1. MEMBRANE LIFE London May 2003

2. Objectives: Link the preservation of membrane integrity with maintaining patients on PD for longer periods of time Link the chemistry of solutions with the structural changes within the peritoneal membrane, and associated clinical changes

3. What determines the biocompatibility of PD Solutions Ph Buffer System Osmolality Concentration of Glucose Potential for formation of advanced glycation end products (AGE) Presence of glucose degradation products (GDP’s) The combination of these factors for a particular PD solution is said to define its ‘biocompatibility profile’

4. Advanced Solutions Portfolio

5. Extraneal is a peritoneal dialysis solution with Icodextrin instead of glucose as the primary osmotic agent - THE LONG DWELL SOLUTION -

6. Isosmolar (282 mOsm/kg) Reduced AGE Low GDP’s Reduced carbohydrate load Sustained UF Characteristics of Icodextrin: an alternative osmotic agent

7. Extraneal™ PD Solution Icodextrin (g/dl) Sodium (mEq/L) Chloride (mEq/L) Calcium (mEq/L) Magnesium (mEq/L) Lactate (mEq/L) Osmolality (mOsm/kg) pH 7.5 132 96 3.5 0.5 40 282 5.2-5.6 Composition   Isosmolar to serum (282 mOsm/kg)   UF equivalent to 4.25% dextrose   Sustained positive net UF for >14 hr   “Gentle” ultrafiltration   One bag per day (long dwell) Characteristics

8. Ultrafiltration Kinetics of Icodextrin Exchanges Ho-dac-Pannekeet et al, KI, 1996 Time (minutes) Net Ultrafiltration (ml) -800 -400 0 400 800 1200 1600 060120180240300360420480540 Dextrose 4.25% Dextrose 1.5% Icodextrin 7.5%

9. Reduced carbohydrate absorption 0 20 40 60 80 100 Carbohydrate Absorption (%) Glucose n=7 Icodextrin n=11 Glucose n=7 Icodextrin n=11 Carbohydrate Absorption (g) Glucose Concentration : 3.86% Mistry C D, et al., KI 1994; 46: 496-503 Reduced carbohydrate absorption with icodextrin at 8 hours 0 20 40 60 80 100

10. Ultrafiltration (ml) UF with Icodextrin and Glucose within and without peritonitis episodes * p<0.01 * Gokal et al, PDI 15: 226-230, 1995.

11. - -DIALYSIS AND NUTRITION IN ONE BAG -

12. What is Nutrineal™? Nutrineal™ is a peritoneal dialysis solution with amino acids instead of glucose which integrates dialysis and nutritional supplementation

13. Nutrineal™: Characteristics Amino acids as osmotic agent No glucose No change in dialysis procedures More physiological pH (6.7) Osmolality equivalent to 1.36% glucose Clearance equivalent to 1.36% glucose PD 4 formulation

14. Nutrineal™: Composition Essential Amino Acids Non-Essential Amino Acids Electrolytes Valine - 1.393 gArginine* - 1.071 g Na: 132 mmol/L Leucine - 1.020 gAlanine - 0.951 g Ca: 125 mmol/L Isoleucine - 0.850 gProline - 0.595 g Mg: 0.25 mmol/L Methionine - 0.850 gGlycine - 0.510 g Cl: 105 mmol/L Lysine - 0.955 gSerine* - 0.510 g Histidine** - 0.714 gTyrosine* - 0.300 g Threonine - 0.646 g Phenylalanine - 0.570 g Tryptophane - 0.270 g

15. Nutrineal ® is primarily a dialysis solution designed :   To act as an amino acid supplement for protein malnourished PD patients   To replenish normal peritoneal amino acid / protein losses which may prevent protein malnutrition in the longer term   As a non-glucose based dialysis solution which could be useful for diabetic and obese patients What is the Purpose of Using Nutrineal ® ?

16. In just one exchange, an amino acid-based PD solution can deliver 25% of the target Daily Protein Intake * 1 Jones MR, et al., PDI, 1998;18(2):210-216 With an absorption rate of 70-80% over 4-6 hours, one exchange of 2L Amino Acid solution provides approximately 18g of AAs to an average, stable, 60kg patient: that is 0.3 g/kg body weight/day, which represents 25% of the 1.2 g/kg body weight/day target intake 1 * Recommended dosage for adults: one 2L or 2.5L bag/day 25% Target DPI Delivering 25% of daily protein intake

17. The relationship between malnutrition and increased morbidity and mortality in dialysis is well known >73% 63-73% <63% 100 90 80 70 60 61218 24 0 % Surviving Months % Lean body mass (% of standard weight) and patient survival Adapted from McCusker FX, et al., Kidney Int Suppl, 1996;56:S56-61 Nutrineal: Recommended dosage for adults: one 2L or 2.5L bag/day Delivering 25% of daily protein intake

