Date: 24 –April-2007 Trial ID: GHTUR/E/2 Version: 2 Integrated Clinical Trial Report Status: Final ICTR Synopsis

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Synopsis

TITLE OF TRIAL INDUCTION OF PUBERTY WITH 17 Β-ESTRADIOL IN GIRLS WITH TURNER SYNDROME. AN OPEN RANDOMISED TRIAL TRIAL CODE: GHTUR/E/2 INVESTIGATORS IN TOTAL, 35 PRINCIPAL INVESTIGATORS IN SPAIN WERE INCLUDED INITIALLY IN THE TRIAL. ONLY 19 RECRUITED PATIENTS. NO SIGNATORY INVESTIGATOR HAS BEEN APPOINTED YET. TRIAL SITES THE TRIAL WAS CONDUCTED IN SPAIN INITIALLY AT 35 CENTRES; ONLY 19 CENTRES INCLUDED PATIENTS IN THE TRIAL. PUBLICATIONS NONE TRIAL PERIOD 23 JULY 1998 – 26 JULY .2004

DEVELOPMENT PHASE PHASE 3

OBJECTIVES PRIMARY OBJECTIVE: • TO INDUCE NORMAL PUBERTAL DEVELOPMENT IN TURNER GIRLS IN ACCORDANCE WITH THAT OF THEIR

PEERS (AND THEIR INDIVIDUAL STATE OF PSYCHOSOCIAL MATURATION) ASSESSED BY TANNER SCORE BY TREATMENT WITH 17β-ESTRADIOL, EITHER DOSED PER KILO BODY WEIGHT/DAY (GROUP I) OR IN A FIXED DOSE (GROUP II).

SECONDARY OBJECTIVE: • TO SECURE OPTIMAL UTILIZATION OF THEIR GROWTH POTENTIAL AND ASSESS WHETHER TREATMENT

WITH 17Β-ESTRADIOL CAN INCREASE GROWTH VELOCITY (CM/YEAR) WITHOUT COMPROMISING FINAL HEIGHT PREDICTION.

METHODOLOGY • THE TRIAL WAS A MULTI-CENTRE, RANDOMISED, OPEN-LABELLED, PARALLEL GROUP TRIAL OF 2 YEARS

DURATION. • SUBJECTS ATTENDED A SELECTION VISIT AND WERE RANDOMIZED, WITH INITIATION OF TRIAL PRODUCT

TREATMENT, AND 8 SUBSEQUENT REGULAR VISITS (EVERY THREE MONTHS) UNTIL THE END OF THE TRIAL (24 MONTHS).

• SUBJECTS WERE RANDOMISED TO EITHER OF TWO TREATMENT REGIMENS: − GROUP I (INDIVIDUAL DOSE): TABLETS OF 17Β-ESTRADIOL (NOVO NORDISK A/S), DOSAGE 5-15

ΜCG/KG/DAY TO BE TAKEN ORALLY IN THE MORNING FOR 2 YEARS. THE DOSAGE WILL BE READJUSTED AT EACH VISIT TO THE CLINIC AND ROUNDED OFF TO 0.05 MCG (HALF A TABLET)

− GROUP II (FIXED DOSE): • FIRST TREATMENT YEAR: TABLETS OF 17Β-ESTRADIOL (NOVO NORDISK A/S) 0.2 MCG (2 X 0.1

MCG TABLETS) TO BE TAKEN DAILY WITHOUT INTERRUPTION. • SECOND TREATMENT YEAR: TABLETS OF 17Β-ESTRADIOL (NOVO NORDISK A/S) 0.5 MCG TO

BE TAKEN DAILY WITHOUT INTERRUPTION. NUMBER OF SUBJECTS PLANNED AND ANALYSED IT WAS PLANNED TO INCLUDE 140 RANDOMISED SUBJECTS. ONLY 50 SUBJECTS WERE FINALLY INCLUDED IN THE TRIAL: ———————————————————————————————————————————————————————————————————————————————— Group I Group II Total N (%) N (%) N (%) ———————————————————————————————————————————————————————————————————————————————— Screened 50 (100) Screening failures 2 (4) Randomised 24 24 48 (96.0) Exposed 24 (100.0) 24 (100.0) 48 (96.0)

