that of packed red blood cell preparations to support resuscitation and improve survival. Consequently, patients will be administered a lipid dose that exceeds the amount contained in a single dose of a normal liposome preparation. Therefore, the safety of NMU-HbV needs to be evaluated. Previously, we conducted the NMU-HbV-101 trial, a single-centre, open-label, dose-escalation study, at Hokkaido University Hospital.¹¹ In that phase I trial, participants were administered NMU-HbV as follows: cohort 1, 10 mL; cohort 2, 50 mL and cohort 3, 100 mL. A total of 11 healthy volunteers were enrolled. All adverse events, including liposome-induced infusion reactions, were well-tolerated. Moreover, no clinically significant changes were observed in any vital signs, including blood pressure.

In the planned phase Ib trial, to gain clinical usefulness, we will increase the amount of NMU-HbV to 400 mL. To mitigate the risk of adverse events such as fever, rash and infusion reactions, premedication with dexamethasone, acetaminophen, diphenhydramine and famotidine will be administered in this study.

We are publishing this study design for the following reasons. First, since the previous phase I study did not establish the safety of HbV at volumes exceeding 100 mL, this phase Ib study is a necessary prerequisite for advancing to well-designed clinical trials in patients with anaemia of various aetiologies. Publishing the study protocol will enhance research transparency and scientific credibility. Second, the primary objective of this study is to evaluate whether higher volumes of HbV may lead to cardiac strain, elevated blood lipids or severe allergic reactions in healthy volunteers.

Methods and analysis

Trial design

This will be a single-centre, open-label, phase Ib dose-escalation study of NMU-HbV in 16 healthy volunteers. NMU-HbV will be administered at Nara Medical University Hospital in Nara, Japan.

Eligibility criteria

Eligible participants must meet all of the following inclusion criteria¹: be a healthy Japanese adult²; provide written informed consent prior to the screening test³; be between 18 and 49 years of age at the time of consent acquisition⁴; have a body weight between 50 and 85 kg at the time of the screening test⁵; be able to abstain from smoking during the admission period⁶; have a blood haemoglobin concentration at screening within the range of 12.0–16.5 g/dL (men) or 10.0–14.5 g/dL (women) and⁷ be able to attend all required tests and complete the clinical trial.

The exclusion criteria at screening include the following¹: a clinically relevant history of surgery or current surgical treatment within 4 weeks prior to registration²; a clinical history or current treatment of hepato-renal-cardiac dysfunction³; a history or presence of drug or liposome allergies⁴; a history of alcohol or drug dependence⁵; donation of 400 mL of whole blood within 4 months, 200 mL of whole blood within 2 months or a blood component within 14 days prior to the screening test⁶; use of prescription or over-the-counter drugs (including crude drugs) within 14 days prior to the screening test (agents such as topical disinfectants or eye drops with no systemic exposure are permitted if the principal investigator deems them to have no effect)⁷; planning to participate concurrently in another clinical trial⁸; participation in another clinical trial within 4 months (starting from the last administration date) prior to the screening test and receiving the study drug (NMU-HbV)⁹; prior receipt of the study drug (NMU-HbV)¹⁰; any suspected need for therapy due to current symptoms or organ dysfunction indicated by abnormal vital signs or laboratory data at screening (however, the principal investigator(s) may override an exclusion if they judge a participant’s condition appropriate for the study)¹¹; having a positive infectious disease test (HBs antigen, syphilis test (Treponema pallidum latex agglutination (TPLA) method), HCV antibody, HIV antibody) or urinary drug abuse test,¹² women who are likely to become pregnant and who have not received contraceptive treatment, who cannot consent to contraception (the following criteria are used to define those who do not have a pregnancy potential: evidence of a prior hysterectomy, bilateral salpingectomy, or bilateral oophorectomy, premenarcheal status, postmenopausal status (defined as at least 12 months since the last menstrual bleeding without another medical cause)¹³; men who have female partners of childbearing potential who are not using effective contraception, and the participant is unwilling or unable to consent to contraceptive use from the time of study enrolment until 12 weeks after study completion¹⁴; women who have male partners who could cause pregnancy, where neither partner is using effective contraception, and the participant is unwilling or unable to consent to contraceptive use from the time of study enrolment until 12 weeks after study completion and¹⁵ those judged by the principal investigator(s) to be unsuitable for the trial (eg, due to anticipated non-adherence or difficulty attending visits).