19. Physioneal  offers a:   physiological pH   physiological bicarbonate concentration   physiological pCO 2   reduced level of GDPs Coles GA., et al., NDT 1998; 13:3165-3171 Mactier RA., et al., KI 1998; 53:1061-1067 McKenzie R., et al., JASN 1998; 9:1499-1506 Topley N., et al., JASN 1999; 10, 230A (A1169) Bicarbonate/Lactate PD Solutions A Natural Solution for a Natural Membrane

20. - Product Description Dextrose CaCl 2 MgCl 2 NaHCO 3 Na Lactate NaCl   Ca and Mg separated from bicarbonate during sterilization to prevent precipitate formation   Dextrose separation during sterilization results in very low levels of GDPs   pH 7.4 Physioneal® Bicarbonate-buffered PD Solution

21. Physioneal & biocompatibility   Physioneal and peritoneal host defence Physioneal and membrane integrity Jones S., et al., Kidney Int 2001;59:1529-1538 N=106 Duration: 6 months HA: Hyaluronic Acid CA125: Cancer Antigen 125

22. McGill Pain Questionnaire: Total Weighted Pain Rating Index 0 2 4 6 8 10 12 Conventional lactate solution Experimental 38mM bicarbonate solution Physioneal solution PD solution tested Total weighted pain rating index Mactier RA., et al., KI 1998; 53:1061-1067 Physioneal for improved therapy comfort

23. Mechanisms for Bio-incompatibility 1. 1.The modulation of peritoneal cell function 2. 2.Cellular stress mechanisms Carbonyl stress – breakdown products of glucose react with proteins to form a series of biologically active compounds 3. Inhibition of peritoneal cell function and reduction in cell viability

24. PD Solution Biocompatibility Biocompatibility Factors pH and buffer system GDP cytotoxicity AGE Hyperosmolality

25. Peritonitis Bioincompatible PD solutions High glucose tissue injury? Increased glucose absorption Decreased Osmotic gradient Increased use of hypertonic solutions UF  Systemic inflammation High peritoneal transport rate Local peritoneal inflammation Chung, et al Perit Dial Int 2000;20(5):S57-S67 Glucose exposure and inflammation

26. How do GDP’s impact on peritoneal membrane function Glucose Glucose Degradation Products (GDP’s) Fibrosis Angiogenesis Membrane structural and functional alterations Heat Sterilisation Proteins AGE Formation Modulation of Cell function Inflammation FilterHeat

27. Extraneal™ and GDPs Ueda et al, 2000 Kidney Int 58: 2518-2524 1.36% GlucoseExtraneal 1.36% GlucoseExtraneal Glyoxal6.2 ±0.51.7 ±0.2 Glyoxal6.2 ±0.51.7 ±0.2 Methyl-glyoxal 7.8 ±0.61.8 ±0.2 Methyl-glyoxal 7.8 ±0.61.8 ±0.2 3-deoxyglucosone47.2 ±4.33.5 ±0.4 3-deoxyglucosone47.2 ±4.33.5 ±0.4 Total reactive Total reactive carbonyl compounds64.7 ±8.725.7 ±4.1 carbonyl compounds64.7 ±8.725.7 ±4.1 Extraneal™ contains significantly lower levels of carbonyl stress compounds even versus conventional 1.36% glucose solution

28. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Glucose 3.86% Glucose 2.27% Glucose 1.36% Physioneal 3.86% Physioneal 2.27% Physioneal 1.36% IcodextrinAmino Acids Methylglyoxal umol/L Schalkwijk et al, Perit Dial Int, 2000;20(6):796-798 In vitro GDP concentration «Methylglyoxal » In vitro GDP concentration «Methylglyoxal »

29. How does glucose impact on peritoneal membrane function GlucoseOsmolality Cellular activation Fibrosis Angiogenesis VEGF TGF-  1 Membrane structural and functional alterations 0 100 200 300 400 500 600 700 510204060100 Concentration (mM) Glucose Mannitol * * * * * TGF-  1 (pg/10 5 HPMC)

30. Glycation of Albumin with Extraneal™ 0 25 50 75 100 102040 1.36% glucose Extraneal™ Extraneal™ Increase in % Glycation Above Control Time (Days) In vitro study of glycation of albumin with Extraneal™ Millar et al., J Am Soc Nephrol, 1995

