Dr. Milton Leong Director IVF CENTRE Hong Kong Sanatorium & Hospital

Dr. Milton LeongDirector

IVFCENTREHong Kong Sanatorium & Hospital

2

The Gonadotrophin Releasing Hormone Antagonists

3

Synthesis of GnRH

pGlu1-His2-Trp3-Ser4-Tyr5-Gly6-Leu7-Arg8-

Pro9-Gly10NH2

by Schally in 1968

GnRH could restore ovulatory functions in hypogonadotrophic amenorrheas.

Schally AV, Arimura A, Bowers CY et al. 1968

4

Structure of GnRH agonists

modifications of natural GnRHto have GnRH agonistic properties

1 2 43 65 98 107

pyro (Glu) – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2

activation of the GnRH receptor

regulation of GnRHreceptoraffinity

regulation ofbiologic activity

5

Premature LH surge

Poor quality

No fertilization or very poor pregnancy rate

Cancel egg retrieval

5-20%

All cycles treated in 1980’s

6

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 et al., 1984

7

The long luteal protocol

22nd dayof previous

cycle

14 days

1st dayof gonado-

tropins

gonadotropin administrationin an individualized dosage

ovulationinduction

oocytepick up

embryotransfer

luteal phase support

start ofGnRH agonist

8

Action of GnRH agonists

LH + FSH

post-receptor-cascade

GnRH - receptor

GnRH

GnRH - agonistflare up effect

downregulation

pituitary suppression

9

GnRH agonist

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.

10

to achieve antagonistic properties of natural GnRH moremodifications than only in position 6 and 10 are necessary

1 2 43 65 98 107

pyro (Glu) – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2

activation of the GnRH receptor

regulation of GnRHreceptoraffinity

regulation ofbiologic activity

Structure of GnRH antagonists

11

Action of GnRH antagonists

LH + FSH

post-receptor-cascade

GnRH - receptor

GnRH

GnRH - antagonistpituitary suppression

12

name amino acid sequenceGnRH pGlu – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2

1st generation4F Ant NAc1,1Pro – D4FPhe – DTrp – Ser – Tyr – DTrp – Leu – Arg – Pro – GlyNH2

2nd generationNalArg NACD2Nal – D4lFPhe=pTrp – Ser – Tyr – DArg – Leu – Arg – Pro – GlyNH2

Detirelix NACD2Nal – D4ClPhe – pTrp – Ser – Tyr – DHarg(Et2) – Leu – Arg – Pro – DAlaNH2

3rd generationNalGlu NACD2Nal – D4C7Phe – D3Pal – Ser – Arg – DGlut(AA) – Leu – Arg – Pro – DAlaNH2

Antide NACD2Nal – D4ClPhe – D3Pal – Ser – Lys(Nic) – DDLys(Nic) – Leu – Lys(Isp)Pro – DAlaNH2

Org30850 NACD4ClPhe – D4ClPhe – DBal – Ser – Tyr – DLys – Leu – Arg – Pro – DAlaNH2

Ramorelix NACD2Nal – D4ClPhe – DTrp – Ser – Tyr – DSet(Rha) – Leu – Arg – Pro – AzaglyNH2

Cetrorelix NACD2Nal – D4ClPhe – D3Pal – Ser – Tyr – DCit – Leu – Arg – Pro – DAlaNH2

Ganirelix NACD2Nal – D4ClPhe – D3Pal – Ser – Tyr – DHarg(Et2) – Leu – Harg(Et2) – Pro – DAlaNH2

A-75998 NACD2Nal – D4ClPhe – D3Pal – Ser – NMeTyr – DLys(Nic) – Leu – Lys(Isp) – Pro – DAlaNH2

Azaline B NACD2Nal – D4ClPhe – D3Pal – Ser – Aph(atz) – DAph(atz) – Leu – Lys(Isp) – Pro – DAlaNH2

Antarelix NACD2Nal – D4ClPhe – D3Pal – Ser – Tyr – DHcit – Leu – Lys(Isp) – Pro – DAlaNH2

Structure of GnRH antagonists

13

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.

