PART II: SCIENTIFIC INFORMATION

PHARMACEUTICAL INFORMATION

Drug Substance

Proper Name Chemical Name Molecular Formula Molecular Mass

L-Arginine 2-Amino-5-guanidinopentanoic acid

L-Histidine 2-Amino-3-(1H-imidazol-4-yl)propanoic acid

C

C

6

6

H

H

14

9

N

N

3

4

O

O

2

2

174.2

155.16 acid acid

C

6

H

13

NO

2

131.18

L-Lysine HCl 2,6-Diaminohexanoic acid

L-Methionine 2-Amino-4-(methylthio)butanoic acid

C

6

H

14

C

5

H

11

N

2

O

NO

2

2

182.65

S 149.21

L-Proline acid

C

9

H

11

NO

2

165.19

Pyrrolidine-2-carboxylic acid C

5

H

9

NO

2

115.13 acid acid

C

4

H

9

NO

3

119.12

C

11

H

12

N

2

O

2

204.23 L-Tryptophan 2-Amino-3-(lH-indol-3-yl)- propanoic acid

L-Tyrosine 2-Amino-3-(4-hydroxyphenyl)propanoic acid

C

9

H

11

NO

3

181.19

Calcium Chloride Dihydrate acid

Calcium Chloride Dihydrate

Magnesium Chloride Hexahydrate Magnesium chloride hexahydrate

Sodium Chloride

Sodium Lactate

Sodium chloride

Sodium 2-hydroxypropanoate

CaCl

2

MgCl

2

2H

2

6H

NaCl

2

O

0

147.01

203.30

58.44

C

3

H

5

NaO

3

112.07

DETAILED PHARMACOLOGY

Nutrineal™ PD4 (1.1% Amino Acid Peritoneal Dialysis Solution) contains a mixture of amino acids, lactate, and electrolytes (sodium, chloride, calcium, and magnesium), which are currently used in approved parenteral nutrition solutions and peritoneal dialysis solutions. The amino acids in Nutrineal™ PD4 are not new chemical entities, but are naturally occurring compounds whose distribution and metabolism are well known, and whose urinary excretion is negligible in patients with end stage renal disease (ESRD)

8

. Due to the volume of information concerning amino acid use in humans (orally and parenterally), and the long history of research on amino acids, the nonclinical pharmacology, pharmacokinetic, and toxicology studies traditionally done

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as part of new drug development have not been conducted as part of the development program for Nutrineal™ PD4. Several pharmacology and nonclinical toxicology studies have been conducted with Nutrineal™ PD2. In addition, several nonclinical studies have been conducted with other peritoneal dialysis solutions or intravenous solutions that are similar in composition to

Nutrineal™ PD4 and provide valuable supporting data on the safety of amino acid based peritoneal dialysis solutions. All solution formulations are presented in Table 1.

Table 1 - Quantitative Composition of Nutrineal™ PD4 Compared to Other Peritoneal Dialysis Solutions and

Intravenous Solutions (g/litre)

Total amino acids

3%EAA

1

(iv)

30

5%SAAS

2

(iv)

50

1%AA +

PD2

3

(ip)

10

2%AA +

PD2

3

(ip)

20

Nutrineal™

PD2 (ip)

11

Dianeal® PD2 w/dextrose

4

(ip)

none

0.35 none

L-glutamic acid

L-aspartic acid

Dibasic potassium phosphate

Calcium chloride dihydrate

Magnesium chloride hexahydrate

Sodium acetate

Sodium lactate

Sodium chloride none none none none none none none none none none

2.61 none

0.051

3.4 none

0.585 none none none

0.257 none none none

0.257 none none none none none 42.5 none none

0.257† 0.257

0.051 0.051 0.051 none none none

4.48

5.38

4.48

5.38

4.48

5.38

EAA = essential amino acids; SAAS = synthetic amino acid solution; AA = amino acids; iv = intravenous;ip=intraperitoneal

1

Solution used in Study No. R.D. 01-101, CC1101B

2

Solution used in Study No. R.D. 1-101A, CC1101A

3

Solution used in Study No. RO62830524

4

Solution used in Study No. RO62830524 contained 42.5 g/L dextrose

*Lysine added to the formulation as lysine HCl but analytically measured and reported as lysine

