1. ART products and usage TOTAL CYCLE CONTROL
Leong Ka Hong MDCM DSc
Adjunct Professor Dept of Obs/Gyn
McGill University Montreal Canada
Specialist Reproductive Medicine Hong Kong
Shanghai May 2004

2. Highly purified urofollitropin Ferring Pharmaceuticals
BRAVELLETM
Highly purified urofollitropin
Ferring Pharmaceuticals

3. BRAVELLE Highly purified urinary FSH NOT a generic Metrodin-HP
NDA in USFDA
Indications
Ovulation induction
IVF

4. BRAVELLE vs Metrodin-HP
Urine sources
Argentina (BSA no risk)
Italy (BSA high risk)
FSH dosage/amp IU 75
Active ingredient purity (% of total protein)
95%
LH residual
(% of total protein or IU)
≤ 2%
< 0.1 IU
Purification technology
Multiple Ion exchange chromatography
Immunochromatograph (monoclonal anti-FSH)

5. BRAVELLE vs rFSH Sources Purification steps Other industrial steps
Bravelle: pooled human urine
rFSH: rFSH containing culture media with Fetal Bovine serum
Purification steps
Bravelle: ion exchange chromatography
rFSH: immuno-chromatography
Other industrial steps
More or less the same

6. Risks of Prion Contamination
Bravelle:
Low possibility, from pooled human urine
Urine from Argentina (CJD low risk)
rFSH
Possibility 1, Fetal Bovine Serum used in culturing CHO cells (FBS were from US or Canadian sources in which BSE cases found recently)
Possibility 2, immunopurification step which employed monoclonal FSH antibody. Mab was also made from cultured cells which grown in media with FBS

7. Dickey et al., 2003

8. Dickey et al., 2003

9. Dickey et al., 2003

10. Dickey et al., 2003 Conclusions
Bravelle(R) and Follistim(R) had comparable efficacy in controlled ovarian hyperstimulation in women undergoing IVF-ET. There were no differences in the nature or number of adverse events between the treatment groups although Bravelle(R) injections were reported to be significantly less painful.

11. Feigenbaum et al., 2001

12. Feigenbaum et al., 2001

13. Feigenbaum et al., 2001

14. Feigenbaum et al., 2001 Conclusion
The efficacy and treatment toleration of Bravelle™, a new, highly purified, human-derived FSH, is comparable to that of recombinant follitropin beta in patients undergoing ovulation induction.

15. BRAVELLE HKIVF DATA # of OPU 19 11 <40, 8 ≥40 Total dose 887 (46)
Range 26 – 68
# of eggs retrieved
193
MTII 126 (65%)
MTI 38 (20%)
Fertilization rate %
59%
Total # of Embryos Transfer 54
Average # of ET 2.8/opu
# of pregnancy 9
Pregnancy rate %/opu
47%
Implantation rate
22.6%

16. Progesterone Sites of production Corpus luteum Placenta Functions
Increase endometrial receptivity
Maintain pregnancy

17. Oral vs Vaginal Administration
Liver first-pass metabolism
Low serum level (low bioavailability)
Side effects due to metabolites
Vaginal
Avoids liver first-pass metabolism
Fewer systemic side effects
Low serum level
Uterine first-pass effect
High tissue conc. (High bioavailability)

18. Progesterone - Natural vs Synthetic
Androgenic activity  
Teratogenicity
Lipoprotein metabolism
Beneficial to BV
Half-life
4 – 8 min. -

19. Structure & Synthesis of Progesterone

20. Plasma Progesterone Levels
Follicular phase: <2 ng/ml
Luteal phase: ng/ml
1st trimester of pregnancy: ~ ng/ml
Near term: ~ ng/ml
15% drop: 1 hr after a meal & in the early morning

21. Endometrin® Product Description
Progesterone vaginal tablet (with applicator)
Developed in Israel
Micronized progesterone (100 mg)
Indication
Progesterone supplementation or replacement in cases such as treatment of infertile women and IVF

