State of the Art and Challenges in Blood Substitutes Research: A Case Study on Perﬂuorocarbon-Based Oxygen Carriers

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State of the Art and Challenges in Blood Substitutes Research: A Case Study on Perﬂuorocarbon-Based Oxygen Carriers

Juan Carlos Briceño

Summary. In 1995 a research project on perfluorcarbon-based oxygen carriers (PFC-OCs) was started. Initial interest was prompted by reports of improved tissue oxygenation during cardiopulmonary bypass and by the advances of some formulations that were undergoing preclinical or clinical trials at that time. During Phase I of the project (1995–1999) knowledge of the manufacturing process of PFC-OCs was acquired and the PFC-OC Oxyfluor (HemaGen/PFC, St Louis, MO, USA) was tested in animal models of cardiopulmonary bypass and hemorrhagic shock. Phase II of the project (2000–2003) has led to the design and construction of a pilot plant for pro- duction of PFC–OCs, the procurement of the equipment and materials required for manufacturing sterile, injectable PFC emulsions, and the prepa- ration of the first PFC-OCs. The completion of Phase II of the project allows the research team to manufacture new PFC-OCs and to evaluate them in the experimental models of suitable clinical procedures. A better knowledge of the physiological effects of the infusion of PFC-OCs is expected.

Key words. Blood substitutes, Oxygen carriers, Perﬂuorocarbon microemulsions

Introduction

In 1995 investigators initiated research on perﬂuorocarbon-based oxygen car- riers (PFC-OCs) at the Fundación CardioInfantil (FCI) and the University of Los Andes (UA). Initial interest on the topic was prompted by the previous

229 Blood Substitutes Laboratory, Fundación CardioInfantil Calle 163A No. 28-60. PO Box 091479 and University of Los Andes, Bogotá, DC, Colombia

University of Los Andes, Carrera 1 No. 18A10, PO Box 4976, Bogotá, DC, Colombia

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participation of the author in the development and evaluation of a hyper- osmolar perfluorocarbon-based oxygen carrier (PFC-OC). This PFC-OC (perfluorodecalin, 20% w/v) was at that time tested for experimental car- diopulmonary bypass at the University of Texas [1]. These studies demon- strated that the PFC-OC increased cerebral blood flow, brain PO

2

, P

v

O

2

, and reduced brain tissue hypercapnia. However, the PFC-OC did not prevent brain tissue acidosis, metabolic acidosis or anasarca. During the Phase I of the FCI- UA Blood Substitutes Project, Oxyﬂuor (HemaGen/PFC, St Louis, MO, USA), a 40% v/v perﬂuorodichlorooctane emulsion was evaluated in animal models of cardiopulmonary bypass and hemorrhagic shock. The objective of this chapter is to report the advances of Phase II of the project, regarding both the production and the evaluation of PFC-OCs.

The FCI-UA Blood Substitutes Project Phase I: 1995–1999

Research on the potential use of perﬂuorocarbon-based oxygen carriers (PFC-OCs) started at the Fundación CardioInfantil and the University of Los Andes in 1995 [2]. The oxygen-carrying properties of PFCs and the intense activity of companies with products undergoing preclinical or clinical trials drew the attention of the clinicians in the research group. Increasing the knowledge of the physiological effects of the infusion of PFC-OCs was also a source of interest. Accordingly, the Phase I of the FCI-UA Blood Substitutes Project was started. It was divided into three subprojects: production of PFC- OCs, evaluation of efﬁcacy and evaluation of safety. A description of these subprojects is presented below.

Production of PFC-OCs

Objective

To study the feasibility of setting up the facilities for the experimental pro- duction of PFC-OCs.

Methodology

A feasibility study of local production of PFC-OCs was performed. This study included analysis of the methods of production of PFC-OCs, and the prelim- inary design of a laboratory facility for the production of PFC-OCs. In addi- tion, procedures were established for the selection and procurement of the PFCs, emulsiﬁers, and surfactants most appropriate for clinical applications.

Results

From this activity the group acquired signiﬁcant knowledge of the manufac-

turing process, materials, technology involved in producing PFC-OCs. It was

realized the high costs of the equipment and materials required, and the need

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for external ﬁnancial support for setting up the facilities for production of PFC-OCs.

Evaluation of the Efﬁcacy of PFC-OCs on an Animal Model of Cardiopulmonary Bypass

Given the impossibility of producing PFC-OCs at that time, several compa- nies with PFC-OCs products undergoing clinical and preclinical trials were contacted and asked for samples of PFC-OCs to be tested in experimental models of clinical procedures. One PFC-OC was thus obtained (Oxyﬂuor, 40%

v/v perﬂuorodichlorooctane, HemaGen/PFC, St Louis, MO, USA).

