Abstract
Since around 1985, a liposome encapsulated hemoglobin vesicle (HbV) has been developed in Waseda University as an artificial red cell. Subsequently, in 1995, Terumo Co. produced a prototype of HbV for clinical trial, the so–called Neo Red Cell. We tested this in preclinical study and believed firmly that infusion of the HbV could substitute for ordinary blood transfucion [Citation]. Subsequently, further improvement was done on the HbV by investigators of Waseda and Keio University supported by a grant of Health & Welfare Ministry, Japan, and recent physicochemical properties of the HbV are evaluated, mostly applicable for clinical trial [CitationCitation].
SAFETY AND EFFICACY OF ARTIFICIAL BLOOD
If a new product will be recognized and accepted as artificial blood, two requirements should be satisfied. One is proper physicochemical activity and another is its clinical efficacy.
Physicochemical activity required for HbV is listed in . Oxygen transporting function is the most crucial thing, which depends on an amount of hemoglobin (Hb) contained in vesicle and oxygen dissociation curve of the Hb. Secondly, conversion rate of Hb to metHb in vitro (during storage) and in vivo (after infusion) should be minimized. In the third, dispersibility of vesicle in the solution and blood should be kept constant and homogenous. In the fourth, size of the vesicle is required to be 0.1–0.2 microns of diameter. In the fifth, adequate viscosity of the solution is also important. In the sixth, homogeneity & stability of the vesicles should be maintained. Finally, adequate pH of the solution is necessary.
Table 1 Physiochemical activity for efficacy
On the other hand, clinical efficacy should be assessed by items shown in . Satisfactory oxygen supply to tissues depends on good pulmonary oxygenation of artificial red cell, sufficient oxygen release at the tissues, less conversion to Hb to metHb in the blood, and maintenance of normal cardiac output, which may be supported accompanied with the normal circulating blood volume. Finally, good clinical efficacy of artificial red cell depends on adequate retention of the vesicles in the circulating blood.
Table 2 Clinical efficacy
In addition, any adverse effects should never occur in subjects in whom the HbV will be infused. Concerning drug safety, particulary for artificial blood, several criteria have been issued [Citation[4-7]]. Recently, certain check points, listed in , were proposed, particularly for the HbV. On the other hand, criterion concerned with the efficacy of artificial blood is scanty.
Table 3 Safety of HbV solution
PHYSICOCHEMICAL PROPERTIES FOR SAFETY
In the process for encapsulation of Hb into liposome vesicle, it is required to once convert Hb to carbon-monoxide-Hb. Therefore it must be confirmed whether or not there will be some residual carbon-monoxide-Hb and carbon-monoxide in the solution. Likewise it must be checked if some excess phospholipid of lipsosome and polyethylene glycol remained in the solution. Obviously, retention of free Hb and contamination of endotoxin cannot be allowed. And also its sterility must be kept absolute.
CLINICAL SAFETY OF THE HBV
It must be proved that the HbV do not cause any adverse effect on our vital functions, as shown in . Therefore those items must be examined in preclinical studies using animals. For example, on the heart and circulatory function, the research groups of Waseda and Keio University have obtained a definite evidence that normal circulation is maintained after replacement with the HbV for severe blood loss. Most of those vital functions have been tested in small animal experiments and no adverse effect has been observed. It is very hard to make a good proof, however, that the HbV will never affect psychological or behavior function. In addition, it must be confirmed that neither teratogenicity nor tumorgenicity (not only in the animal in which the HbV is infused but also will be found, in his offspring) at least within three generations.
Preclinical study for recently developed HbV, however, has not been done in middle–sized and large animals as yet, since we could not obtain a sufficient amount of the HbV for those experiments due to its producing capacity. Fortunately, a few pharmaceutical campanies, such as Terumo Co., are going to associate for mass production of HbV. Therefore, preclinical studies, including in middle–or large–sized animals, will be achieved in the near future.