22. Endometrin Characteristics
Prolong release for 12 hours (100 mg)
Bid
Uterine 1st pass effects
Ensures maximal uterine/endometrial exposure
Advanced formulation
Easy administration with specific applicator
Without messy discharge
Unaltered vaginal pH – minimized risks of infection

23. Progesterone supplement
ENDO
UTRO
CYCLO
1 supp BD
48.6ng
34ng
62.2ng
2 supp BD
55.7ng
87ng
54.7ng

24. General Principles of combined pituitary suppression / ovarian stimulation therapy (From Insler and Lunenfeld)

25. The structure of GnRH agonistic analogues

26. Outcome results according to type of protocol and gonadotrophin used (n=13426)

27. Crude pregnancy rates using different GnRH agonist protocols in IVF (From Daya)

28. Schematic representation of different protocols using GnRH agonists in combination with gonadotrophins for ovarian stimulation in IVF

29. Trade names, plasma half-lives, relate potency, route of administration and recommended dose for the clinically available gonadotrophins

30. Structure of GnRH agonists
Modifications of natural GnRH
to have GnRH agonistic properties 1 2 4 3 6 5 9 8 10 7
pyro (Glu) – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2
activation of the
GnRH receptor
regulation
of GnRH
receptor
affinity
regulation of
biologic activity
Modification of position 6 and 10 is typical for all GnRH agonists. This leads to a change in
- GnRH receptor affinity (which is increased)
- regulation of biologic activity (which is increased due to a longer half life)

31. Action of GnRH agonists
Down
regulation
Action of GnRH agonists
1. Binding of GnRH leads to a post-receptor-cascade and this consecuitively to the release of LH and FSH
2. Adding of GnRH agonists will lead - because they have a higher affinitiy to the receptors and have a higher biologic potency - to binding of the agonists to the receptors instead of the natural GnRH.
3. Initially, this will lead to an increase in the receptor action, number and post-receptor-cascade with a consecutive increase in the release of LH and FSH (flare up effect).
4. After that, however, receptors are internalized, lysed, the number of receptors decreases, the post-receptor-cascade is downregulated and the stimulus to release LH and FSH will also be suppressed.
5. Downregulation and pituitary suppression will result.
GnRH
LH + FSH
GnRH - receptor
post-receptor-cascade
flare up effect
GnRH - agonist
pituitary suppression

32. Premature LH surge Poor quality
No fertilization or very poor pregnancy rate
Cancel egg retrieval
5-20%
All cycles treated in 1980’s

34. Results of first application of GnRH-agonists in the long protocol
11 patients eligible for IVF
GnRH agonist s.c. (buserelin) started at day of menstruation or one day before
ovarian stimulation started with HMG or purified FSH when all ovarian follicles and the endometrial lining has disappeared on ultrasound (average: 15 days)
one ongoing pregnancy achieved
Porter RN, Smith W., Craft IL., Abdulwahid NA., JAcobs HS (1984) Induction of ovulation for in-vitro fertilization using buserelin and gonadotropins. Lancet 2;
Porter et al., 1984

35. The long luteal protocol
ovulation
induction
oocyte
pick up
embryo
transfer
gonadotropin administration
in an individualized dosage
start of
GnRH agonist
In the long luteal protocol a GnRH agonist depot preparation is administered during the mid-luteal phase of the preceeding cycle, or a GnRH agonist is started with a daily administration at that time.
Two weeks later, in between menstruation will start, pituitary suppression is achieved. At that time point a transvaginal ultrasound should be done to exclude cyst formation, since the flare up effect of the agonist may lead to ovarian cyst formation. If pituitary suppression has been achieved and the ovaries do not show cysts, gonadotropin stimulation can be started at that day. It will go on until hCG can be administered for ovulation induction.
Luteal phase support is necessay for these protocols.
22nd day
of previous
cycle
1st day
of gonado-
tropins
luteal phase support
14 days