†Nutrineal PD4 (with 1.1% amino acids) has 0.183 g/L calcium chloride dihydrate

0.051 none

4.48

5.38

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The amino acids and electrolytes contained in Nutrineal™ PD4 are present naturally in the foods consumed by humans. These ingredients have been tested in numerous biological systems and been the focus of biological research for several centuries. In foods, amino acids are chemically bonded together to form proteins. After oral ingestion, the proteins are hydrolyzed to amino acids in the digestive tract and these are then absorbed into the portal blood stream. During intraperitoneal administration, the amino acids from Nutrineal™ PD4 are absorbed across the peritoneal membrane and are transported via the portal circulation first through the liver and then out into the peripheral tissues. Nutritional studies conducted during the period of approximately

1900-1960 defined the amino acid requirements for various species, the metabolic processes by which the body handles them, and their acceptable levels of intake. The composition of

Nutrineal™ PD4 is based on this well-established data.

In a controlled clinical study in an outpatient setting (Baxter Clinical Study No. RD-92-

CA-042), there was a statistically significant (p<0.05) overall improvement in nutritional status over a three month study period among the Nutrineal™ PD2 treated patients compared to the control patients, based on the proportion of patients showing improvement from baseline in two or more of the five efficacy parameters (e.g., albumin, prealbumin, total protein, transferrin, and mid-arm muscle circumference). The proportion of patients showing overall improvement was

70% for the Nutrineal™ PD2 group and 45% for the control group. The difference in nutritional status between the Nutrineal™ PD2 patients and the control patients was most pronounced at month 1; differences between the two groups were significant (p< 0.056 or well below) for all four parameters (albumin, prealbumin, total protein, and transferrin) measured.

Additionally, analysis of insulin-like growth factor-1 (IGF-1), another indicator of nutritional status, showed a statistically significant (p<0.003) difference between the Nutrineal™ PD2 and control patients at month 3.

The peritoneal equilibration test (PET), which measures the transport characteristics of the peritoneal membrane, showed no statistical or clinically significant differences between the

Nutrineal™ PD2 and control groups in ultrafiltration volume or peritoneal membrane mass transfer area coefficients for glucose, urea, creatinine, and total protein after three months, as well as at six months in eight patients who received Nutrineal™ PD2 for that long. Therefore, the use of Nutrineal™ PD2 for up to six months had no deleterious effects on the peritoneal membrane.

In an uncontrolled clinical study in an outpatient setting using Nutrineal™ PD4 with 2.5 mEq/L calcium electrolyte concentration (Baxter Clinical Study No. PRO-NIV-AO-048C), statistically significant increases in serum albumin (p<0.01) and transferrin (p<0.05) were observed at month

3 (end of treatment).

In a 35 day controlled metabolic balance study in patients with protein calorie malnutrition

(Baxter Clinical Study No. DT88002), a significant improvement in nitrogen balance occurred during the use of Nutrineal™ PD2. The significant improvement in nitrogen balance was accompanied by statistically significant increases in serum total protein and serum transferrin, increases in serum albumin and a trend toward normalization of plasma amino acids.

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Significant decreases from baseline were observed for both phosphorus (p=0.050 and

0.006, respectively) and potassium (p=0.045 and 0.031, respectively) in the 35 day metabolic balance study and in a controlled, three month outpatient study. Clinical research has shown that phosphorus and potassium balances usually change in parallel with nitrogen balance. In the absence of significant dietary changes, serum concentrations of potassium and phosphorus decrease when nitrogen balance increases.

To evaluate the amount of amino acids taken up from Nutrineal™ PD2, dialysate amino acid concentrations and dialysate effluent volumes were measured at the end of a 4-hour exchange with Nutrineal™ PD2 on the 1st, 9th and 19th days of treatment. Net absorption was obtained by subtracting the mass remaining at four hours (volume multiplied by concentration) from the mass infused (volume infused multiplied by concentration in Nutrineal™ PD2). Percent net absorption (percent uptake) was calculated as the mass absorbed divided by the mass infused multiplied by 100%.

The results are shown in Table 2. The mean percent net absorption (or percent uptake) at four hours ranged from 77% for lysine and histidine to 85% for methionine. This value for any amino acid was essentially the same on all three days in which it was assessed. The reproducibility of these numbers attests to the lack of any effect of the solution upon the transport characteristics of the peritoneal membrane.

