American Society of Nephrology Kidney Week 2017 October 31 – November 5 • New Orleans, LA, USA

References: 1) Mandel NJ. J Urology (2004); 2) Kaplon DM. J Endourol (2010); 3) Holmes R. Urolithiasis (2016); 4) Hoppe B. Ped Nephrology (2016); 5) Maalouf N. Nephrol Dial Transplant (2011)

Acknowledgements: Rita Pease (Allena Pharmaceuticals) kindly provided assistance with abstract and poster preparation. We thank Nasal Lama (New York University) for PCR analysis of pig samples.

ALLN-177, a Novel Oral Enzyme Therapy, Reduces Urinary Oxalate Excretion and Plasma Oxalate

in a Porcine Dietary Model of Severe Hyperoxaluria

Danica Grujic1, Paulina Szczurek2, Nadia Mosiichuk2, Liuda Lozinska2, Stefan Pierzynowski2

1 Allena, Newton, MA, United States 2 Lund University, Lund, Sweden

Poster TH-PO1071 • Presented at the American Society of Nephrology Kidney Week 2017 November 2, 2017 • New Orleans, LA, USA

Danica Grujic1, Paulina Szczurek2, Nadia Mosiichuk2, Liuda Lozinska2, Stefan Pierzynowski2

1 Allena, Newton, MA, United States; 2 Lund University, Lund, Sweden

ALLN-177, a Novel Oral Enzyme Therapy, Reduces Urinary Oxalate Excretion and Plasma Oxalate in a Porcine Dietary Model of Severe Hyperoxaluria

Hyperoxaluria

Porcine Model of Hyperoxaluria

ALLN-177: Oxalate Decarboxylase

Assessments of ALLN-177 Mechanism of Action

Study Design

• Oxalate is an end-product of carbohydrate and amino acid metabolism, and it is also absorbed from the diet. There is obligatory renal excretion of the metabolic and dietary oxalate load

• Hyperoxaluria (HOx) is a major risk factor for kidney stones, nephrocalcinosis, and oxalate nephropathy, which may lead to chronic kidney disease and end-stage renal disease

• Secondary HOx is caused by excess oxalate absorption from diet and can be:

– Enteric: Primarily associated with bariatric surgery, resection of the small intestine, diseases of the small intestine, or pancreatic insufficiency

– Idiopathic: Unknown etiology • There are no pharmacological agents approved to treat any type

of HOx

• Crystalline, recombinant oxalate-degrading enzyme

• Orally-administered, not absorbed systemically

• Degrades oxalate present in the gastrointestinal (GI) tract and reduces the amount of oxalate available for absorption, thereby decreasing urinary oxalate (UOx) excretion

• Pigs are more suitable than rodents for modeling HOx based on greater similarity to the human GI and genitourinary tract.1,2

• Pigs willingly eat a human-like high-fat diet with rhubarb as a source of oxalate

Key Similarities• Multi-pyramidal kidneys

• Similar glomerular filtration rate

• Similar renal blood flow characteristics and creatinine clearance

Conclusions

ALLN-177 Therapy Normalizes UOx Excretion and Plasma Oxalate (POx)

References: 1) Mandel NJ. J Urology (2004); 2) Kaplon DM. J Endourol (2010); 3) Holmes R. Urolithiasis (2016); 4) Hoppe B. Ped Nephrology (2016); 5) Maalouf N. Nephrol Dial Transplant (2011)

Acknowledgements: Rita Pease (Allena Pharmaceuticals) kindly provided assistance with abstract and poster preparation. We thank Lama Nasal (New York University) for PCR analysis of pig samples.