Date: 24 –April-2007 Trial ID: GHTUR/E/2 Version: 2 Integrated Clinical Trial Report Status: Final ICTR Synopsis

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Withdrawals 2 (4) 0 (0.0) 0 (0.0) Completed 22 (91,6) 24 (100.0) 46 (92.0)

DIAGNOSIS AND MAIN CRITERIA FOR INCLUSION INCLUSION CRITERIA 1. TURNER SYNDROME VERIFIED BY CHROMOSOMAL TEST. 2. TREATMENT WITH GROWTH HORMONE. 3. BONE AGE ≥12 YEARS AND <14 YEARS. 4. CLEAR SIGNS OF OVARIAN INSUFFICIENCY (FSH MARKEDLY ELEVATED (POSTMENOPAUSAL RANGE)). 5. WELL DOCUMENTED GROWTH RATE DURING THE LAST 12 MONTHS (HEIGHT DATA APPROXIMATELY MONTH -12 AND MONTH -6) PRIOR TO INCLUSION. 6. PARENTS/GUARDIANS AGREE TO GIVE SIGNED, INFORMED CONSENT. EXCLUSION CRITERIA: 1. SIGNS OF SPONTANEOUS PUBERTY (TANNER BREAST STAGE > M2) 2. KNOWN OR SUSPECTED HYPERSENSITIVITY TO TRIAL PRODUCT. 3. ACUTE OR CHRONIC LIVER DISEASE. 4. PREVIOUS TREATMENT WITH ESTROGEN. 5. UNDIAGNOSED ABNORMAL GENITAL BLEEDING. 6. KNOWN THYROID DISEASES NOT ADEQUATELY TREATED. 7. PORPHYRIA. 8. CARDIAC DISEASES THAT DO NOT ALLOW THE TREATMENT. 9. CURRENT LIVER ENZYME INDUCING MEDICATION, EG. RIFAMPICIN, BARBITURATES. 10 .KNOWN OR SUSPECTED ESTROGEN DEPENDENT NEOPLASIA. 11. TREATMENT WITH ANABOLIC STEROID HORMONES NOW AND IN THE PAST. 12. DIABETES MELLITUS DEFINED ACCORDING TO THE WHO CRITERIA. 13. ACTIVE DEEP VENOUS THROMBOSIS THROMBOEMBOLIC DISORDERS OR A DOCUMENTED HISTORY OF

THESE CONDITIONS. 14. END-STAGE RENAL DYSFUNCTION. 15. HYPERTENSION (MEAN OF TWO DIASTOLIC BP MEASUREMENTS IN TWO SUCCESSIVE VISITS ≥ 90MMHG, O

WHEN THE MEAN OF TWO SYSTOLIC BP MEASUREMENTS IN TWO OR MORE SUCCESSIVE VISITS IS > 140MMHG).

16. SEVERE ASTHMA. 17. A PERSONAL HISTORY A FAMILY HISTORY (SISTERS, BROTHERS AND PARENTS) OF VENOUS

THROMBOEMBOLISM. 18. SEVERE OBESITY (BMI > 30 KG/M2). 19. PROLONGED IMMOBILIZATION, MAYOR ELECTIVE OR POSTTRAUMATIC SURGERY OR MAJOR TRAUMA. TEST PRODUCT, DOSE AND MODE OF ADMINISTRATION, BATCH NUMBER TEST PRODUCTS: - TABLETS (WHITE) OF 0.1 MCG 17Β-ESTRADIOL DELIVERED IN DUMA PLASTIC CONTAINERS OF EACH 100

TABLETS; BATCH NUMBERS: 706601, HBBR245, KBBN055, HBBR246 - TABLETS (WHITE) OF 0.5 MCG 17Β-ESTRADIOL DELIVERED IN DUMA PLASTIC CONTAINERS EACH

CONTAINING 100 TABLETS, BATCH NUMBERS: FK5G401, KBBN057, 528392, FK5G402 DOSE: - GROUP I: (INDIVIDUAL DOSE)