36. GnRHa 剂量组 曲普瑞林 (mcg) 5 15 50 100 病例数 11 10 12 刺激天数 9 7.5 10.5 FSH使用量24
22.5 27
28.5
S7-LH
3.3
1.5
1.3
0.8
HCG-LH
2.5
2.3
1.8
0.9 排卵 3
Source: Janssens et al, 1998

39. DECAPEPTYL DOWN REGULATION 2000-2003
< 40
≥ 40
# of patients 90
76 (32.9)
14 (40.8)
# of pregnancy 42 40 2
Pregnancy %
46.7
52.6 14
# of twins+ 10
# of babies 43 1
Miscarriage rate
16%
50%

40. DECAPEPTYL DOWN REGULATION 2000-2003 LABORATORY DATA
# of eggs
831
MTII 539 (67%)
MTI 139 (16.7%)
# of eggs ICSI
551
# of fertilized
427
Fert. % 76.4
# of E.T.
244
Mean transferred 2.7
# of preg. (F.H.) 46
Implantation rate 20.5%

41. GnRH agonists Over-suppression:
LH becomes so low that it affects the production of estrogen, and possibly progesterone in the luteal phase
Leads to poor response, poor pregnancy outcome due to early abortion
Also it is:
Too long and too much drug use, cost, cancelled cycles and it is unnatural.

42. Ovulation Stimulation Scheme
BCP to control cycle (or ovulation monitor)
BCP D16/ovulation +7day DECAPEPTYL
Decapepyl 100mcg/day x 7 days
D2 FSH, LH, E
D3 Bravelle IU/day with monitor
Choragon 10000IU for induction
Endometrin BD (+ Progesterone)

43. BRAVELLE HKIVF DATA # of OPU 19 11 <40, 8 ≥40 Total dose 887 (46)
Range 26 – 68
# of eggs retrieved
193
MTII 126 (65%)
MTI 38 (20%)
Fertilization rate %
59%
Total # of Embryos Transfer 54
Average # of ET 2.8/opu
# of pregnancy 9
Pregnancy rate %/opu
47%
Implantation rate
37%

44. OVULATION STIMULATION
Comparison of protocols:
Decapeptyl Down Regulation
Long Lucrin Down Regulation
Antagonist, no down regulation

45. Structure of GnRH antagonists
to achieve antagonistic properties of natural GnRH more
modifications than only in position 6 and 10 are necessary 1 2 4 3 6 5 9 8 10 7
pyro (Glu) – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2
activation of the
GnRH receptor
regulation
of GnRH
receptor
affinity
regulation of
biologic activity
In contrast to GnRH agonists, there are more changes necessary in the structure of the natural GnRH molecule, to achieve antagonistic properties. These antagonistic properties mean:
- no intrinsic effect
- competitive action

46. Action of GnRH antagonists
1. Binding of GnRH leads to a post-receptor-cascade and this consecutively to the release of LH and FSH
2. Adding of GnRH antagonists will lead to a competitive action of GnRH and GnRH antagonists - which do not have any intrinsic activity.
3. A sudden downregulation of the post-receptor-cascade is the result with a consecutive decrease in the stimulus to release LH and FSH.
4. Pituitary suppression is achieved within a few hours without any initial flare up effect.
GnRH
LH + FSH
GnRH - receptor
post-receptor-cascade
pituitary suppression
GnRH - antagonist

47. Comparison of the long protocol and the antagonist protocols
more
physiologic
no cyst
formation
no hormonal
withdrawal
antagonist administration
gonadotropin administration
multiple dose protocol
less gona-
dotropins
early
pregnancy?
Advantages of the antagonist protocol:
- no cyst formation (since there is no flare up effect)
- no hormonal withdrawal symptoms (since pituitary suppression is achieved after ovarian stimulation has been started and not before)
- the antagonist cannot be given in early pregnancy, since this can be excluded by a pregnancy test
- less gonadotropins necessary
- shorter treatment duration
- more physiologic, since it can be integrated in the spontaneous menstrual cycle
flare up
effect
pituitary
suppression
agonist administration
gonadotropin administration
long protocol
longer
treatment
pre-treatment cycle
treatment cycle