Exclusion criteria at the time of predose examination include the following¹: participation in another clinical trial with medication administration between screening test and hospitalisation²; use of other drugs within 14 days before hospitalisation³; receiving medical treatment from another physician at the time of hospitalisation⁴; consumption of foods or beverages containing caffeine or alcohol within 3 days prior to study drug administration; and⁵ other items that the investigator deems inappropriate (eg, if it is judged that the patient is ineligible to participate in the clinical trial as a result of the pre-administration examination, or if it is expected that it will be difficult to comply with the visit or manage the subject). The participant consent form is available as online supplemental material. The flow diagram of this study is shown in figure 1.

Figure 1. Phase Ib study design. Each participant will receive intravenous administration of NMU-HbV at a dose and maintenance infusion rate specific to their cohort: cohort 1, 100 mL at 2.5 mL/min; cohort 2, 100 mL at 5.0 mL/min; cohort 3, 200 mL at 5.0 mL/min and cohort 4, 400 mL at 5.0 mL/min. The patients will be premedicated with dexamethasone, acetaminophen, diphenhydramine and famotidine. In cohort 1 (n=4), NMU-HbV will be administered to one participant each week (weeks 1–4). In each of cohorts 2–4 (n=4 per cohort), NMU-HbV will be administered to one participant in week 1 and to three participants in week 2. After completion of the administration, the participants will be monitored until day 4 in the hospital and on days 8 and 15 as hospital visits. A review of safety and tolerability by the Data and Safety Monitoring Committee will be conducted before transitioning to the next cohort.

Study procedures

A total of 16 healthy volunteers will be enrolled in four cohorts, with four participants each. The physical, blood and urine test schedules are presented in onlinesupplemental tables 1 2. Each volunteer will receive dexamethasone (6.6 mg; intravenous injection), acetaminophen (500 mg; oral), diphenhydramine (10 mg; oral) and famotidine (20 mg; oral) as premedication between 1 and 2 hours before administration of the NMU-HbV.

From the time of admission to discharge, participants may not use any medications other than the investigational drug and the premedication administered before it. In addition, the participants will not receive concomitant therapies from the time of admission to discharge. However, this does not apply if the principal investigator (or a subinvestigator) judges that treatment is necessary for the adverse events that occur during this clinical trial.

Patient and public involvement

Neither patients nor the public were involved in the design, conduct, reporting or dissemination plans of this research.

Intervention: administration of NMU-HbV

Each participant will receive intravenous administration of NMU-HbV at the dose determined for each cohort at 1.0 mL/min for 10 min, with the remainder delivered at a maintenance rate specific to the cohort: cohort 1 (100 mL) at 2.5 mL/min; cohort 2 (100 mL) at 5.0 mL/min; cohort 3 (200 mL) at 5.0 mL/min and cohort 4 (400 mL) at 5.0 mL/min. Only when the observation period of each cohort is completed and confirmed by the safety evaluation committee, administration to the next cohort and observation will be initiated.

Criteria for discontinuing interventions

The investigators will discontinue the allocated interventions if¹ the participant requests to discontinue the clinical trial or withdraws consent²; the principal investigator (or a subinvestigator) judges that the participant has experienced an adverse event serious enough to warrant the discontinuation of the clinical trial³; the participant no longer meets inclusion criteria or now meets exclusion criteria⁴; the participant does not attend the hospital, cannot be contacted, or other circumstances make it difficult to observe or investigate safety or⁵ the principal investigator (or a subinvestigator) judges that the clinical trial should be discontinued for other reasons.