TABLETS OF 17Β-ESTRADIOL (NOVO NORDISK A/S), DOSAGE 5-15 ΜCG/KG/DAY TO BE TAKEN ORALLY IN THE MORNING FOR 2 YEARS. THE DOSAGE WAS READJUSTED AT EACH VISIT TO THE CLINIC AND ROUNDED OFF TO 0.05 MCG (HALF A TABLET). ESTROGEN TREATMENT STARTED WITH A DOSE OF ABOUT 5 ΜCG/KG/DAY ACCORDING TO THE DOSE TABLE. IF PRETREATMENT BA WAS RETARDED MORE THAN 2 YEARS THE DOSE SHOULD NOT HAVE BEEN INCREASED DURING THE FIRST TREATMENT YEAR. IF PRETREATMENT BA WAS NOT RETARDED MORE THAN 2 YEARS, A DOSE INCREASE TO 10 ΜCG/KG/DAY WAS CONSIDERED AFTER 6-12 MONTHS. MAMMA AND BONE AGE DEVELOPMENT ON PREVIOUS DOSAGE WAS TAKEN IN CONSIDERATION CONCERNING DOSE INCREMENT. AFTER 18 MONTHS THE DOSE COULD HAVE BEEN INCREASED TO 15

Date: 24 –April-2007 Trial ID: GHTUR/E/2 Version: 2 Integrated Clinical Trial Report Status: Final ICTR Synopsis

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ΜCG/KG/DAY. EACH TABLET WAS TAKEN ORALLY, SWALLOWED WITHOUT CRUSHING, DIVIDED IF NECESSARY, WITH A DRINK OF WATER.

- GROUP II: (FIXED DOSE) FIRST TREATMENT YEAR: TABLETS OF 17Β-ESTRADIOL (NOVO NORDISK A/S) 0.2 MCG (2 X 0.1 MCG TABLETS) TO BE TAKEN DAILY WITHOUT INTERRUPTION.

SECOND TREATMENT YEAR: TABLETS OF 17Β-ESTRADIOL (NOVO NORDISK A/S) 0.5 MCG TO BE TAKEN DAILY WITHOUT INTERRUPTION. EACH TABLET SHOULD BE TAKEN ORALLY, SWALLOWED WITHOUT CRUSHING WITH A DRINK OF WATER.

DURATION OF TREATMENT 2 YEARS REFERENCE THERAPY, DOSE AND MODE OF ADMINISTRATION, BATCH NUMBER NO REFERENCE THERAPY WAS USED IN THIS TRIAL CRITERIA FOR EVALUATION – EFFICACY PRIMARY EFFICACY VARIABLE: TANNER SCORE FOR PUBERTAL STAGE, FSH SECONDARY EFFICACY VARIABLE: HEIGHT VELOCITY/PREDICTED FINAL HEIGHT. BA ASSESSMENTS ACCORDING TO G&P AND TANNER WHITEHOUSE II (RUS). ENDPOINTS: • PHYSICAL EXAMINATION, INCLUDING PUBERTY STAGE (TANNER SCORE: BREAST, PUBIC HAIR, AXILLARY

HAIR) (EVERY 3 MONTHS) • LABORATORY TESTS FOR FSH (EVERY 3 MONTHS) • BONE AGE ASSESSMENTS: X-RAY OF THE LEFT HAND AND WRIST FOR BONE AGE DETERMINATION (EVERY

12 MONTHS) • HEIGHT WAS MEASURED EVERY 3 MONTHS CRITERIA FOR EVALUATION – SAFETY ENDPOINTS: • PHYSICAL EXAMINATION INCLUDING PALPATION OF THE BREASTS. BLOOD PRESSURE (SUPINE POSITION)

AND WEIGHT (EVERY 3 MONTHS). • ADVERSE EVENT REGISTRATION INCLUDING BLEEDING/SPOTTING EPISODES (EVERY 3 MONTHS). • LABORATORY TESTS FOR HEMOGLOBIN, S-CREATININE, S-ALT/AST AND ALKALINE PHOSPHATASE (ONCE A