The total grams of amino acids absorbed can be calculated from the data in Table 2 and the amino acid content of Nutrineal™ PD2, as shown in Table 3. The percent net absorption of amino acids over a four hour exchange was (17.53/22.00)x100% equals 79.7%, or approximately

80%.

Table 2 - Mean Percent Net Absorption of Amino Acids*

Amino Acid Day of Treatment with Nutrineal™ PD2 [Day of Study]

Histidine

1 [16] 9 [26] 19 [35]

77 76 77

Isoleucine 83 81 83

Leucine

Lysine

84 82 84

77 74 76

Methionine 85 84 85

Phenylalanine 79 77 78

Threonine 80 78 80

Tryptophan 80 78 79

Valine

Tyrosine

Alanine

Arginine

80 78 79

78 76 78

81 79 81

78 76 77

Glycine

Proline

81 79 81

79 76 79

Serine 82 80 82

*Results from Baxter Clinical Study No. DT88002

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Table 3 - Net Amino Acid Absorption during a 4-hr Exchange with Nutrineal™ PD2

Amino Acid g/2 L % Net Absorption* g Absorbed

Histidine 1.42 77 1.09

Isoleucine 1.70 82 1.40

Leucine 2.04 83 1.70

Lysine 1.52 76 1.15

Methionine 1.70 85 1.44

Phenylalanine 1.14 78 0.89

Threonine 1.30 79 1.03

Tryptophan 0.54 79 0.43

Valine 2.78 79 2.20

Tyrosine 0.60 77 0.46

Alanine 1.90 80 1.53

Arginine 2.14 77 1.65

Glycine 1.02 80 0.82

Proline 1.18 78 0.92

Serine 1.02 81 0.83

Total 22.00 17.53

*Mean of Days 16, 26, and 35

The percent absorption was directly correlated with the dialysate/plasma (D/P) creatinine ratio, a measure of membrane transport characteristics for small molecules measured by a specific protocol in a four-hour peritoneal solute equilibration test. The D/P ratio is a measure of the extent to which equilibration between blood and dialysate is achieved over four hours, and this quantity varies from patient to patient. The data indicate that, in general, absorption of amino acids from the peritoneal cavity in an individual patient was governed by the transport properties of that patient's peritoneal membrane. Generally, the higher the D/P ratio, the higher the percent amino acid uptake. A similar relationship exists between D/P creatinine and the dextrose absorption from the dextrose-based dialysis solutions.

The percent net absorption of amino acids from Nutrineal™ PD2 is essentially the same as that found in studies with other similar amino acid peritoneal dialysis solutions. The studies reported that amino acids are rapidly absorbed from the peritoneal cavity during dialysis with such a solution and that there is, on the average, net absorption of 70 - 90% of the amino acids infused intraperitoneally during a 4 - 6 hour dwell.

The acute effect of Nutrineal™ PD2 on plasma amino acid concentrations was assessed by sampling plasma before and at the end of a four-hour morning exchange with Nutrineal™ PD2.

Pre- and post-exchange plasma amino acid concentrations for Day 16, which was the first day in which patients received Nutrineal™ PD2, are shown in Table 4. Overall, the mean increase in amino acid concentration above pre-exchange for the amino acids included in the solution was about 63% at the end of the four-hour exchange. The increases ranged from 21% for lysine to

196% for methionine. The table also includes increases or decreases in concentrations of amino acids that were not present in Nutrineal™ PD2.

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Table 4 - Pre- and Post- Exchange Plasma Amino Acid Concentrations (Day 16) During Treatment with

Nutrineal™ PD2*

N=21 change from baseline)

N=21 p-value

Pre vs. Post

Essential Amino Acids

Histidine

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Threonine

Tryptophan

Valine

66 ± 12

59 ± 14

86 ± 20 a

160 ± 32

24 ± 10

56 ± 17

117 ± 34

20 ± 11

141 ± 30

91 ± 17 (38)

105 ± 28 (78)

126 ± 33 (45)

194 ± 31 (21)

71 ± 23 (196)

83 ± 19 (48)

166 ± 44 (42)

26 ± 13 (30)

341 ± 82 (142)

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

<0.05

<0.01

Semi-Essential Amino Acids

Cystine

Tyrosine

Nonessential Amino Acids

59 ± 24

34 ± 10

62 ± 26 (5)

43 ± 13 (26)