Primary and secondary outcomes

The primary outcome will be safety in terms of¹ the presence or absence of adverse events and frequency of occurrence up to 14 days after administration of the study drug; and² clinically significant changes from the baseline up to 3 days (72 hours) after administration of the study drug based on subjective and objective symptoms, vital signs, electrocardiograms and clinical test values. The secondary outcome will be pharmacokinetics. Changes in blood NMU-HbV concentration will be evaluated from immediately after the administration of NMU-HbV through day 4. In addition, the maximum blood concentration (C_max), time to reach the maximum blood concentration (T_max), area under the blood concentration-time curve (AUC) and elimination half-life (T_1/2) of NMU-HbV will be estimated.

Sample size and recruitment

Based on the number of cases per cohort in a phase I study of NMU-HbV, we set the number of participants to four in each cohort.¹¹ Healthy volunteers will be recruited via the 3H Medi Solution website. Information on this phase Ib trial will be posted at Nara Medical University Hospital. Investigators will select eligible participants and explain the study to them. The planned study period is from 2 January 2025 to 30 June 2026. The planned recruitment periods are as follows: cohort 1, 26 May–1 July 2025; cohort 2, 29 September–28 October 2025; cohort 3, 12 January–17 February 2026 and cohort 4, 11 May–16 June 2026.

Data collecting methods

The principal investigator (or a subinvestigator) will conduct the screening tests (onlinesupplemental tables 1 2) for consenting participants within 30 days prior to the administration of the study intervention. If the principal investigator (or subinvestigator) deems it necessary, a retest will be conducted. The principal investigator (or subinvestigator) will determine the eligibility of each participant based on the screening test and inclusion and exclusion criteria.

The investigator (or a subinvestigator) will conduct the observations and tests listed in onlinesupplemental tables 1 2 during hospitalisation and at scheduled visits after discharge (on days 8 and 15). Participants will be discharged following the observations and tests on day 4, unless adverse events requiring extended hospitalisation have occurred.

For blood coagulation and biochemistry tests after administration of the investigational drug, samples will be processed by adding dextran, and the obtained measurement values will be used for safety evaluation as standard values (dextran-added standard values) obtained by correcting the facility standard values with a specified coefficient (correction coefficients and standard values are described in the ‘Operational Procedures for Blood Test Value Measurement’).

Additional observations and tests will be conducted, as necessary. If any concerns arise regarding the participant’s clinical signs, tests not prespecified for safety evaluation will be performed, and appropriate measures will be taken, as necessary.

An adverse event is defined as any undesirable or unintended disease or symptom (including abnormalities in clinical test values) that occurs in a participant from the start of investigational drug administration until the end of the observation period or trial discontinuation, regardless of its causal relationship to the investigational drug. Furthermore, symptoms or diseases present prior to the administration of the investigational drug are classified as pre-existing conditions and are not considered adverse events.

Data management, monitoring and auditing

The head of the medical institution conducting the clinical trial and the person conducting the clinical trial (principal investigator) will perform quality control and quality assurance of the clinical trial data in accordance with the standard operating procedures for the conduct of clinical trials and mutually agreed on documents, as well as the standard operating procedures for monitoring and auditing established by the principal investigator.

Statistical analysis

The following statistics will be calculated: for categorical data, the number of cases and corresponding percentages; for quantitative data, the number of cases, arithmetic mean, SD, median and minimum and maximum values. Safety analyses will be performed for cases subjected to safety evaluation.

The occurrence or absence of adverse events due to the investigational drug will be tabulated according to the administration group, and a frequency table will be created. When tabulating events by severity, if a participant experiences the same type of adverse event multiple times, only the instance with the highest severity will be counted. If it is necessary to interpret adverse events differently, the International Medical Terminology MedDRA developed by the International Council on Harmonisation of Technical Requirements for Pharmaceuticals for Human Use will be used, and tabulation will be performed using system organ classes and preferred terms.

For vital signs (blood pressure (systolic and diastolic), pulse rate, body temperature and respiratory rate) and clinical test values, summary statistics will be calculated for the measured values at each timepoint.