YEAR). • LABORATORY TESTS FOR HbA1C AND RANDOM BLOOD GLUCOSE (EVERY 3 MONTHS). STATISTICAL METHODS THE EFFICACY ANALYSIS IS PERFORMED SEPARATELY ON THOSE PATIENTS WHO WOULD HAVE RECEIVED LESS 17Β-ESTRADIOL THAN THEY WOULD HAVE RECEIVED ON AN INDIVIDUAL DOSAGE AND THOSE PATIENTS WHO WOULD HAVE RECEIVED MORE 17Β-ESTRADIOL THAN THEY WOULD HAVE RECEIVED ON AN INDIVIDUAL DOSE. THE PRIMARY OUTCOME OF INTEREST IS THE EVENT OF ATTAINING A TANNER BREAST SCORE OF 4 OR MORE. THE COMPARISON OF THE TWO GROUPS IS MADE USING 2X2 FISHER'S EXACT TEST OR A 2X2 CHI-SQUARED TEST IF THE USUAL ASSUMPTIONS ARE MET. SECONDARY EFFICACY ANALYSES COMPARE FSH VALUES, THE PROPORTION REACHING A TANNER BREAST SCORE OF 3 OR MORE AND TANNER PUBIC HAIR STAGE OF 3+ OR 4+. SAFETY ANALYSES INCLUDE THE COMPARISON OF BONE AGE AND OTHER SAFETY VARIABLES. SAFETY ANALYSIS WILL ALSO BE DONE ON TANNER GIRLS WHO WOULD HAVE RECEIVED MORE 17Β-ESTRADIOL ON THE FIXED DOSE THAN THEY WOULD HAVE RECEIVED ON AN INDIVIDUAL DOSAGE. FOR REASONS OF SIMPLICITY, THIS GROUP IS DEFINED AS THOSE GIRLS WEIGHING LESS THAN 35 KG AT ENTRY INTO THE STUDY. DEMOGRAPHY OF TRIAL POPULATION 50 GIRLS WITH TURNER SYNDROME WERE SCREENED IN THE TRIAL AND 48 RANDOMISED. THE AGE AVERAGE OF RANDOMISED GIRLS WAS 13.8 ± 1.3 YEARS. PER GROUPS, THE AGE AVERAGE WAS:

• GROUP I: 14.3 ± 1.4 YEARS; CI = 13.7 – 14.8

Date: 24 –April-2007 Trial ID: GHTUR/E/2 Version: 2 Integrated Clinical Trial Report Status: Final ICTR Synopsis

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• GROUP II: 13.4 ± 1.0 YEARS; CI = 13.0 – 13.9

THERE WAS A STATISTICALLY SIGNIFICANT DIFFERENCE BETWEEN GROUPS (P = 0.019) EFFICACY RESULTS PRIMARY ENDPOINTS (TANNER SCORE FOR PUBERTAL STAGE, FSH) (ASSESSED AT THE END OF TRIAL)

• PUBERTAL STAGE IS REACHED WHEN TS GIRLS ACHIEVED ≥ 4 FOR BREAST TUNNER SCORE. ACCORDING TO OUR DATA, THE TS GIRLS ENROLLED TO GROUP I (INDIVIDUAL DOSE) REACHED THE PUBERTAL STATUS IN 42 % VERSUS TS GIRLS ENROLLED TO GROUP II (FIXED DOSE), THAT REACHED A 65% PUBERTAL STATUS AT THE END OF TRIAL. THIS DIFFERENCE IS NOT SIGNIFICANT (P=0,068).

• FSH LEVELS WERE MEAUSURED ALONG THE STUDY IN BOTH GROUPS. THERE WERE NO SIGNIFICANT

DIFFERENCES BETWEEN GROUP I (INDIVIDUAL DOSE) AND GROUP II (FIXED DOSE) WITH REGARD TO FOLLICLE-STIMULATING HORMONE (FSH). ACCORDING TO OUR DATA WE OBSERVE THAT FSH LEVELS DECREASED DURING THE STUDY, ACHIEVING LOWER LEVELS AT THE END OF STUDY. THERE ARE SIGNIFICANT DIFFERENCES (P≤0.05) BETWEEN BASELINE AND VISITS 3, 6, 7, 8 AND 9 IF BOTH GROUPS ARE ASSESSED TOGETHER.