The Society of Blood Substitutes, Japan provided a guidline for clinical trial with new artificial blood six years ago. The project team of Ministry Health & Welfare, Japan, decided to make some protocols for clinical trial of HbV in accordance with accumulation of data from preclinical studies.
DESIGN OF CLINICAL TRIAL
For planning the clinical trial, two important things are that the trial should fully satisfy ethical requirements and also draw definite evidence from its results.
To satisfy the medical ethics, we have to keep in mind three points, as listed in . First of all, any clinical trial should be done as treatment or therapy for patients. Secondly, the treatment or therapy performed in clinical trials should be equivalent or superior to conventional treatment or therapy. Thirdly, clinical trials must be done holding authorized and proper, informed consent.
Table 4 Consideration on medical ethics for clinical trial
Considering medical ethics, two main requirements for selection of subject are considered. One of them is a definite reason that this artificial blood (HbV) is beneficial for treatment or therapy of the subject. In another, certain time will be obtained to receive an informed consent from the subject before action of clinical trial, as shown in .
Table 5 Selection of subjects for the clinical trial regarding reception of informed consent
For assessment of clinical efficacy of the HbV as a substitute of blood, two crucial items are focused on, namely stabilized circulatory dynamics and sufficient oxygen supply to tissues:
For achievement of definite and satisfactory results, four items should be considered, as follows.
Setting for proper control for procedure (treatment or therapy);
Complete achievement of initial determination (control) of physiological parameters;
Application of routine laboratory test and simple technique for measurement;
Selection of subjects who do not suffer severe illness or are not placed in unstable or complicated pathological conditions.
With those understandings, first clinical trial should be recommended to be done for replacement of surgical blood loss of 16–20 ml/kg, listed in . Namely, it will be done for hemodilutional autologous blood transfusion, blood transfusion for emergent surgery or unexpected bleeding during surgery in which proper blood for transfusion is not provided, and blood transfusion for patients with an uncommon blood type whose surgery should be done relatively urgently.
Table 6 First selection for clinical trial
Some other applications as shown in that might be proposed for the clinical trial. In our opinion, however, it should be better to avoid those, since those pathological conditions or illness and situations per se influence parameters measured. The data obtained from those patients in those situations will mislead and hinder us from making an accurate evaluation on the HbV as artificial blood and the decision to progress further to practical application for the HbV.
Table 7 No program projected in clinical trials for new artificial blood
The physiological parameters measured in the first clinical trial should be limited in routine laboratory tests if possible and technique should be simple and easy. Those physiological parameters measured are listed in . Insertion of CVP catheter is possibly acceptable for cases whose surgical bleeding is anticipated to exceed over 12 ml/kg, but Swan Ganz catheter is not always acceptable. If the tip of the CVP catheter is placed in front of the tricuspidal valve, we can judge blood sampled there as the mixed venous blood. [Citation[8]]. Then we can determine its oxygen partial pressure, which could estimate mean oxygen partial pressure of whole body tissue. Blood lactate level can be used as an indicator for tissue hypoxia.
Table 8 Physiological parameters measured in the clinical trial
Incidentally, an actual protocol for the trial with hemodilutional autologous blood transfusion is illustrated in . Namely, after receiving patient's written informed consent, the patient will be anesthetized and an arterial and central venous catheter will be inserted. All physiological parameters listed in will measured. Then his autologous blood of 8 ml/kg will be collected and simultaneously the HbV plus colloid solution, such as hydroxyethyl strach solution, or colloid solution alone, will be infused. Selection of the HbV or the colloid solution alone should have been done randomly before the anesthesia. Second physiological measurements as same as the first are performed immediately after the replacement. Second collection of autologous blood and infusion are carried out in the same manner. Again, third physiological measurements will be performed. Then surgery will be commenced and bleeding associated with surgery will be replaced with the autologous blood collected. If total blood loss will not exceed over 20 ml/kg, no additional blood transfusion or any other treatment may be required. Fourth and fifth measurements will be performed at two hours after the surgery and 24 hours after, respectively. However, if the bleeding will exceed over 20 ml/kg and hemodynamic condition will become unstable, necessitating some drugs or additional homologous blood transfusion, data obtained above will be excluded from the study.