NS

<0.01

Alanine

Arginine

Asparagine

Aspartic acid

Citrulline

Glutamic acid

Glutamine

Glycine

Ornithine

Proline

394 ± 130

79 ± 20

45 ± 11

14 ± 6

88 ± 25

53 ± 22

674 ± 129

341 ± 135

53 ± 12

181 ± 45

472 ± 143(20)

118 ± 28(49)

43 ± 20 (-4)

14 ± 8 (0)

99 ± 26 (12)

51 ± 28 (-4)

677 ± 127 (1)

338 ± 131 (-3)

86 ± 19 (62)

279 ± 74 (54)

<0.01

<0.01

NS

NS

<0.01

NS

NS

NS

<0.01

<0.01

Serine

Taurine

Hydroxyproline

-Aminobutyrate

69 ± 18

69 ± 30

37 ± 12

7 ± 5 a

* Results from Baxter Clinical Study No. DT88002.

Mean standard deviation; units are

mol/L

86 ± 25 (25)

62 ± 26 (-10)

40 ± 13 (8)

11 ± 4 (57)

<0.01

<0.01

NS

<0.01

It is also known from previous studies in the literature that amino acids absorbed from the peritoneal cavity appear rapidly in blood, with a peak in concentration at one to two hours after instillation of the solution into the peritoneal cavity. It may be presumed that much of the absorption is through the visceral peritoneum via the mesenteric vessels to the liver, i.e., similar to the physiologic route of delivery of diet-derived amino acids through the gut and via the portal vein. After the maximum is reached the concentration declines rapidly, but remains above preexchange concentrations at four hours. In Study No. DT88002, which also evaluated the blood concentration of amino acids pre-exchange and 4 hours post-Nutrineal™ PD2 exchange, it was determined that all amino acids originally present in the solution, except glycine, were greater post-exchange when compared to pre-exchange blood concentrations (Table 4). Similarly, other amino acids/metabolites, such as cystine and ornithine, were also greater post-Nutrineal™ PD2 exchange, as compared to pre-exchange concentrations. These increases may also reflect the

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effect of amino acids absorbed from food proteins, as patients ate during the early portion of the exchange.

The effects of treatment with Nutrineal™ PD2 on the amino acid profile during the 20 days of treatment were also investigated. Plasma samples were obtained on Day 16 (prior to the first instillation of Nutrineal™ PD2 on the first day of treatment, Day 26 (after ten days of treatment), and Day 35 (on the 20th day of treatment). The results are shown in Table 5. Between Days 16 and 26, there were significant increases in histidine, lysine, threonine, valine, cystine, arginine, citrulline, ornithine, and proline and a decrease in taurine. Between Days 26 and 35, there was a further increase in cystine and a further decrease in taurine.

Table 5 - Pre-exchange Plasma Amino Acid Concentrations* during Treatment with Nutrineal™ PD2