SECONDARY ENDPOINTS (ASSESSED AT LAST VISIT, AFTER 24 MONTHS)

• TIME TO PUBERTAL ONSET (DESCRIBED AS ≥ 4 FOR BREAST TUNNER SCORE). ACCORDING TO OUR DATA, THE MEDIAN TIME TO REACH THE PUBERTAL STATUS IS SIGNIFICANTLY SHORTER IN GROUP II (FIXED DOSE) (P= 0,046). TS GIRLS ENROLLED TO GROUP I (INDIVIDUAL DOSE) SPENT A MEDIAN OF 818 DAYS (AVERAGE 775) TO REACH THE PUBERTAL ONSET, AND THE TS GIRLS ENROLLED TO GROUP II (FIXED DOSE) A MEDIAN OF 733 DAYS (AVERAGE OF 699).

• TANNER SCORE FOR BREAST ≥ 3 (ASSESSED AT LAST VISIT): 83% ENROLLED TO GROUP I

(INDIVIDUAL DOSE) REACHED 3 OR MORE FOR TUNNER BREAST SCORE VERSUS 96% TS GIRLS ENROLLED TO GROUP II (FIXED DOSE). THIS DIFFERENCE IS NOT SGNIFICANT (P=0,098).

• TANNER SCORE FOR PUBIC HAIR ≥ 4 (ASSESSED AT LAST VISIT): THE GIRLS INCLUDED IN GROUP I

(INDIVIDUAL DOSE) REACHED THE TANNER SCORE 4 OR MORE FOR PUBIC HAIR IN 71% VERSUS 87 % ACHIEVED FOR THE GILS INCLUDED IN GROUP II (FIXED DOSE). THIS DIFFERENCE IS SIGNIFICANT (P= 0.043).

• TANNER SCORE FOR PUBIC HAIR ≥ 3 (ASSESSED AT LAST VISIT): 100% OF THE TS GIRLS ENROLLED

AT GROUP II (FIXED DOSE) REACHED TANNER SCORE FOR PUBIC HAIR ≥ 3, AND 92% IN GROUP I, BUT THE DIFFERENCE IS NOT STATISTICALLY SIGNIFICANT (P = 0.985).

• GROWTH RATE (CM/YEAR): THERE ARE NO SIGNIFICANT DIFFERENCES BETWEEN THE TWO GROUPS;

ONLY SIGNIFICANT DIFFERENCES ARE OBSERVED BETWEEN VISITS 1 AND 9 WHEN ALL DATA ARE GROUPED (P<0.05).

NO SIGNIFICANT DIFFERENCES FOR GROWTH RATE IN SDS FOR CRONOLOGIC AGE AND BONE AGE.

• HEIGHT VELOCITY/PREDICTED FINAL HEIGHT: NO STATISTICAL SIGNIFICANCE OBSERVED

BETWEEN GROUPS IN PREDICTED FINAL HEIGHT IN CM, AND IN SDS FOLLOWING STANDARD OF FUNDACION ANDREA PRADER (P>0.5). SIGNIFICANT DIFFERENCES BETWEEN VISITS 1 – 9 AND 5-9 IN GROUP II WHEN MEASURED IN CM/YEAR (P=0.016 AND 0.007 RESPECTIVELY).

• BONE AGE: SIGNIFICANT DIFFERENCES INTRAGROUP AMONG VISITS 1-5-9 MEASURED BY

INCREMENT (YEARS) FROM BASAL VISIT (P<0.001). NO DIFFERENCES BETWEEN GROUPS. WHEN MEASURED BY RATIO BONE AGE/CRONOLOGICAL AGE, SIGNIFICANT DIFFERENCES ARE OBSERVED AMONG GROUPS IN VISITS 1 AND 5 (P=0,011 AND P=0,018 RESPECTIVELY). WHEN MEASURED BY RETARDATION OF BONE AGE COMPARED TO CRONOLOGICAL AGE (YEARS), VALUES IN GROUP II WERE SIGNIFICANTLY LOWER THAN GROUP I IN VISITS 1 AND 5 (P=0.008 AND 0.016 RESPECTIVELY).