Baseline Characteristics:• Twelve juvenile pigs, 8 male and 4 female; 8-10 weeks of age

(Polish Landrace x Yorkshire x Hampshire x Petreum)

• Mean ± SD body weight at randomization: 21.8 ± 6.2 kg

• Mean ± SD food intake during study: 1.04 ± 0.3 kg/day

• Mean ± SD UOx before exposure to high-fat diet + rhubarb: 61.3 ± 14.5 mg/gCr/day

Pre-treatment: • Severe HOx induced and maintained with a high-fat diet mixed

with frozen rhubarb (2.5:1 [w/w])

• Feed (approximated): fat (20%), protein (15%), carbohydrate (45%), oxalate (2.8 g/kg feed), Ca (2 g/kg feed)

ALLN-177 Degrades Oxalate Primarily in Stomach Absence of Oxalabacter formigenes Colonization in Study Pigs

Urine Oxalate (mg/gCR/d)

Plasma Oxalate (µmol/L)

Before HOD, normal levels 61.34 ± 14.51 8.1 ± 3.8

Pre-treatment, HOD 100.1 ± 20.1 13.9 ± 0.9

Treatment HOD + ALLN-177 61.5 ± 14.3 9.9 ± 2.3

Reduction from pre-treatment (%) 39%; p < 0.001 29%; p < 0.001

* HOD = High-fat diet + rhubarb

Poster TH-PO1071 • Presented at the American Society of Nephrology Kidney Week 2017 • November 2, 2017 • New Orleans, LA, USA

Total chyme samples were collected 3h after feeding from 3 segments of the GI tract. Shown is a mean (SD) of total oxalate (mg)/total amount of chyme (g) per specific GI segment. The percent difference between the control group fed a high oxalate diet and the ALLN-177 group fed the same diet +22,500 u of ALLN-177 is depicted. The colon samples are not presented since they contain chyme content from several days. Analysis was performed using an IC HPLC method with a modified protocol.3

Shown is PCR analysis from stool samples collected on the last day of the treatment period from representative pigs (actual pig numbers at the top of gel picture). Lane M, 100 bp DNA molecular weight ladder; (-) negative control; (+) positive control. The primers used are designed to detect multiple species of Oxalobacter formigenes known to colonize humans and pigs. ALLN-177 was given to all pigs for 7 days except pig 10, which was provided with food only. In this study, none of the pigs were colonized with Oxalobacter formigenes, and 7 days of treatment with ALLN-177 did not affect colonization.

Shown in the table above (and graphically represented in the figure) are mean ± SD UOx and POx at baseline, pre-treatment on high-oxalate diet, and on high-oxalate diet + ALLN-177 treatment, as well as mean and percent change from pre-treatment; calculated from a single collection at the end of each period. Analysis was performed using IC HPLC for both UOx and POx as described previously.4,5

Treatment: • 22,500 u ALLN-177 with feed, (3x 7,500 u/meal; same dose used

in the clinical trials, poster #TH-PO1073)

Primary endpoint: • Mean within-pig difference from pre-treatment to on-treatment

in 24h UOx expressed as mg/gCr/day calculated from a single collection at the end of pre-treatment and treatment periods

Secondary endpoints:• Change in plasma oxalate (POx)

• Oxalobacter formigenes colonization assessed by PCR

Adaptation

2 weeks

High-Fat Diet

(N = 12)

Pre-treatment

High-Fat Diet + Rhubarb

(N = 12)

1 week

Treatment

High-Fat Diet + Rhubarb+ ALLN-177

(N = 12)

1 week

Urin

e O

xala

te (

mg

/gC

r/24

h)

Pla

sma

Oxa

late

(µm

ol/

L)

140

120

100

80

60

40

20

0 0

2

4

6

8

10

12

14

16

High-Oxalate Diet

Normal UOx

NormalPOx

High-Oxalate Diet+ ALLN-177

Stomach

39%

Duo/jejunum

166%

Ileum

20%

Control

ALLN-177

Oxa

late

(m

g)

/ To

tal c

hym

e (

g) 1200

1000

800

600

400

200

0

• A high-fat diet enriched with rhubarb induced hyperoxaluria and hyperoxalemia in the pigs, with a > 50% increase in both urine and plasma oxalate levels

• ALLN-177 therapy for 7 days normalized urinary oxalate excretion and reduced plasma oxalate to the normal range

• Changes in oxalate levels occurred in the absence of Oxalobacter formigenes colonization

• Oral ALLN-177 was well-tolerated, with no observable effects on growth, food or water intake, or macroscopic changes in the GI tract or kidneys

• The reductions demonstrated in both plasma and urine oxalate with ALLN-177 treatment in a large animal model of severe hyperoxaluria provides proof of concept for a potential new therapy for severe oxalate-related disease

M – + 18 5 27 23 11 10 12 19 4 15 31 21 24