Day 16 Day 26 Day 35 Normals**

N=21 N=19 N=18

Essential Amino Acids

Histidine 66 76 + 14 a

Isoleucine 59 60 + 10

Lysine 160 + 32

89 + 18

173 + 39

+ 22 + 4 a

Threonine

Valine

17 53 + 19

117 + 34 132 + 36 a

+ 21 + 11

141 + 30 187 + 30 a

Semi-Essential Amino Acids

63 + 20 a

75 + 13 a

56 + 11

89 + 21

174 + 31 b

23 + 4

56 + 16

136 + 40 b

21 + 12

186 + 42 a

68 + 23 a,c

34 + 8

88 + 10

64 + 16

127 + 27

197 + 38

28 + 5

56 + 9

155 + 41

NA

232 + 51

61 + 10

62 + 13

Nonessential Amino Acids

Alanine 394 + 130 402 + 128

95 + 23 a

Aspartic Acid

Glutamic Acid

Glutamine

Glycine

Proline

-Aminobutyrate

14 + 6

53 + 22

16 + 7

98 + 26 a

40 + 19

674 + 129

341 + 135

181 + 45

689 + 123

288 + 102

62 + 18

203 + 47 a b

71 + 14 b

12 33 + 13

7 + 5 11 + 4 a

414 + 155

93 + 19 a

45 + 10

14 + 7

98 + 29 a

46 + 26

660 + 92

318 + 119

59 + 15

205 + 55 b b

71 + 12

45 + 20 a,c

35 + 13

10 + 6

433 + 166

99 + 22

48 + 13

6 + 3

46 + 22

39 + 2

480 + 133

265 + 118

66 + 28

210 + 65

108 + 24

48 + 18

16 + 13

NA

Laboratory, University of Iowa

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In Study No. RD-92-CA-042, the chronic effects of Nutrineal™ PD2 on the plasma amino acid profile were compared to those with dextrose Dianeal®. Over a three month treatment period, patients in the Nutrineal™ PD2-treated group showed significant increases in pre-exchange plasma concentrations of histidine, threonine, valine, ornithine, and serine compared to patients in the Control Group who received only dextrose dialysis solution (Table 6). There was also a marginal increase in lysine concentration (p=0.069). As in the previous study (Study No.

DT88002) all but ornithine had been abnormally low before treatment, and these increases brought them closer to the normal range. Ornithine increased because it is a normal metabolite of arginine, which was present in Nutrineal™ PD2. Also, as was observed in the nitrogen balance study (Study No. DT88002), plasma taurine decreased during treatment with Nutrineal™

PD2.

Table 6 - Changes in Pre-exchange Plasma Amino Acid Concentrations in Controls and in Patients Treated with Nutrineal™ PD2 (Study No. RD-92-CA-042) (% Change at Three Months from Baseline)

Amino Acid

Histidine

Threonine

Controls (N=55)

+ 3.7

+ 1.2

Nutrineal™ PD2 (N=57)

+ 13.1

+ 22.4

p-value

0.006

0.004

Serine + 3.1 + 16.2 0.003

Taurine - 2.5 - 23.0 0.018

Since the peritoneal membrane is more permeable to large molecules than the synthetic or semisynthetic membranes used in hemodialysis, losses of proteins and amino acids into peritoneal dialysate are significant. Various studies with amino acid dialysis solutions have shown conclusively that amino acids are efficiently absorbed from the peritoneal cavity. Since the fractional absorption is approximately 80% over 4 - 5 hours, one exchange with 2 L of a solution

1.0% or greater in amino acid content can easily replace the 3 - 4 g of amino acids and 8 - 10 g of proteins normally lost during peritoneal dialysis. Due to the loss of proteins during peritoneal dialysis, the protein requirement of an average sized Continuous Ambulatory Peritoneal Dialysis

(CAPD) patient is increased by about 0.2 g protein per kg body weight per day. Peritonitis can transiently increase these peritoneal protein losses by 2-fold or more.

TOXICOLOGY

Human Studies

High-dose intravenous amino acid infusions were studied in humans and found to have no adverse reactions or gross abnormalities with respect to amino acid metabolism when single essential amino acids (tryptophan, leucine and methionine) were administered at up to 2.5 g/kg body weight in a 6 - 10 hour period

9

.

Animal Studies

Acute Toxicity

Acute toxicity studies of highly purified amino acids were conducted as part of a 5-day subacute toxicity study in Sprague-Dawley rats. The data are summarized in Table 7 (see below). The

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results showed that four of ten male rats injected intravenously at a dose of 120 mL/kg body weight and a rate of 4 mL/min of a 3% amino acid solution died immediately following the injection. There were no deaths in the female rats. Deaths were attributed to pulmonary hemorrhage resulting from the large injection volume. There was no local damage at the site of injection. The mechanism of toxicity was judged to be attributable to volume overload of the vascular system. Since the total blood volume of a rat is approximately 7% of body weight, infusion of 120 mL/kg at a rapid rate is equivalent to increasing the blood volume to 190 mL/kg from 70 mL/kg or 2.7-fold. The outcome of pulmonary hemorrhage and death is expected because of the limited capacity of the vascular system and rupture at the weakest vascular walls, the capillaries of the lung. This study shows the maximum non-lethal dose is greater than 80 mL/kg and the minimum lethal dose is less than 120 mL/kg.

The margin of safety is determined by comparing the no observed effect dose in the animal toxicity study with the clinical dose. The usual clinical dose of 1.1% amino acids is 5.24 mg/kg/min assuming 2 L of 11 g of amino acids/L given intraperitoneally to a 70 kg patient are absorbed in 60 min ([(11 g/L x 2 L) ÷ 70 kg] ÷ 60 min = 5.24 mg/kg/min). The maximum nonlethal dose in rats of 3% amino acids given a dose of 80 mL/kg at a rate of 4 mL/min and average body weight of 200 g is equivalent to a dose of 600 mg/kg/min. Thus the margin of safety is 600 mg/kg/min ÷ 5.24 mg/kg/min = 114. This margin of safety is an underestimate because the acute mechanism of toxicity, vascular hypervolemia, is not achievable by the intraperitoneal route of administration.