14Ditkoff et al., 1991

Estradiol [pg/ml]

050

100150200250300350

-5 -4 -3 -2 -1 0 1

LH [mU/ml]

0

20

40

60

80

100

-5 -4 -3 -2 -1 0 1

Follicular diameters [mm]

14

16

18

20

22

-5 -4 -3 -2 -1 0 1

FSH [mU/ml]

0

5

10

15

20

25

30

-5 -4 -3 -2 -1 0 1

Days relative to ovulationcontrolNal-Glu cycles

Antagonists in controlled ovarian stimulation - the first steps

15

Cetrotide®

NACD2Nal – D4ClPhe – D3Pal – Ser – Tyr – DCit – Leu – Arg – Pro – DAlaNH2

Cetrorelix

16

Dose finding studies to identify the minimal effective dose in the multiple dose and single dose protocol

17

The development of the multiple dose antagonist protocol

20 patients in IVF cycles

gonadotropins were started on cycle day 2

Cetrorelix in a daily dosage of either 3 mg or 1 mg started on day 7 of stimulation

no spontaneous LH surge was observed

Diedrich et al., 1994

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dose finding study using Cetrorelix in a daily dosage of 3 mg, 1 mg, and 0.5 mg

Felberbaum et al., 1996

dose 3 mg 1 mg 0.5 mgpatients (n) 12 12 11mean number of gonadotropin ampoules 30 27 26estradiol level on day of hCG (pg/ml) 852.25 325.19 1022.50 602.86 2164.91 2102.93

oocytes (n) 106 94 127fertilization rate (%) 45.3 53.2 67.7embryos per transfer (n) 30 28 27

no premature LH surge

19


dose finding study using Cetrorelix in a daily dosage of 0.5 mg, 0.25 mg, and 0.1 mg

premature LH surge occured in the 0.1 mg group

Albano et al., 1997

dose 0.5 mg 0.25 mg patients (n) 32 30 gonadotropin ampoules (mean SD) 35.1 11.8 33.4 8.1 estradiol level on day of hCG (pg/ml) 2122 935 2491 819 oocytes (n) 396 487 fertilization rate (%) 69.52 72.94 clinical pregnancy rate per embryo transfer 30.0 31.0

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Cetrotide® 0.25 mg is the minimal effective dose in the multiple dose antagonist protocol

21

The development of the single dose antagonist protocol

11 patients for IVF

first injection of 3 mg Cetrorelix always on day 8 of the cycle

second injection, if no hCG injection latest 72 hours later possible

3 patients received a second injection of Cetrorelix

these 3 patients had low estradiol levels on day of first Cetrorelix administration

Olivennes et al., 1995

22

The development of the single dose antagonist protocol

Cetrotide® 3 mg is the minimal effective dose in the

single dose antagonist protocol

23

Dose Finding Studies

With Ganirelix :

2 mg, 1 mg, 0.5 mg, 0.25 mg, 0.125 mg and 0.0625 mg were used

0.25 mg daily was the preferred dosage

With Ganirelix, increasing dosage related with drop in pregnancy rate and increase in abortion rate.

2 mg daily dosage slowed follicular growth as well as almost stopping ay increase in estradiol secretion.

The Ganirelix dose-finding Study Group, Hum Reprod 1998;13:3023-31

24

Comparison of the long protocol and the antagonist protocols

agonist administrationagonist administration

gonadotropin administrationgonadotropin administration

long protocol

antagonist administrationantagonist administration

gonadotropin administrationgonadotropin administration

multiple dose protocol

flare upeffect

pituitarysuppression

no cystformation

no hormonalwithdrawal

longertreatment

less gona-dotropins

earlypregnancy?

morephysiologic

pre-treatment cycle treatment cycle

25

Comparison of antagonist protocols and the long luteal protocol

Prospective, randomized trials

26

The multiple dose antagonist protocol compared to the long luteal protocol

Prospective, randomized phase III study

7 European centres

273 patients for IVF or IVF/ICSI

Stimulation procedures long luteal protocol: buserelin nasal spray (4 x 150µg) multiple dose antagonist protocol: Cetrotide® 0.25 mg start with 150 IU FSH per day in the antagonist and

agonist group

Albano et al., 2000

27


Albano et al., 2000

inclusion criteria age: 18 - 39 years normal menstrual cycle (range: 24 - 35 days) with an intraindividual

variation of max. ± 3 days no more than 3 IVF procedures normal uterus and at least one functioning ovary

exclusion criteria severe endometriosis (AFS III/IV) PCO syndrome

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Cetrotide® Buserelin pnumber of patients 188 85 -age (years) 31.9 3.7 31.6 3.8 n.s.days of analogue treatment 5.7 2.3 26.6 3.2 < 0.001number of patients who got hCG (%) 181 (96.3) 77 (90.6) n.s.number of gonadotropin ampoules 23.6 8.5 25.6 7.6 < 0.01days of gonadotropin treatment 10.6 2.3 11.4 1.8 < 0.01estradiol on day of hCG (pg/ml) 1625 836 2082 1049 < 0.01


Albano et al., 2000

n.s. not significant

29

Cetrotide® Buserelin pno. of patients 188 85 -no. of patients with pick-up 178 77 -no. of cumulus oocyte complexes perpatient 8.0 4.9 10.6 6.6 < 0.01no. of 2 PN oocytes per patient 4.5 3.3 6.0 4.1 = 0.01no. of cleaved embryos (% of 2 PN) 671 (89.5) 345 (79.9) -- excellent (n, % of all) 235 (35.0) 94 (28.1) -- good (n, % of all) 321 (47.8) 154 (44.6) -- fair (n, % of all) 115 (83.5) 67 (78.8) -

no. of embryos per transfer 2.2 0.6 2.2 0.6 n.s.