Objective

To evaluate efﬁcacy of use of PFC-OCs during experimental cardiopulmonary bypass.

Methodology

An animal model of cardiopulmonary bypass was implemented. In this model, 30-kg swine were subjected to 2 h of total normothermic nonpulsatile bypass from the right atrium to the ascending aorta. The extracorporeal circuit included a roller pump and a hollow ﬁber oxygenator. Mean arterial pressure was maintained at 50 mm Hg and ﬂow rate at 80 ml·min

^-1

·kg

^-1

. Brain tissue pH, PO

2

, and PCO

2

, were measured using ion specific electrodes. Mixed venous, jugular venous and arterial blood gases, fluid balance, hematological and hemodynamical variables were also monitored before, during, and 30 min post bypass, after which the animals were killed. Whole body oxygen con- sumption was calculated. Tissue samples from brain, lungs, kidney, liver, spleen and muscle were taken for histological analysis. In the control group (n = 8), Ringer’s lactate was used as priming solution and perfusate. In the study group Oxyfluor was used as priming solution.

Results

In these experiments Oxyﬂuor improved tissue oxygenation and total body

oxygen consumption. Oxyﬂuor also reduced FCC

RBC

, increasing oxygen trans-

port reserve of the red blood cell phase. However, tissue ﬂuid accumulation

was not alleviated, and blood and brain acidosis were signiﬁcantly aggravated

by the use of Oxyﬂuor. It was also observed that the fractional contribution

to oxygen consumption from the plasma phase (FCC

PL

) in the Ringer’s lactate

group was similar to the fractional contribution to oxygen consumption from

the PFC phase (FCC

PFC

) in the Oxyﬂuor group. An interesting ﬁnding was that

the secondary effects were not reduced by increasing tissue oxygenation and

it was concluded that further research should be conducted to optimize the

formulation of PFC-OCs for use in cardiopulmonary bypass and to reduce

secondary effects [3].

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Evaluation of the Safety of PFC-OCs on an Animal Model of Hemorrhagic Shock

Objective

To study hemodynamical, hematological and secondary effects of reinfusion with a PFC-OC in a model of hemorrhagic shock.

Methodology

1 .0 kg rabbits were bled 60% of volemia at 1 ml/min through the femoral artery, and immediately the same volume was reinfused at 3 ml/min with Ringer’s lactate (n = 11) or Oxyﬂuor (n = 11). Mean arterial pressure, ECG, and heart rate were monitored. Samples for blood gases, hematology and chemistry analysis were taken at the beginning of bleeding, halfway through bleeding, at end of bleeding, and at end of reinfusion. Animals were killed after one month, and tissue samples were taken for histological examination.

Results

During these experiments of hemorrhagic shock, PaO

2

increased and PvO

2

decreased, increasing oxygen extraction. Reinfusion with Oxyﬂuor did not cause negative changes in hemodynamics, hematology and blood gases of the animals. One-month survival rate was better in the Ringer’s lactate group (6/11), than in the Oxyﬂuor group (1/11), due to low PO

2

of inspired air during and post, and probably to species sensitivity to PFC-induced increased pulmonary residual volume.

Conclusions of Phase I

From this initial phase of the project that lasted four years, the main conclu- sions were: an increased knowledge was acquired of the effects of PFC-OCs during experimental procedures, including the fact that despite improving tissue oxygenation on cardiopulmonary bypass, secondary effects were not alleviated; the need of further research to understand the physiological changes induced by PFC-OCs; the consolidation of a multidisciplinary group of anesthesiologists, cardiovascular surgeons, perfusionists and biomedical engineers working towards the identification and modification of clinical pro- cedures on which PFC-OCs can be successfully used; and the need of exter- nal financial support for the construction of the laboratory for the production of PFC-OCs.

The FCI-UA Blood Substitutes Project Phase II:

2000–2003

A two-year grant from the National Science Foundation of Colombia (COL-

CIENCIAS) was awarded in April 2000.At that time, the objectives of the Phase

II of the project were: to set up lab facilities and equipment for production of

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PFC-OCs; to implement methodology for the formulation and manufactur- ing of PFC-OCs; to evaluate and optimize formulations of PFC-OCs in vitro and in animal models; to study the physiological changes induced by the infusion of PFC-OCs; to design the clinical trials for the PFC-OCs; and to transfer the technology of production and evaluation of PFC-OCs to the phar- maceutical industry. The project was divided into production and evaluation of PFC-OCs, which are presented next.

Production of PFC-OCs

The objective of this part was to manufacture PFC microemulsions that are stable, sterile, and able to transport signiﬁcant amounts of oxygen when used as injectable oxygen carriers (PFC-OCs).