Table 7 - Intravenous Acute Toxicity of 3% Essential Amino Acids Solution

Species and Strain Number of Animals & Dosage* (mL/kg) Number of Deaths

Rat, Sprague-Dawley

Rat, Sprague-Dawley

Rat, Sprague-Dawley

Sex/Group

10 M + 10 F

6 M + 10 F

10 M + 10 F

40

80

120

Reference: Final Report, R.D. 01 - 101, CC1101B, Oct. 31, 1973

*3% essential amino acid solution was injected intravenously at a rate of 4 ml/min. none none

4 M

The acute toxicity of amino acids administered individually or in mixtures was studied in order to develop an amino acid solution suitable for parenteral injection in humans

10

. Male rats were given intraperitoneal injections of aqueous solutions or suspensions of crystalline amino acids.

The dosages required for 50% lethality in rats range from 3 - 7g per kg body weight except for

L-tryptophan, which has a LD

50

of 1.6 g per kg body weight (see Table 8). Large dose increments which often approximated 50% of the LD

50

were required for the LD

99

. Symptoms of toxicity appeared from 10 minutes to 2 hours after the intraperitoneal injection; all amino acids caused dyspnea, hypothermia and extreme prostration. Body temperature was often reduced to

35°C before death. The prostration was usually associated with uncoordinated movements.

Pathologic changes were most obvious in the kidney and liver. The glomeruli were congested and there were degenerative changes and vacuolization in renal tubular and hepatic parenchymal cells. The lesions were completely reversible if the animal survived.

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Acute toxicity studies of mixtures of amino acids were also studied extensively

17 of mixtures of amino acids is not strictly additive and the LD

50

. The toxicity

of such mixtures is always less than would be calculated from the toxicities of each constituent single amino acid.

Table 8 - Intraperitoneal LD

50

Doses of L-Amino Acids in Rats

Compound LD

50

mmoles/kg body weight* LD

50

mg/kg body/weight†,§

L-Alanine 37† 5078

L-Arginine HCl 18 ± 3 3793

L-Glycine 47† 3528

L-Histidine HCl

L-Isoleucine

L-Leucine

L-Lysine HCl

23 ± 3

52 ± 6

41 ± 9

22 ± 4

3569

6821

5378

4019

L-Methionine

L-Phenylalanine

L-Proline

L--Threonine

L-Tryptophan

L-Tyrosine

L-Valine

*Gullino, et al; 1956

(10)

† Milne; 1968

(11)

§ Lewis RJ, Tatken RL; 1982

(12)

29 ± 9

32 ± 3 no data

26 ± 2

8 ± 1

No data

46 ± 7

4328

5286 no data

3097

1634 no data

5389

Repeat Dose Toxicity

The repeat-dose toxicity of Nutrineal™ PD2 and similar peritoneal dialysis and intravenous solutions was assessed in a series of studies including intraperitoneal administration and intravenous administration.

In a 28-day intraperitoneal study in rats (Baxter Study No. R062830524), groups of 10 animals received doses of a 1% or 2% purified amino acid solution (nearly twice that in the proposed drug product) with electrolytes (in a Dianeal® PD-2 formulation) at a dose of 30 mL/kg. The solution was administered intraperitoneally four times per day for a total daily dose of up to 120 mL/kg. A control group received a solution without amino acids or dextrose (e.g. sterile water plus electrolytes in a Dianeal® PD-2 formulation), and a reference group included a solution consisting of Dianeal® PD-2 with 4.25% dextrose. A fifth group received the 2% purified amino acid solution with electrolytes (in a Dianeal® PD-2 formulation) twice a day. The infused solutions were not drained as is the procedure in the clinical setting. No adverse toxicological findings were observed. A slightly higher spleen weight was seen in the animals treated with the solution with dextrose (reference group). Abdominal adhesions noted to some extent in all groups were attributed to the use of indwelling peritoneal catheters used to deliver the solutions.

