Expanded Access Use of Sanguinate Saves Lives: Over 100 Cases Including 14 Previously Published Cases

Abstract

Background: PP-007 (SANGUINATE^®, PEGylated carboxyhemoglobin, bovine) is under development to treat conditions of ischemia/hypoxia. Hemorrhagic/hypovolemic shock (H/HVS) becomes a life-threatening comorbidity due in part to hypotension and hypoxia. Blood transfusions are indicated, but supply and compatibility issues may limit subject access or when blood is not an option due to religious restriction or concern for clinical complications. PP-007 is universally compatible with an effective hydrodynamic radius and colloidal osmotic pressure facilitating perfusion without promoting extravasation. Methods: A review of previous clinical trials was performed and revealed an Open-Label Phase 1 safety study of acute severe anemia (hemoglobin ≤ 5 g/dL) in adult (≥18 y) patients unable to receive red blood cell transfusion (NCT02754999). Primary outcomes included safety events with secondary efficacy measures of organ function and survival at 1, 14, and 28 days. Additionally, a retrospective review of published, peer-reviewed case reports was performed, evaluating the administration of Sanguinate for Expanded Access in those patient populations where blood was not an option over the past 12 years. Results: A total of 103 subjects were enrolled in the Phase I safety study with significant co-morbidities that most commonly included hypertension (n = 43), acute and chronic kidney disease (n = 38), diabetes mellitus (n = 29), gastrointestinal bleeds (n = 18), and sickle cell disease (n = 13). Enrollment characteristics included decreased hemoglobin and severe anemia (mean baseline hemoglobin of 4.2 g/dL). Treatments included an average of three infusions [range 1–17]. Secondary efficacy measures were mean Hb levels, respiratory support, and vasopressor requirements, all demonstrating clinically relevant improvements. Fourteen additional cases were identified in the literature. Though one patient died due to pre-treatment conditions, all patients but one were discharged home in stable condition. Conclusion: Collectively, these observations are encouraging and provide support for the continued evaluation of PP-007 in advanced clinical trials in severe anemia including H/HVS. The review of published case reports underscored the potential of Sanguinate to reduce early mortality. Adverse effects included transient hypertension, lethargy, dizziness, and troponin elevation. These findings highlight the need for continued research and funding of blood alternatives to improve outcomes when standard blood transfusions are unavailable or contraindicated.

1. Introduction

Sanguinate is a polyethylene glycol (PEG) modified bovine hemoglobin saturated with carbon monoxide (CO). It has an oxygen affinity intermediate to that of human hemoglobin and hypoxic tissues, enabling selective oxygen transfer to hypoxic tissues, particularly in the microvasculature. During hypoxic stress, the microvasculature may constrict, inhibiting red blood cell flow and oxygen delivery. Consequently, Sanguinate’s ability to traverse the microvasculature is crucial. With a molecular weight of 120,000 Daltons, approximately 80% that of an IgG antibody, Sanguinate can easily traverse the microvasculature, off-load oxygen, and continue binding and releasing oxygen for ≥ 72 h. It offers several additional novel features, making it a potential therapeutic option for patients with impaired tissue oxygen delivery or for volume resuscitation (

Table 1. Properties of Sanguinate. HBOC: hemoglobin-based oxygen carrier; Hb: hemoglobin; and NO: nitric oxide. See references [1,2].

HBOC Class	(PEGBvHb)CO
Product Name	Sanguinate
Hb Type	Bovine hemoglobin conjugated with PEG on surface lysines
Non-Hb Components	PEG
Molecular Weight	120 kDa
P50	9.56 mmHg
Cooperativity Coefficient	1.56
Oxygen Dissociation Rate Constant	16.7
Auto-oxidation Rate Constant	0.19/hr
NO Consumption	49.7 µM−1s−1
Additional Notable Properties	Pegylated: Increases shelf life to over 4 years. Enhances circulating half-life. Delivers CO, which is anti-inflammatory and anti-vasoconstrictive. Acts as a plasma expander. p50 ensures oxygen is only delivered to hypoxic tissue.

).