Methodology

Facilities

A clean-air pilot plant for preparation, emulsiﬁcation and quality control of injectable PFC-OCs was designed and built.

Equipment and Materials

Equipment and materials for preparation, emulsiﬁcation and quality control of injectable PFC-OCs were speciﬁed and purchased.

Human Resources

Personnel to design and supervise plant construction, equipment and mate- rials purchasing, to operate the pilot plant and to manufacture PFC-OCs were recruited and trained.

Microemulsion Formulation and Preparation

PFC-OC microemulsions are being prepared with different combinations of PFCs and surfactants.

Results

Facilities

A 90-m

²

clean air pilot plant for manufacturing sterile, injectable solutions has been constructed at FCI. It started activities on December 2002.

Equipment and Materials

The PFC-OCs manufacturing equipment consists of a microﬂuidizer (model

M110-Y, Microﬂuidics, Newton, MA, USA), a microdiffusor and an autoclave

sterilizer. The PFC-OCs process control equipment includes a combined par-

ticle size and zeta potential analyzer (model 90Plus, Brookhaven Instruments,

Holtsville, NY, USA). All basic materials for PFC-OCs production including

the PFCs and surfactants have been purchased. The PFCs under evaluation

are perﬂuorodecalin and perﬂuorooctyl bromide (F2 Chemicals, Preston,

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UK), along with egg-yolk and soybean lecithin (Reference Epikuron 200, Ovothin 160 and Epikuron 170, Lucas Meyer, Hamburg, Germany).

Microemulsion Formulation and Preparation

The ﬁrst microemulsions have been prepared. Stability and particle size appear to be within speciﬁcations.

Evaluation of PFC-OCs

The objective of this part was to evaluate the safety and efﬁcacy of the PFC–OCs in vitro and in animal models of clinical procedures.

Methodology

In vitro evaluation consists of stability, biocompatibility and toxicity tests. In vivo studies include the evaluation of efﬁcacy, safety (toxicity), and effect on microcirculation on animal models. After this evaluation, optimization of the formulation of the PFC-OCs is planned.

Results

These experiments are expected to begin once the stability evaluation is successfully completed.

Challenges

During Phase II of the project several challenges were faced which are dis- cussed here. Regarding equipment and facilities the main difﬁculty was that the set up-costs of the pilot plant exceeded the provisions. The strict speciﬁ- cations of the plant resulted in increased costs of the facilities. Furthermore, as the production process was further detailed, additional equipment was required, even for small-scale output. These additional costs also resulted on a delay in the program of the project. Concerning the materials required for the project, the project budget was also strained by the high costs of the PFCs and the surfactants used in the formulations. Availability of PFCs is short, and data on their performance in biomedical use is scarce.

Challenges about human resources derived from the considerable effort that had to be dedicated to recruit the qualiﬁed personnel to design and supervise the construction of the pilot plant, to purchase the equipment and materials, to operate the pilot plant, and to manufacture the PFC–

OCs. Another obstacle for the success of the entire project is the long-

term sustainability of these essential personnel. In relation to microemulsion

formulation and preparation, the most important challenge of the project

at the present time is to consistently obtain microemulsions whose

stability complies with the requirements for in vivo studies and for long-term

storage.

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Conclusions

When this project started in 1995 there was significant research and com- mercial interest in PFC-OCs. Products like Oxyfluor (HemaGen/PFC, St. Louis, MO, USA) and Oxygent (Alliance Pharmaceutical, San Diego, CA, USA) were undergoing clinical trials. Eight years later and despite reports that PFC-OCs increase tissue oxygenation in animal models of hemorrhagic shock [5], and cardiopulmonary bypass [3,6], no PFC-OC has yet been approved for clinical use by the FDA. During Phase I of this project, Oxyfluor

^TM

(HemaGen/PFC) was tested in animal models of cardiopulmonary bypass and hemorrhagic shock. In Phase II of the project, a pilot plant for production of PFC–OCs has been designed and constructed. The equipment and materials required for manufacturing sterile, injectable PFC-OCs have been speciﬁed and pur- chased. The pilot plant started operations by the end of 2002. The ﬁrst microemulsions have been produced and are currently being tested in vitro and in vivo. The research team has now the ability to design and produce new PFC-OCs, and to evaluate these PFC-OCs in animal models of suitable clini- cal procedures. Important challenges have been faced during this phase of the project, regarding facilities and equipment, materials, personnel and in the formulation and preparation of the microemulsions. These ongoing studies will contribute to understand the physiological changes and the secondary effects induced by the infusion of PFC–OCs.

Acknowledgments. The author wishes to acknowledge the assistance of his colleagues and students at the Fundación CardioInfantil and the University of Los Andes, and the ﬁnancial support of these institutions and of COLCIENCIAS.

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2

, PCO

2

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