31. Osmolality of Standard vs. Advanced PD Solutions Mistry et al, 1994 Normal Plasma Osmolality: 282 - 295 mOsm/Kg Osmolality (mOsm/Kg) 382 485 282 344 0 100 200 300 400 500 600 Glucose 1.36% Glucose 2.27% Glucose 3.86% Extraneal

32. The transport of solutes and fluid High transporters: An increase in surface area/permeability leading to greater glucose absorption and a more rapid dissolution of the osmotic gradient 10  m Net result Normal peritoneal vessel 10  m Vascular alterations 02-25>25 Peritonealsclerosis Months CAPD number of vessels/field 80 70 60 50 40 30 20 10 0 Mateijsen et al. PDI, 19: 517-525, 1999

33. High transport & outcome High transporters: Efficient membranes for small solute clearance but may have difficulty with ultrafiltration, especially during the long dwell 60 70 80 90 100 06121824 % Surviving Time in Months High High Average Low Average Low Recent studies (Davis 1 and Churchill 2 ) have shown that high transporters had a worse prognosis probably due to more difficulty with fluid balance management Impact on outcomes in PD 1 Davis et al. KI 1999 Vol 54 p 2207 – 2217 2 Churchill et al JASN 1998 - Vol 9 1285 - 1292

34. Which of these factors is thought to influence/cause the peritoneal membrane to become more permeable ? 1 time on PD 2 glucose 3 GDPs 4 hyperosmolality 5other dialysate components

35. Morphological changes in the peritoneal membrane 100  m Normal parietal peritoneum Parietal peritoneum >5 years PD The Peritoneal Biopsy Registry ® 100  m

36. Median thickness of sub-mesothelial compact zone 2000 1000 Membrane Thickness (microns) 161511245437238 n = 97 + months 73-96 months 49-72 months 25-48 months 25-48 months 0-24 months HDUraemic Donor 0 Donor v All p=0.00001 (Kruskal-Wallis) HD v PD p=0.0049 (Kruskal-Wallis) PD p=0.0049 (Kruskal-Wallis) PD p=0.0049 (Kruskal-Wallis) 1500 500 The Peritoneal Biopsy Registry ®

37. Loss of Ultrafiltration Caused by the following changes in the Peritoneum’s structure: Degenerative changes in blood vessels and vascular walls (neo-angiogenesis & occlusion) Fibrosis of sub-mesothelial layer Loss of mesothelium

38. Glucose absorption Increased circulating insulin Hypertonic glucose solutions Continual absorption of glucose Atherogenesis Hyperlipidaemia PD patients are estimated to absorb 100-300 g of glucose/day 1 1 Martis et al, in Owen et al, Dialysis & Transplantation: A companion to Brenner & Rector’s The Kidney, 2000;179-198.

39.   Structure Vascular changes Membrane tissue Function High peritoneal transport rate Loss of UF capacity Nutritional status & appetite Fluid balance>BP>CVD Metabolic alterations Atherosclerosis Comfort (reducing POI) Improving membrane & patient life Local effects High glucose Low pH Lactate Hyperosmolarity Systemic effects Glucose absorption Fluid & solute removal

40. Modeled peritoneal glucose exposure 0 2 4 6 8 10 12 14 16 3 X 1.36% G3 X 1.36% P 2 X 1.36% P 1 X 3.86% G1 X 3.86% P1 X ICO1 X AA 1 X ICO Peritoneal glucose exposure (g.min/ml) Holmes et al. PDI 2000;20(2):S37-S41 CAPD Dwells Day time = 3 X 5 hours Overnight = 1 X 9 hours Additive effects that can be achieved by using a combination of Physioneal , Icodextrin & Amino Acid Solutions

41. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Glucose 3.86% Glucose 2.27% Glucose 1.36% Physioneal 3.86% Physioneal 2.27% Physioneal 1.36% IcodextrinAmino Acids Methylglyoxal umol/L Schalkwijk et al, Perit Dial Int, 2000;20(6):796-798 In vitro GDP concentration «Methylglyoxal » In vitro GDP concentration «Methylglyoxal »

42. Holmes et al PDI 2000;20(2):S37-S41 0 20 40 60 80 100 120 140 3 X 1.36% G3 X 1.36% P 2 X 1.36% P 1 X 3.86% G1 X 3.86% P1 X E1 X N 1 X E Total CHO absorption (g/24/h) Modeled total carbohydrate absorption CAPD Dwells Day time = 3 X 5 hours Overnight = 1 X 9 hours Additive effects that can be achieved by using a combination of Physioneal , Extraneal  & Nutrineal 

43. QUIZ TIME!!!

44. Improved PD solution biocompatibility Biocompatibility Factors pH and buffer system GDP cytotoxicity Glycation, Amadori and AGE Hyperosmolality