Tissue hypoxia secondary to acute blood loss or thrombotic events remains a major life-threatening complication in various medical scenarios. Thus, hemoglobin-based oxygen carrier (HBOC) therapies, such as Sanguinate, are emerging as potential blood alternatives for urgent clinical use [1]. HBOCs are particularly valuable in unique patient populations where crossmatched blood may not be available or for those whose religious beliefs preclude the use of blood products [2].

Sanguinate is currently being evaluated in the HEMERA-1 clinical study for safety and efficacy in acute ischemic stroke. Sanguinate treatment has demonstrated improvements in both short-term National Institutes of Health Stroke Scale (NIHSS) scores and 90 day modified Rankin scores in combination with mechanical thrombectomy, intravenous thrombolytics, or both [3].

We performed a retrospective analysis of published case reports regarding the use of Sanguinate in those patient populations over the past 10 years and a report on 101 patients receiving Sanguinate for Expanded Access indication, with 14 of these patients published in case reports (

Table 2. Case reports of Sanguinate usage over the last 10 years. SCD = sickle cell disease; JW = Jehovah’s Witness; HCC = hepatocellular carcinoma; AML= acute myeloid leukemia; ACS = acute chest syndrome; TTP = thrombotic thrombocytopenic purpura; and HHS = hyperhemolysis syndrome.

Author, Publication Year	# of Patients	Population	Nadir Hgb (g/dL)	Units of PEGylated Carboxyhemoglobin Bovine (Sanguinate®) Received (500 mL)	Outcome
Nalley et al., 2014 [4]	1	SCD with HHS	3.5	2	Survived to discharge
Kato et al., 2015 [5]	1	JW with Hgb SC SCD and ACS	N/A	1	Survived; no adverse events reported
Parmar et al., 2015 [6]	5	AML: Hemolytic reaction, ACS and sickle cell crisis	<3.5	2–8	Survived; no adverse events reported
Thenuwara et al., 2017 [7]	1	JW; post-partum hemorrhage	3.0	3	Survived to discharge
Sam et al., 2017 [8]	1	JW with TTP	3.8	4	Survived to discharge
Brotman et al., 2019 [9]	1	JW undergoing cystoprostatectomy and nephrectomy	4.5	1	Survived to discharge
Holzner et al., 2018 [10]	2	JW; 1 liver transplant, 1 HCC	3.9–7.8	1	All survived to discharge
McConachie et al., 2018 [11]	2	JW; 1 GI bleed and 1 sepsis	2.6–4.9	1–5	1 survived, discharged in stable condition; 1 died

) [4,5,6,7,8,9,10,11,12,13,14,15]. While this is a small cohort of patients, which is less robust than prospective blinded study data, it may be a meaningful introduction to the potential value of Sanguinate and identify further directions in its development. Since this documents Expanded Access use, the guiding hypothesis is that Sanguinate may be developed for use in cases when blood is not an option.

It is clear that an oxygen carrier is needed and that HBOCs may address unmet medical needs. However, a modest cohort of patients without deep and complete physiologic data may provide some insight, yet it is not definitive. However, the transfusion of banked blood may not provide benefits. In over 100 randomized controlled trials of more transfused blood compared to decreased blood transfusion, no recommendations may be concluded, and no physiological data that banked blood supplies oxygen to tissues or increases oxygen delivery (rather, the opposite seems to be true) [12].

2. Materials and Methods

A systematic search to identify uses of Sanguinate in a broad patient population was conducted using a public government database (clinicaltrials.gov). Inclusion criteria included performance of the trial within the United States, completion of the trial, and use in patients who otherwise would be unable or unwilling to receive conventional blood products. In performing this search, 12 studies were identified—5 were performed within the United States, and only 4 have been completed. Only one enrolled patient was unable or unwilling to use conventional blood products. This study is also the largest Phase I trial involving Sanguinate to date [3].

Additional cases of Sanguinate use not captured by clinical trial data were identified in the literature. A PubMed.gov search was performed of cases where blood transfusions were not possible and Sanguinate was used as a blood alternative for therapeutic intervention of any underlying cause in the last 10 years. We identified eight published case reports and small case series that document the clinical context and nadir Hgb level at which intervention with blood products would have been necessary, but, instead, Sanguinate was used. The drug product was provided for use in the life-threatening cases issued under the FDA Expanded Access program.