The dose of 120 mL/kg/day of 2% amino acid solution with electrolytes can be considered a no effect level. Noting that, in most cases, human exposure is one to two exchanges per day and that clinical usage involves removal of the solution after a prescribed dwell time, provides enhanced margins of safety.

In separate additional studies, amino acid solutions were also administered intravenously to rats for five or 30 days (Baxter Report Nos. R.D. 01-101,CC1101B, R.D. 1-101A,CC1101A). In the

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five day study, doses of 40 mL/kg of a 3% essential amino acid solution resulted in no significant signs of toxicity or histopathological effects, and, despite a mild transient hypokinesia, this dosage was considered a no effect level. The highest dose in this study was initially 120 mL/kg, which resulted in mortality on the first day. A reduction in this dose to 80 mL/kg produced signs that were limited to hypokinesia that was more pronounced than that seen at the lower dose, but was nevertheless tolerated for the remaining four days. The dose of 80 mL/kg represents the average blood volume per kg of body weight in mammalian species, i.e. this dosage volume results in doubling the total vascular volume by the end of the infusion period.

In the 30-day intravenous study, rats received daily doses of 40 and 80 mL/kg of a 5% synthetic amino acid solution (SAAS). At 40 mL/kg, signs were limited to transient hyperkinesia and hyperpnea, with no significant signs of toxicity. This dose was considered a no effect level, since the observations were considered related to the volume of solution administered. At 80 mL/kg, effects included hyperemia, decrease in hemoglobin and hematocrit values, decreased serum calcium and uric acid values and increased serum sodium and potassium. One animal died following infusion. However, there were no histopathological changes noted in tissue evaluation.

Studies in dogs were carried out by the intravenous route of administration for five and 30 days

(Baxter Report Nos. R.D. 1-101A, CC1101A, R.D. 1-101-3 (N255)). The five-day study involved the infusion of a solution of 5% synthetic amino acids (SAAS) plus 25% dextrose at

100 mL/kg/day (two different groups received 100 mL/kg/day at 7.5 and 3 mL/min) and 40 mL/kg of the same 5% synthetic amino acid solution plus 5% dextrose. At 40 mL/kg, there was no mortality. Clinical observations and increases in serum glucose, lactic acid and urea nitrogen and increases in urinary excretion were largely attributed to the volume and quantity of material administered. There were no histopathological changes in any tissue. However, the administration of 100 mL/kg resulted in mortality, and involved all dogs that received the solution at a rate of 7.5 mL/min and some dogs that received 3 mL/min. The cause of death was attributed to the rapid increase in blood osmolality consistent with an increase in plasma glucose.

Lactic acid and urine nitrogen were also increased, as was urinary glucose. There were no effects discernible that would have been associated with the amino acids, although they could have contributed to the increased urea nitrogen levels due to the nitrogen content of amino acids.

In the 30-day study with dogs, a 5% synthetic amino acid solution (SAAS) was administered intravenously at 40 and 100 mL/kg five days per week. In this study, both of these doses were well tolerated; the only indication of a treatment related effect was an increase in serum urea nitrogen that was likely related to the administered nitrogen content associated with amino acid administration. There were no clinical, hematological, organ weight, body weight, or histopathological changes attributable to the administered amino acid solution.

Reproductive Toxicology

Reproduction and teratology studies have not been conducted with Nutrineal™ PD4. This is because the stress associated with an intraperitoneal route of administration would obscure appropriate scientific evaluation of these studies.

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The effect of an oral diet of highly purified amino acids on growth, reproduction, embryotoxicity and lactation was studied in rats

13

. The purified amino acids were mixed with salts (minerals and electrolytes), vitamins (B and C), and glucose prepared in 50% aqueous solution and offered together with a separate supplement of fat-soluble vitamins in corn oil to weanling rats. At maturity, the animals were mated and produced normal litters with no evidence of reduced fertility or embryotoxicity. The F1 litters were maintained on the purified amino acid diets and, at maturity, yielded satisfactory litters (F2 generation) that were nursed to weaning. These studies indicate a purified amino acid diet, when supplemented with appropriate vitamins, salts, fat, and carbohydrate, does not have adverse effects on reproduction and fetal survival.