48. The Cetrotide® 0.25 mg multiple dose protocol
ovulation
induction
oocyte
pick up
embryo
transfer
gonadotropin administration
in an individualized dosage
1st day
of menstruation
In the multiple dose antagonist protocol ovarian stimulation is started with the second or third day of the menstrual cycle.
Cetrotide® 0.25 mg is started on the 6th day of ovarian stimulation in the morning or on the 5th day in the evening. And is administered up to and including the day of hCG, if given in the morning.
1st day
of gonado-
tropins
luteal phase support
Cetrotide® 0.25 mg administration
daily s.c. starting on day 6 of stimulation

49. Possibilities to individualize the multiple dose protocol
To avoid a premature LH rise the administration of cetrotide® 0.25 mg on day 6 of stimulation should be the standard procedure
Using the standard procedure, a mean of 6.3 injections are necessary
This is in accordance with the package size of 7 ampoules cetrotide® 0.25 mg per patient

50. Possibilities to individualize the multiple dose protocol
Individualized administration of Cetrotide® 0.25 mg can be done
According to follicle size: only if leading follicle is  14 mm
Thereby, the multiple dose protocol can also be adapted to patients with a lower response

51. Personal experience with multiple dose of Cetrorelix 0.25 mg
Patient group:
Over suppression with agonist long protocol (LH < 1mlU)
Patient over 40
Poor response to agonists suppression

52. Cetrorelix 0.125mg Flexible Dose Trial
Selection Criteria:
1. Previous over-suppression with agonist
2. Previous poor response
3. Previous LH surge if no agonist

53. Cetrorelix 0.125mg Flexible Dose Trial
Methods:
FSH LH E2 on day 2
U/S on day 3, start Gonal-F 225IU/day
Stimulation day 4, check E2 LH U/S

54. Cetrorelix 0.125mg Flexible Dose Trial
Treatment Criteria
LH > 1.5IU/L
Leading follicle = 15mm diameter
Cetrorelix 0.125mg/day given until day of HCG injection
Monitor by E2 LH U/S everyday

55. Age Distribution
Mean = 36.6 (range 29-44)

56. Cetrorelix 0.125mg Flexible Dose Trial
RESULTS

57. BMI Distribution
Mean = 21.8 (range 19-30)
Mean = 21.8 (range 19-30)

58. Cetrorelix Start Cycle Day
FSH 225 IU/day SC starts on day 3

59. # Days Cetrorelix Used
Mean = 2.2 days (range 1-3)

60. LH and Cetrorelix 0.125mg/day
Range mIU/ml
Pre
Day 1 post
Day HCG

61. Clinical Data Age BMI 36.6 (29-44) 21.8 (19-30) 15<40yr 5>40yr
Ova # per OPU
%MT II
% Fertilization
9 (1-16)
77%
74.4%
Transfer # embryos
Pregnancy rate
Implantation rate
2.6 <40yr >40yr
60%<40yr 40% >40yr
25.4% (16/63)

62. Experience with Cetrotide 0.125mg

63. Ovulation Stimulation
<40
(41)
≥40
(6)
(60)
(2)
(117)
(58)
Average age
33.9 41
33.4
40.5
35.2
41.9
# of OPU 6 60 2
117 78
# of egg retreived
458 34
718 21
1272
443
# of MTII
307, 67%
22, 65%
510, 71%
12, 57%
881, 69%
313, 71%
# of MTI
81, 19%
8, 24%
126, 18%
4, 19%
202, 15%
60, 14%
Deca
Long Luc
Cet