3. Case Studies

A review of the literature over the past 12 years revealed eight manuscripts that described the outcomes of fourteen patients who had received at least one unit of Sanguinate through the FDA Expanded Access program. These ranged from a case report involving a single patient to the largest case series describing five patients (See Figure A1 and Figure A2, and

Table A1. eIND subject individual demographic and treatment data.

Subject No	Age	Sex	Condition	BNAO	Units Infused	Hb Pre-Infusions	Hb Post-Infusions
1	29	F	Chemo/cytopenia/AML	JW	4	3.6	4
2	27	F	Chemo/cytopenia/AML	JW	1	3	2.8
3	65	M	Chemo/cytopenia/AML	JW	1	5	unk
4	25	F	CVA/Pancytopenia	JW	3	4.5	4.7
5	43	F	Procedure Bleed/Abdm	JW	3	3.8	4.2
6	56	M	Procedure Bleed/GI	JW	1 ½	2.8	3.1
7	22	F	Procedure Bleed/GI	JW	2	1.6	unk
8	77	F	Procedure Bleed/GI	JW	1	3.7	5.3
9	44	M	Procedure Bleed/GI	JW	3	6.6	7.7
10	91	M	Procedure Bleed/GI	JW	1 ½	4.1	3.9
11	47	M	Procedure Bleed/GI	JW	3	4.7	2.9
12	78	F	Procedure Bleed/GI	JW	2 ½	5.2	3.9
13	42	F	Procedure Bleed/Gyno	JW	6	3.3	3.9
14	53	M	Procedure Bleed/Gyno	JW	4	4.1	3.7
15	60	F	Procedure Bleed/Gyno	JW	2	2.7	unk
16	75	F	Procedure Bleed/Gyno	JW	2	3.5	3.3
17	61	F	SCD/AChS	JW	2	2.9	4.1
18	19	F	SCD/AChS	hemolysis	4	2.1	unk
19	10	F	SCD/AChS/Pneumonia	JW/hemolysis	1	9.3	8.9
20	28	F	SCD/BMT	hemolysis	8	3.5	2.5
21	19	M	SCD/TTP	JW/hemolysis	4	2.4	4
22	23	F	SCD/VOC	hemolysis	4	3.4	3.1
23	51	F	SCD/VOC	JW/hemolysis	2	1.7	unk
24	49	M	Septic Shock/Abscess	JW	3	8.8	unk
25	55	F	Trauma/Fall	JW	2	3.1	3.1
26	62	F	Trauma/Fall	JW	1	2.9	2
27	59	F	Trauma/MVA	JW	2	2.5	2.1
28	73	F	Trauma/MVA	JW	1	4.4	unk
29	21	M	Trauma/MVA	JW	1	6.5	7.2

). Six of the eight manuscripts addressed usage in JW patients, and the remaining two addressed use in sickle cell disease (SCD) patients with complications. The number of patients in each group was fairly similar—there were eight JW patients and seven SCD patients (one was both JW and had SCD). These two patient demographics represent the two most common scenarios in which blood would not be an option—for religious reasons in JW patients and for either difficulties in matching blood due to multiple alloantibodies or for concern for hyperhemolysis after transfusion in SCD patients.

The level of anemia was uniformly severe in each patient—twelve patients had an initial hemoglobin level of <5 g/dL. The only one higher than this (7.8 g/dL) was in a liver transplant patient. One case did not have a reported nadir hemoglobin. This is notable in and of itself because the transfusion of conventional blood products is considered at a cutoff higher than this—usually around Hgb 7–8 g/dL in accordance with other co-morbidities and current clinical status. The reported side effects ranged from none reported to hypertension, transient increase in troponin (attributed to demand ischemia), and grade 1 parasthesias.