The following analyses will be performed on the NMU-HbV values:

1. Blood drug concentration: Descriptive statistics will be calculated for each administration group and test timepoint.

2. Pharmacokinetic parameters: Drug concentration parameters (C_max, T_max, AUC, T_1/2, apparent oral clearance and mean residence time) will be calculated from blood drug concentrations using non-compartment analysis, and descriptive statistics for each parameter will be calculated for each administration group.

There will be no interim analyses in this clinical trial. When making changes to the analysis plan, the person conducting the clinical trial (principal investigator) will consider the appropriateness of the changes and their impact on the evaluation of the clinical trial before deciding whether to make the changes. In addition, the person conducting the clinical trial (principal investigator) will clearly document the content of the discussion, whether changes were made, and the reasons for these changes.

Discussion

HbV has been developed as a blood transfusion alternative to spare red blood cell consumption at medical institutes and it offers life-saving resuscitation for patients with fatal haemorrhages in emergencies. The multiple advantages of HbV address the key limitations of donor-derived blood products. Notably, HbV is blood type independent, eliminating the need for blood group matching and enabling universal administration. As HbV is produced using a purified human haemoglobin solution, it carries no risk of transmitting infectious agents. Its long-term stability at room temperature (up to 2 years) allows for flexible storage and rapid deployment in emergency or resource-limited settings. Its high oxygen-carrying capacity, achieved through the encapsulation of concentrated haemoglobin, supports effective tissue oxygenation. In addition, it exhibits no potential for biological accumulation, because its components are designed to be safely metabolised and cleared from the body. The submicron-scale particle size of HbV facilitates its unhindered passage through capillaries. Moreover, the formulations are free of potassium ions, thereby eliminating the risk of hyperkalaemia, which is a common concern with stored red blood cells, especially in patients with end-stage renal failure. The product can be manufactured in compliance with the good manufacturing practice (GMP), ensuring consistency and safety at scale. A phase I clinical trial has confirmed its preliminary safety and pharmacokinetic profile in healthy volunteers.

A dose-escalation study is required to overcome the following limitations: First, in the development of blood transfusion alternatives, vasoconstriction and hypertension have been identified as major issues following the administration of purified haemoglobin with chemical modifications.¹² In contrast, HbV encapsulates a haemoglobin solution, which reduces its reactivity with nitric oxide, an endothelium-derived relaxing factor.^{13 14} As demonstrated in a previous clinical trial, no increase in blood pressure was observed at doses up to 100 mL.¹¹ However, as HbV volumes greater than 400–800 mL are expected to be required in clinical practice, in this dose-escalation study, we will evaluate the safety and potential efficacy at high infusion volumes. Second, the lipid components of HbV products include PEGylated lipids. Hence, it is important to monitor whether immune reactions occur in response to pre-existing antipolyethylene glycol (PEG) antibodies.⁵ The presence of anti-PEG antibodies in the blood of the participants will also be monitored at three different points in an ancillary study. Given that PEGylated lipids account for only 0.3 mol% of the formulation and the particle surface carries a negative charge, such reactions are considered unlikely.¹⁶ Third, a high volume (up to 400 mL) and transfusion rate (up to 300 mL/hour) may trigger unexpected adverse events in healthy volunteers that were not observed in the previous phase I trial including cardiac strain, elevated blood lipids or severe allergic reactions.

Ethics and dissemination

Dissemination plans

Dissemination plans include publishing in peer-reviewed scientific journals and presenting at international conferences.

Ethics approval

The study protocol was approved by the Institutional Review Board of Nara Medical University on 10 December 2024. This study will follow the Declaration of Helsinki and adhere to the Clinical Trials Act. Written informed consent will be obtained from all participants. Additional consent will be obtained when data and biological specimens are used in ancillary studies.

Protocol amendments

If the protocol requires amendments, all changes and their reasons will be reported to the Institutional Review Board. Following approval of the amendments by the Institutional Review Board, the patient instructions will be updated accordingly.

Ancillary and post-trial care

The investigator will have insurance to compensate the participants for any harm related to the trial.