STORAGE STABILITY OF LARGE VOLUME PARENTERAL RINGER’S KETONE SOLUTIONS PREPARED BY FILTER STERILIZATION

1. STORAGE STABILITY OF LARGE VOLUME PARENTERAL RINGER’S KETONE SOLUTIONS PREPARED BY FILTER STERILIZATION RAMON BONEGIO, GINA RAGNO, RICHARD L. VEECH AND C. ROBERT VALERI NAVAL BLOOD RESEARCH LABORATORY, BOSTON UNIVERSITY SCHOOL OF MEDICINE, BOSTON, MA This work was supported by the U.S. Navy (Office of Naval Research Contract N00014-00-1-0352). The opinions or assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the Navy Department or Naval Service at large.

2. BACKGROUND: Lactated Ringer's solution produces detrimental effects because the D, L-lactate in the solution causes cardiac arrhythmias in experimental animals and human patients. In addition, the metabolism of D-lactate generates oxygen free radicals while the metabolism of L-lactate inhibits glycolysis. Ringer's ketone solution was designed in which the lactate in Ringer's lactate solution is replaced with 27 mM of D-betahydroxybutyrate. Ringer's ketone solution also contains a physiological concentration of glucose (5 mM).

3. STUDY DESIGN & METHODS A commercial company prepared one liter Ringer’s ketone solution in Ethox plastic bags under GMP conditions and this solution was evaluated for storage stability by measurement of chemical composition, physical stability, biological compatibility, sterility, and endotoxin content.

4. RESULTS The Ringer’s ketone solution which contains 27 mM of Na D-betahydroxybutyrate and 5 mM glucose cannot be sterilized either by conventional heat sterilization or by gamma irradiation due to the decomposition of both the betahydroxybutyrate and glucose (Table 1). Sterile and endotoxin-free Ringer’s ketone solution has been prepared using filter sterilization. Chemical composition and high pressure liquid chromatography testing demonstrated that this solution is stable for at least 1 year stored at 25C and 40C (Table II). This filter sterilized solution was tested in tissue cultures of mouse proximal tubular cells that are sensitive to endotoxin exposure. Filter sterilized Ringer’s ketone solution did not adversely affect the survival of these cells when cultured in either aerobic or anaerobic conditions(Figure 1A and 1B). This solution has been tested in vivo by Drs. Hasan Alam and Peter Rhee who administered Ringer’s ketone to rats subjected to hemorrhagic shock. They demonstrated that Ringer’s ketone caused significantly less apoptosis in lung than D,L lactate containing solutions (reference 1 and 2).

5. CONCLUSIONS Filter-sterilized, large volume parenteral Ringer’s ketone solutions containing betahydroxybutyrate and glucose were sterile and endotoxin-free and remained stable at room temperature for at least one year when tested for chemical composition, physical stability and biological compatibility. We now plan to test this resuscitative solution in larger animals and to this end, 10 liters of Ringer’s ketone has been provided to Drs. Hasan Alam and Peter Rhee for testing in pigs, Dr. Susan Stern for testing in pigs, and Dr. George Kramer for testing in sheep.

6. REFERENCES • Alam HB, Austin B, Koustova E, Rhee P. Resuscitation-induced pulmonary apoptosis and intracellular adhesion molecule-1 expression in rats are attenuated by the use of ketone ringer’s solution. J Am Coll Surg 2001:193;255-263. • Koustova E, Rhee P, Hancock T, Chen H, Inocencio R, Valeri CR, Alam HB. Ketone and pyruvate Ringer’s solutions decrease pulmonary apoptosis in a rat model of severe hemorrhagic shock and resuscitation. J Am Coll Surg (in press).

7. Table I: The stability of glucose and b-hydroxybutyrate in Ringer’s ketone solution after radiation sterilization.

8. TABLE II: Long-term stability of filter sterilized Ringer’s ketone solution at 25 C (room temperature) and 40 C

9. 1 Figure 1a: Survival of BUMPT cells exposed to filter sterilized Ringer's lactate or Ringer's Ketone solutions under aerobic conditions 180 160 140 120 Serum 100 Ringer's L-Lactate 80 Percent survival (% of baseline MTT) Ringer's ketone 60 DL lactate 40 DMEM 20 0 -20 -40 Days of Exposure Figure 1: Renal proximal tubular cells cultured under normoxic (1a) and hypoxic (1b) conditions survive well in filter sterilized Ringer’s Ketone solutions stored at room temperature for > 6 months MEAN + SEM N=3 0 1 2 3

10. Figure 1b: BUMPT survival after 48hrs of hypoxia 120 100 BUMPT survival (% baseline) 80 60 40 20 0 FBS10% DMEM L-Lactate DL lactate Ringer's ketone MEAN+SEM, n=3