Albano et al., 2000


30

Cetrotide® Buserelin pno. of embryo transfers (% cycles) 157 (83.5) 67 (78.8) n.s.no. of clinical pregnancies (% cycles) 42 (22.3) 22 (25.9) n.s.no. of miscarriages 7 2 -no. of ectopic pregnancies 1 0 -no. of deliveries (% cycles) 34 (18.1) 19 (22.4) n.s.no. of children born (% embryosreplaced) 42 (12.2) 21 (14.3) -


Albano et al., 2000


31

Cetrotide® Buserelincumulus oocytes complexes per patient 8.0 4.9 10.6 66

estradiol on day of hCG (pg/ml) 1625 836 2082 1049

embrys transferred per patient 2.2 0.6 2.2 0.6

hospitalized OHSS °I & °II (%)* 2 (1.1) 5 (6.5)- II° 2 4- III° - 1

clinical pregnancies per cycle (%) 42 (22.3) 22 (25.9)

The multiple dose antagonist protocol compared to the long luteal protocol:

significant reduction of OHSS

Ludwig et al., 2000

* p = 0.03, Fishers exact test, relative risk: 6.2 (95% CI: 1.4 - 27.1)

32


Ludwig et al., 2000

Significantly less OHSS °II and °III(RR 6.2, 95% CI: 1.4 - 27.1, p = 0.03)Less patients with threatened OHSS (no hCG - administration when 12 follicles 15 mm and/or estradiol 4.000 pg/ml)

Cetrotide® 0.25: 3 patients (1.6%)buserelin: 5 patients (5.9%)

One more patient in the buserelin group did not have an embryo transfer because of a threatened OHSS

33


Ganirelix vs. Buserelin

Ganirelix Buserelin

No. of patients 463 237

No. of patients who reached the day of hCG

448 224

No. of patients with oocyte pick-up

440 221

No. of patients with embryo transfer

399 208

Cancellation rate (%) 13.8 12.6

34


Ganirelix vs. Buserelin

Ganirelix Buserelin

LH rises during treatment (%)

2.8 1.3

Days of analogue treatment

5 26

Fertilization rate (%) 62.1 62.1

Clinical pregnancy rate per embryo transfer (%)

25.1 31.7

Overall incidence of OHSS (%)

2.4 5.9

35

Cetrorelix Triptorelin depot

No. of patients 115 39

hCG administered (%) 98.3 92.3

Patients with OPU (%) 98.3 92.3

Patients with embryo transfer (%)

86.1 84.6

Incidence of LH surges 2.6 2.6

Days of stimulation 9.4 10.7


Cetrorelix vs. Triptorelin depot

36

Cetrorelix Triptorelin depot

No. of hMG ampules 24.3 35.6

E2 (pg/ml) on the day of hCG

1786 + 808 2549 + 1194

COC per patient 9.2 10.7

Fertilization rate (%) 50.5 54.7

Clinical pregnancy rateper embryo transfer (%)

21.2 27.3

Babies born per embryos replaced (%)

10.6 13.3

OHSS grades II-III (%) 1.8 5.6


Cetrorelix vs. Triptorelin depot

37

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: 1.4 - 27.1, p = 0.03

Single dose protocol rate of OHSS: 11.1% vs. 3.5% (agonist vs. antagonist protocol)

95% CI: - 18.4 to 3.2 patients requiring hospitalisation: 5.6% vs. 1.8% (agonist vs.

antagonist protocol)95% CI: - 11.7 to 4.1

With both Cetrotide® protocols a clear reduction of OHSS was be achieved

38

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

39

The Cetrotide® 0.25 mg multiple dose protocol

1st dayof gonado-

tropins

gonadotropin administrationin an individualized dosage

ovulationinduction

oocytepick up

embryotransfer

luteal phase support

1st dayof menstruation

Cetrotide® 0.25 mg administrationdaily s.c. starting on day 6 of stimulation

40

Results

Check the stimulation day 7th LH level

LH > 1.5 mIU/ml, 0.25 mg daily was given

LH < 1.5 mIU/ml, reduce to 0.125 mg daily

Switching to a half dosage of 0.125 ml per day gives:

Normal LH levels

Expected follicular growth

Better ovum quality

No premature LH surge or progesterone rise

Documents

Dr. Milton Leong Director IVF CENTRE Hong Kong Sanatorium & Hospital