The Expanded Access (Compassionate Use) program for Sanguinate enrolled 103 patients over 14 months, with an additional 29 patients treated under eINDs. The patients were predominantly female, racially diverse, with a significant portion identifying as Black/African American and age ranging from 19 to 90 years old. Most patients (67%) received five or fewer units of Sanguinate, and 28% received two or fewer, with a maximum single-day dosage of four units and a cumulative maximum of 17 units. Clinical observations revealed that one patient, who had severe gastrointestinal bleeding and hemorrhagic shock and had refused blood transfusions, had significantly improved respiratory and cardiovascular conditions after only a few infusions (see Figure A1 and Figure A2).

4. Discussion

Sanguinate is a unique oxygen therapeutic in that it has additional properties that work in concert with oxygen delivery to improve its therapeutic action. A major portion of its therapeutic action comes from the carbon monoxide released by the molecule immediately following infusion. Carbon monoxide is an anti-inflammatory and anti-vasoconstrictive agent. This helps to impede the blood vessel contraction that occurs following trauma and blood loss. The PEG hemoglobin also acts as both a blood plasma expander and oxygen-delivering agent. Furthermore, PEG acts as a drag-reducing polymer [15]. The combination of these properties ensures adequate blood pressure, blood flow, and oxygenation of the tissues.

Comparing Sanguinate to blood would be extremely complex. Defining a specific patient population with both inclusion and exclusion criteria that will provide a clear statistical difference will be difficult and may in fact be unethical. Individuals have different responses to low hemoglobin concentrations, so the hemoglobin value dictating treatment may be questioned [12].

It is likely better to compare Sanguinate to existing treatment for tissue ischemia, especially in the case of stroke where blood is not considered to be an effective treatment. The blockage event releases cytokines, which results in an inflammatory event that decreases the lumen of the vessels, further impairing blood flow and increasing tissue ischemia. The multiple properties of Sanguinate may act in concert to overcome these concerns [1].

Sanguinate is a carbon monoxide-releasing molecule. Carbon monoxide has long been recognized as a therapeutic gas [16]. It inhibits the release of certain cytokines, thereby stopping the inflammatory response. It is both an anti-inflammatory and anti-vasoconstriction agent. The carbon monoxide is not released but rather displaced by circulating oxygen. That oxygen is then carried by this single PEG-Hb molecule, which is small enough to bypass the vascular blockage and deliver the oxygen to the ischemic tissue. The release of carbon monoxide is a singular event, while the PEG-Hb will continue to load and deliver oxygen as long as it is in the circulation [16].

There is both empirical and direct evidence that, when CO binds to hemoglobin, O2 cannot displace it. In the case of Sanguinate, altering the tertiary structure of hemoglobin with PEG changes the structural and functional properties depending on the chemistry of attachment and type of hemoglobin [17,18,19].

Human hemoglobin does indeed have a greater affinity for CO than for oxygen or CO₂, which is why it is so toxic in high concentrations. Sanguinate uses bovine hemoglobin rather than human hemoglobin for two main reasons. Bovine hemoglobin differs from human hemoglobin. Human Hb requires 2,3-DPG as an allosteric effector, while bovine Hb does not. This is due to a five amino acid difference in the beta chain of bovine Hb [20]. This alters the binding characteristics of the molecule and makes the tertiary structure of bovine Hb more stable than human Hb. The other reason is that bovine Hb is readily available.

Prolong’s method of PEGylation further alters the bovine Hb functional characteristics. The p50 (a measure of oxygen affinity) of bovine Hb is approximately 25, whereas the p50 of PEG-bovine Hb (PEG-HbBv) is approximately 10. This means that Sanguinate has a greater affinity for oxygen than Hb in the red cell. Ischemic tissue has an even greater affinity for oxygen, with a p50 around 2. PEG-HbBv acts as a facilitator of oxygen transport in the plasma by extracting oxygen from the red cell and delivering it to the tissue. This is a cyclical process, and PEG-HbBv does not need to acquire oxygen directly from the lungs.

The affinity for CO is also apparently changed. PEG-HbBv has a greater affinity for oxygen than CO. CO is forced onto the molecule during production to create Sanguinate (PEG-HbBvCO). The CO stabilizes the molecule and allows storage stability of at least 5 years. It is also the anti-inflammatory and anti-vasoconstrictive agent when displaced by oxygen in the circulation.