64. Ovulation Stimulation
<40
≥40
# of ICSI’d
364 29
586 19
1034
349
# of 2PN
280 20
435 15
756
263
Fertilization rate %
77%
69%
74%
79%
73%
75%
# of Embryos Tr
137 17
201 7
337
154
Mean # of ET
3.3
2.8
3.35
3.5
2.9
2.7
# of pregnancy 22 1 30 47
Pregnancy rate% opu
54%
17%
50% 0%
40%
12%
Implantation rate
19% 6%
22%
15%
Deca
Long Luc
Cet

65. Flexible Stimulation When dn reg showed over suppression
Low E, LH, small follicles
D2 E=30.7pg LH=0.54IU/L
D6 E=214pg LH=0.7 IU/L
rLH added to Bravelle dosage
D(HCG) E=1856.5pg LH 1.45IU/L
6/8 cases pregnant

71. Infertility 1/6 couples trying for pregnancy
Medical,social and psychological problems studied
Very few perception studies

72. Perception study in Infertility
Bertarelli Foundation Survey 2000
1st published population perception survey
Polled 6 European Countries, USA and Australia 1998/1999
Telephone survey: random selection households
7036 polled, no mention of % completion

73. Perception Survey in Infertility
6 questions
Asked You/your friend to avoid rejection
52 % claimed personally know somebody who had infertility

74. Perception Study in Infertility
Is Infertility a disease? Mean 38%
Australia, UK, USA 20%
Germany, Italy %
Heard of IVF?
77% (Germany) to 99% (Sweden)

75. Perception study in infertility
Should IVF be reimbursed?
Told respondent cost 3x IVF equals hip replacement
60-80% said to reimburse

76. Perception Study in Infertility
Although 97% of respondents heard of IVF and approved of IVF, only 16% answered correctly that IVF success is similar to that of natural pregnancy.
Most people guessed the success rate, and no correlation can be established

77. YWCA Perception Study 2002 Aims: Infertility problem vs no problem
psycho-social states
medical aspects - treatment profiles

78. Bertarelli vs. Hong Kong Survey
52% know infertility
16% know success rate
89% know IVF
38% said infertility = disease
Hong Kong
50% know infertility
20%know success rate
46% know IVF
50% said infertility=disease

79. YWCA Perception Study 2002 Methodology: Telephone survey
Randomly called numbers
No demographic questions to maximise response
Respondents answer 7,15, or16 questions

80. YWCA Perception Study 2002 Randomly dialed Numbers
Carried out July to October
Successfully Connected:147897
Valid Data: 7028
Success Rate: 5%

81. YWCA Perception Study 2002

82. Why Reject IVF

83. Investigations on Treated Subjects

84. Investigations done Treated subjects

85. Basic Investigations Made

86. Summary of Results 16% of polled has infertility problem
45% view infertility as disease. For infertile couples 52% consider it a disease
35% only has heard of IVF centers
50% know the reason for their infertility
30% know IVF success rate (20-40%)

87. Summary of Results 1173/7208 polled claimed to be infertile
only 265 (34%) have received or under treatment
50.6% had ovulation induction
34% had intra-uterine insemination
24% had IVF/related treatment

88. Summary of Results Diagnosis procedures reported
Ovulation/hormone, laparoscopy 35%
SA, HSG only 45%
Ultrasound only test >50% (58%)
42% answered that none of the above tests were done

89. Summary of Results
Although 50% of respondents who are under treatment accepts IVF
Only 30% actually received IVF treatment
This is 6.5% of respondents who has infertility
Of those rejecting IVF, 45.7% worry IVF begets abnormal babies. 22.5% think too expensive

90. CONCLUSIONS
There is misconception about IVF mainly in the availability, success rate, and the technology itself
There is a problem of education, so that appropriate tests were not done
Funding is not enough, especially third party payment

91. CONCLUSIONS
Patients are not seeking treat-ment. Social factors and other psychological reasons may be present.
May also be that right treatment is not readily offered, so treat-ment abandoned after a while

92. Cost Hip Replacement Vs IVF
OT charge:$40000
Hospital Bed: $20000
Surgical Fee:$30000
Drugs etc: $5000
Total: $95000
IVF
OT charge: $12000
Hospital bed:$3000
Surgical Fee:$15000
Lab Fee:$ 15000
Drugs etc:$15000
Total: $$60000