Genotoxicity Studies

The components in Nutrineal™ PD4 have been shown to be nonmutagenic considering that these components are food sources for all living organisms. All of the in vitro models for determining mutagenic potential utilize culture media composed of the same purified amino acids contained in this product. The amino acids contained in Nutrineal™ PD4 are highly purified, and meet the

U.S. Pharmacopeial standards and are Generally Recognized as Safe (GRAS), as cited in US 21

CFR Parts 172 and 582. Hence, it is unlikely that the product is mutagenic.

Carcinogenicity Studies

Nutrineal™ PD4 has not been tested for carcinogenic potential in animal species because the route of administration is not practical for long-term application in laboratory animals. Oral feeding studies of purified diets with other nutrients have not shown evidence of carcinogenicity attributable to the amino acid content

13,14,15

.

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REFERENCES

1. Slatopolsky E, Weerts C, Norwood K, Giles K, Fryer P, Finch J, Windus D, Delmez J. Longterm effects of calcium carbonate and 2.5 mEq/liter calcium dialysate on mineral metabolism. Kidney Int. 1989 Nov;36(5):897-903.

2. Shaw SN, Elwyn DH, Askanazi J, Iles M, Schwarz Y, Kinney JM. Effects of increasing nitrogen intake on nitrogen balance and energy expediture in nutritionally depleted adult patients receiving parenteral nutrition. Am J Clin Nutr. 1983 Jun;37(6):930-40.

3. Oreopoulos DG, Marliss E, Anderson GH, Oren A, Dombros N, Williams P, Khanna R,

Rodella H, Brandes L. Nutritional aspects of CAPD and the potential use of amino acid containing dialysis solutions. Perit Dial Bull. 1983;3:S10-2.

4. Twardowski Z, Khanna R, Nolph K. Osmotic agents and ultrafiltration in peritoneal dialysis.

Nephron. 1986;42(2):93-101.

5. Goodship TJ, Lloyd S, McKenzie PW, Earnshaw M, Smeaton I, Bartlett K, Ward MK,

Wilkinson R. Short-term studies on the use of amino acids as an osmotic agent in continuous ambulatory peritoneal dialysis. Clin Sci. 1987 Nov;73(5):471-8.

6. Anderson GH, Patel DG, Jeejeebhoy KN. Design and evaluation by nitrogen balance and blood aminograms of an amino acid mixture for total parenteral nutrition of adults with gastrointestinal disease. J Clin Invest. 1974 Mar;53(3):904-12.

7. Fenton SS, Johnston N, Delmore T, Detsky AS, Whitewell J, O’Sullivan R, Cattran DC,

Richardson RM, Jeejeebhoy KN. Nutritional assessment of continuous ambulatory peritoneal dialysis patients. ASAIO Trans. 1987 Jul-Sep;33(3):650-3.

8. Kamin H, Handler P. The metabolism of parenterally administered amino acids. II. Urea synthesis. J Biol Chem. 1951 Jan;188(1):193-205.

9. Quadbeck R, Förster H. Effects of parenteral administration of high dosed single amino acids in human volunteers and in the experimental animal. J Parenter Enteral Nutr. 1982

July;6(4):318.

10. Gullino P, Winitz M, Birnbaum SM, et al. Studies on the metabolism of Amino Acids and related compounds in vivo. I. Toxicity of essential amino acids, individually and in mixtures, and the protective effect of L-arginine. Arch Biochem Biophys. 1956 Oct;64(2):319-32.

11. Milne MD. Pharmacology of amino acids. Clin Pharmacol Ther. 1968 Jul-Aug;9(4):484-

516.

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12. Lewis RJ Sr, Tatken RL, editors. Registry of Toxic Effects of Chemical Substances. In:

U.S. Department of Health and Human Services, DHHS (NIOSH) Publication No. 81-116.

Washington, D.C.: U.S. Government Printing Office; 1982.

13. Greenstein JP, Birnbaum SM, Winitz M, Otey MC. Quantitative Nutritional Studies with

Water-Soluble, Chemically Defined Diets. I. Growth, Reproduction and Lactation in Rats.

Arch Biochem Biophys. 1957 Dec;72(7):396-416.

14. Rose WC. The nutritive significance of the Amino Acids. Physiol Rev. 1938;18:109-36.

15. Conner MW, Newberne PM. Drug-Nutrient Interactions and their Implications for Safety

Evaluation. Fundam Appl Toxicol. 1984 Jun;4(3 Pt 2):S341-56.

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