Figure 1
Similary, as shown in , study of treatment of unexpected bleeding during surgery, and of transfusion for patients with uncommon blood types, is planned for clinial trial using HbV. First measurements before commencement of surgery will be performed and subsequent blood loss during the surgery will be treated with the HbV plus the colloid solution or the colloid solution alone. Second, third, and fourth measurements will be performed at the end of surgery, two hours and 24 hours after surgery, respectively. In those trials, it can be understood easily that the amount of the HbV used should be equal to that of blood lost. On the other hand, it is hard to decide which amount of colloid solution should be infused, namely to be equal or one–half of volume of blood lost. It will be a concern that excess volume loading will be induced immediately after the infusion when the equal amount will be chosen and that hypovolemia will be caused at 2–3 hours after the infusion, when one–half volume will be used. Nevertheless, Hiippala's report [Citation[9]] may lead us to use the one–half volume in the first clinical trial.
Figure 2
CONCLUSION
We have to achieve whole preclinical studies associated with mass production of the HbV. And we hope that the clinical trials mentioned in this paper will be carried out as soon as possible. Artificial blood, such as the HbV, is expected to be applied in many clinical practices, as shown in , in the future. We hope, at least in our country, that the HbV could supplement sufficiently the coming shortage in blood for transfusion.
Table 9 Social needs for artificial blood
REFERENCES
- Takaori, M., Fukui, A. (1996). Treatment of massive hemorrhage with liposome encapsulated human hemoglobin (NRC) and hydroxyethyl starch (HES) in beagles. Artif. Cell Blood Subst.Immobil. Biotech. 24 (6): 643–653.
- Tsutsui, Y., Kimura, T., Ishizuka, T., Oomori, S., Shizawa, T., Goto, H., Ogata, Y., Kaneda, S. (2002). Duration of efficacy of NRC (Neo Red Cell) in arat hemodilution model. Artificial Blood 10: 36–41.
- Kobayashi, K. (2003). Evaluation of artificial oxygen carrier in vivo—History and perspective of Keio-Waseda Joint Project of oxygen infusions. Artificial Blood 11: 37.
- Center for Biologic Evaluation and Research. (1994). Points to consider in the safety evaluation of hemoglobin based oxygen carriers. Transfusion 31: 369–371. [CROSSREF]
- Center for Bilogic Evaluation and Research. (1994). Points to consider in the safety evaluation of hemoglobin-based and perfluorocarbon-based oxygen carriers. Transfusion 34: 712–713. [CSA], [CROSSREF]
- Przybelski, R.L., Daily, E.K., Stern, K.N., MattiaGoldberg, C.A. (1997). A grade scale for assessment of safety of blood substitutes. Transfusion 37: 749–751. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Fratantoni, J.C. (1998). Red Cell Substitutes: Evolution of Approaches for Demonstrating Efficacy in Blood Substitutes - Present and Future Perspectives. (ed.) Tsuchida, E. Amsterdam, Elsevier Sci., pp. 33–39.
- Schou, H., de Sa, V.P., Larsson, A. (1998) Central and mixed venous blood oxygen correlate well during acute normovolemic hemodilution in anesthetized pigs. Acta Anaesthesiol. Scand. 42: 172–177. [PUBMED], [INFOTRIEVE]
- Hiippala, S., Linko, K., Myllylae, G., Lalla, M., Hekali, R., Maekelaeinen, A. (1995). Replacement of major surgical blood loss by hypo-oncotic or conventional plasma substitutes. Acta Anaesthesiol. Scand. 39: 228–235. [PUBMED], [INFOTRIEVE]