Date: 24 –April-2007 Trial ID: GHTUR/E/2 Version: 2 Integrated Clinical Trial Report Status: Final ICTR Synopsis

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SAFETY RESULTS

BLOOD PRESSURE, HEART RATE: TO EVALUATE THE SAFETY OF THE STUDY MEDICATION, THESE PARAMETERS WERE RECORDED EVERY 3 MONTHS. THERE WERE NO SIGNIFICANT DIFFERENCES BETWEEN GROUP I (INDIVIDUAL DOSE) AND GROUP II (FIXED DOSE) WITH REGARDS TO THESE PARAMETERS. ALL VALUES WERE IN THE NORMAL RANGES FOR HEART RATE AND BLOOD PRESSURE. WEIGHT: STATISTICALLY SIGNIFICANT WEIGHT INCREASE DURING THE TRIAL VERSUS BASELINE IN BOTH STUDY GROUPS (FROM VISIT 2 TO 9 IN GROUP I, AND FROM VISIT 3 TO 9 IN II) (P FROM 0.01 TO 0.001). NO DIFFERENCES BETWEEN GROUPS. NO SIGNIFICANT DIFFERENCES FOR LABORATORY TEST (INCLUDING HB, ALT, AST, FA) BETWEEN STUDY GROUPS (EVERY YEAR). FASTING GLUCOSE, IN BOTH GROUPS THE VALUES DECREASED DURING THE TRIAL, WITHOUT DIFFERENCES BETWEEN STUDY GROUPS VERSUS BASELINE.(EVERY 3 MONTHS). ADVERSE EVENTS, ACCORDING TO OUR DATA, THE TS GIRLS ENROLLED IN GROUP I (INDIVIDUAL DOSE) THAT PRESENTED AT LEAST 1 AE WERE 38% FRO GROUP I AND 33% FOR GROUP II (FIXED DOSE), WITH STATISTICALLY SIGNIFICANT DIFFERENCES (P= 0,763). ONLY 1 SERIOUS ADVERSE EVENT WAS PRESENTED IN GROUP I (NO STATISTICALLY SIGNIFICANT DIFFERENCES WITH GROUP II). CONCLUSIONS • FOR THE PRIMARY ENDPOINT “PUBERTAL STAGE AS MEASURED BY BREAST TANNER SCORE ≥ 4”, NO

STATISTICALLY SIGNIFICANT DIFFERENCES ARE OBSERVED IN THE TWO TREATMENT GROUPS (DOSE PER KIL AND FIXED DOSE), ALTHOUGH 65% TS GIRLS IN GROUP II (FIXED DOSE) REACHED THE PUBERTAL STAGE VERSUS 45% IN GROUP I (DOSE PER KILO).

• IN BOTH GROUPS, LEVELS OF FSH DECREASED DURING THE STUDY, WITH STATISTICALLY SIGNIFICANT DIFFERENCES IN THE CONSECUTIVE VISITS WHEN DATA FROM BOTH GROUPS ARE ASSESSED TOGHETER, BUT NO STATISTICALLY SIGNIFICANT DIFFERENCES ARE OBSERVED BETWEEN GROUPS.

• TIME FOR PUBERTAL ONSET IS SIGNIFICANTLY SHORTER IN GROUP II (FIXED DOSE), WITH A MEDIAN OF 733 DAYS VERSUS 818 FOR GROUP I. ALSO TANNER SCORE FOR PUBIC HAIR ≥ 4 IS REACHED IN A HIGHER PERCENTAGE IN GROUP II VERSUS I (87% AND 71% RESPECTIVELY.

• NEVERTHELESS, NO STATISTICALLY SIGNIFICANT DIFFERENCES ARE OBSERVED IN GROTH RATE, PREDICTED FINAL HEIGHT OR BONE AGE.

• SAFETY RESULTS SHOWED NO DIFFERENCES BETWEEN GROUPS IN ADVERSE EVENTS, LABORATORY TESTS, BLOOD PRESSURE OR HEART RATE.

• IN GENERAL, GROUP II (FIXED DOSE) WAS MORE EFFECTIVE THAN GROUP I (DOSE PER KILO), BUT ONLY IN SOME ENDPOINTS.

THE TRIAL WAS CONDUCTED IN ACCORDANCE WITH THE DECLARATION OF HELSINKI AND GOOD CLINICAL PRACTICE.