The concept of a very low p50 is relevant. The p50 of myoglobin is 2–3 mmHg, while the PtO2 of the interstitial space is 30 mmHg. Specific p50 decreases have been studied in multiple third and fourth-generation HBOCs and appear to provide oxygen delivery when absolutely essential, not overloading the organism when unnecessary. It is unknown which exact decreased p50 is optimal. Additionally, this makes sense for oxygen transport to the tissues. Oxygen moves from the lowest affinity to the highest affinity. The lowest affinity is in the red cell, and the highest affinity is in the ischemic tissue. PEG-HbBv is in between, which allows it to act as a conduit to transport oxygen from the red cell to the tissue. The authors do not believe measures such as cooperativity or the Bohr effect are of great impact in the context of these types of products. What is more important are the real-world effects of treatment. All published research, both in vitro and in vivo, demonstrates the proposed effects described above.

Prolong, the manufacturer of Sanguinate, has recently completed a safety study in stroke and has developed a follow-up Phase 2/3 study, which will be the indication contemplated for approval. An indication for stroke and/or tissue ischemia addresses an unmet medical need, rather than specifically replacing red blood cell transfusions.

In order to study whether our hypothesis has value, this patient population provided the quickest inroad to testing the safety and potential efficacy of the product. No protocol is required for emergency use, only the physician making the formal request to the FDA for its use. These were the most extreme patients, as most were almost moribund. Clearly, if Sanguinate were safe, and maybe even effective in these individuals with hypovolemia/ischemia due to different etiologies (accidents, disease, surgery, and religious), then a case could be made for the product’s safety in these patient populations, allowing for further clinical trials. Regrettably, the 107 patients could not be compared by age or sex-matched grouping to look for outcomes, as this was not a standard clinical trial but rather an emergency use, so limited data are available on the patient. No such comparisons could be made.

Review of the Phase 1 study included secondary measures of efficacy that would demonstrate less than 50% survival rates, but this survival rate would be greater than expected versus historical controls [2]. Additionally, increased hemoglobin concentrations with reduced requirements for pressors and respiratory support were possible contributors to survival rates, but additional studies are required to confirm. The overall survival rate of the published cases in the literature is even higher. The potential life-saving benefits emphasize the gap between the importance of having a blood alternative as a therapeutic option and the lack of an FDA-approved option for these patients.

The FDA Expanded Access program provides an alternate option for those who are incompatible with the transfusion of blood components due to religious beliefs, concerns with risks, and other medical reasons. Best used with Patient Management protocols, patients have experienced only mild and manageable side effects. Many case series support the effectiveness of treatment, suggesting potential improvements in patients’ outcomes. However, no oxygen therapeutic agents are approved for human use in the US and can be developed requiring “substantial evidence of effectiveness”, demonstrated through “adequate and well-controlled clinical trials.” The FDA permits the use of certain aspects of real-world evidence to support development, but this approach requires prior discussion with the FDA to mitigate potential bias.

The challenges in ensuring an adequate blood supply emphasize the critical need for substitutes to traditional transfusion products. The approval of oxygen therapeutic agents requires “substantial evidence of effectiveness” through “well-controlled clinical trials.” To support the advancement of treatments for serious or life-threatening conditions, the FDA has established programs that prioritize review and accelerate the approval process for some drugs and biologics. Though programs like the Emergency Use Authorization allow temporary access to unapproved therapies, these programs do not replace the standard approval process and must align with established safety and efficiency requirements. Developing and obtaining FDA approval for the initial blood alternative remains the greatest challenge as it remains a massive logistical and ethical challenge to perform the type of clinical trial that is usually required. It would be nearly impossible to develop a product that is equivalent in both clinical benefit and lack of side effects to traditional blood products—hence, the focus here is on patients who would be unable to receive blood products at all. If the choice is between receiving no blood products versus a blood alternative with a high chance of being lifesaving, then we believe that developing such a product would be highly desirable and a crucial tool for the treatment of these patients [21].