93. ACTION, STRUCTURE AND USE OF GnRH AGONISTS AND ANTAGONIST

94. Structure of GnRH antagonists

95. Comparison: Mode of Actions
Antagonists
Agonists
Immediate onset of actions (shortens treatment durations)
Prevents hormonal withdrawal symptoms
No recovery time of the pituitary
long pre-treatment
Hormonal (estrogen) withdrawal symptoms through desensitization of pituitary
Recovery of the pituitary gonadotrophin secretion, after stopping the treatment takes about 2 weeks.

96. Characteristics of GnRH
Ganirelix
Fully effective within 4 hours, with a half-life of about 13 hours
Cetrorelix
Fully effective within 8 hours, with a half-life of about 36 hours
R.E. Felberbaum and K. Diedrich, 1999.

97. Antagonists in controlled ovarian stimulation - the first steps
Administration of the GnRH antagonist leads to suppression of estradiol, stop of follicular growth and avoidance of a LH or FSH surge.
Ditkoff EC, Cassidenti DL, Paulson RJ, et al. The gonadotropin-releasing hormone antagonist (Nal-Glu) acutely blocks the luteinizing hormone surge but allows for resumption of folliculogenesis in normal women. Am J Obstet Gynecol 1991; 165:
control
Days relative to ovulation
Nal-Glu cycles
Ditkoff et al., 1991

98. Reduction of OHSS using Cetrotide®
Multiple dose protocol
rate of OHSS: 6.5% vs. 1.1% (agonist vs. antagonist protocol)
RR 6.2, 95% CI: , p = 0.03
Single dose protocol
rate of OHSS: 11.1% vs. 3.5% (agonist vs. antagonist protocol) 95% CI: to 3.2
patients requiring hospitalisation: 5.6% vs. 1.8%
(agonist vs. antagonist protocol) 95% CI: to 4.1
With both Cetrotide® protocols a clear reduction of OHSS was achieved

99. Mean number of Cetrotide® 0
Mean number of Cetrotide® 0.25 mg ampoules in the multiple dose protocol
average: 6.3 injections
The data result from the experience in 859 patients, who were treated in an open, prospective, non-controlled phase IIIb trial.
It became clear, that in mean 6.3 injections were necessary, when Cetrotide® 0.25 mg was started on day 5 of stimulation.

100. The GnRH Antagonists Conclusions:
Why treat 100% of patients when we are trying to prevent 5-10% LH surge
Avoid over-suppression and poor response
Effective in preventing LH surge
Reduction of hyper-stimulation
Lower costs

101. Safety Profile Possible side effects No known drug interaction
headache, weakness, mild vaginitis & breast sensitivity etc
No known drug interaction
Overdose
unlikely & no side effects

102. Precautions History of depression, DM
Not taken with other intravaginal products
Not recommended during lactation
Stored in a dry place, <25oC

103. Progesterone Functions Increase endometrial receptivity
Maintain pregnancy
Routes of administration
Injection (IM / IV)
Oral
Rectal
Vaginal, etc

104. Making BRAVELLE (GMP standards)
Screening of eligible health donors
Highly purified material
Documentation on eligible donors
#Ion exchange chromotographic purification
Pharmaceutical Lyophilization & finishing
Aseptic Urine collection
Pooled Post menopausal urine
BRAVELLE
filtrate
Crude filtration & clearance
# Patent technology for FSH purification

105. Making rDNA FSH (Gonal-F, Puregon)
Genetically Eng. CHO cells
Highly purified material
Culturing CHO cells in media with Fetal Bovine Serum (FBS)
Cultivating CHO cells in industrial size culturing tanks
Immuno chromotographic purification
Pharmaceutical Lyophilization & finishing
rFSH secreted by CHO cell into culture media
Gonal-F or Puregon
filtrate
collected & pooled culture media
Crude filtration & clearance