Gonal-f Pen - Pharmaceutical Information, Clinical Trials, Detailed Pharmacology, Toxicology.
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Gonal-f Pen - Scientific Information

Manufacture: EMD Serono, Inc
Country: Canada
Condition: Follicle Stimulation, Hypogonadism, Male, Ovulation Induction
Class: Gonadotropins
Form: Liquid solution, Subcutaneous (SC)
Ingredients: follitropin alfa (r-hFSH), sucrose, meta-cresol, di-sodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate monohydrate, methionine, Poloxamer 188, O-phosphoric acid, sodium hydroxide

Pharmaceutical Information

Drug Substance

Proper name Follitropin alpha (approved INN)
Chemical name recombinant human Follicle Stimulating Hormone for Injection (r hFSH)
Molecular Weight α-subunit: 14 Kda β-subunit: 17 Kda
Molecular Formula C975H1515N267O305S26
Structural Formula Amino acid sequence of follitropin alfa:
Alpha subunit:
Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser 19
Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro 38
Thr Pro Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu Ser 57
Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val 76
Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser 92
Beta subunit:
Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu Glu Cys Arg Phe 19
Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys Tyr Thr Arg Asp Leu Val 38
Tyr Lys Asp Pro Ala Arg Pro Lys Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val 57
Tyr Glu Thr Val Arg Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr 76
Pro Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr 95
Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu 111
Relevant Physicochemical Properties: r-hFSH consists of two non-covalently linked, non-identical protein components designated as the α- and β-subunits. The α-subunit is composed of 92 amino acids carrying two carbohydrate moieties linked to Asn-52 and Asn-78. The β-subunit is composed of 111 amino acids carrying two carbohydrate moieties linked to Asn-7 and Asn-24. r-hFSH is derived from a Chinese Hamster Ovary cell line which has been modified by the addition of the human genes encoding the FSH α- and β-chains.
pH 6-8

Asn: N-glycosylation sites

Clinical Trials

Controlled Ovarian Hyperstimulation in Assisted Reproductive Technologies

In vitro Fertilization

Two-phase III studies were performed to compare the clinical efficacy and safety of GONAL-f (follitropin alpha for injection) 75 IU with that of Metrodin HP 75 IU (uhFSH-HP). The two studies had a similar prospective randomized design.

Study demographics and trial design
Table 1: Summary of patient demographics for clinical trials in ART
Study # Trial design Dosage, route of
administration
and duration
Study
subjects
(n=number
randomized)
Mean age (SD) Gender
Bergh (Study 8237) Prospective, assessor-blind, randomized, parallel-group, multicentre study. 150 IU/day for the first 6 days. Dose adjustments on Day 7 and 9 based on ovarian response 235 32.0 ± 3.5 (r-hFSH)

31.2 ± 5.3 (Metrodin HP )
Female
Frydman (Study 8407) Prospective, double blind, randomized, parallel group, multicentre study 150 IU/day for the first 6 days. Dose adjustments on Day 7 and 9 based on ovarian response 278 31.4 ± 3.5 (r-hFSH)

31.2 ± 4.0 (Metrodin HP)
Female

Patients enrolled in both trials were between 18 and 38 years old with a mean age around 32, with regular menstrual cycles of 25–35 days; no ecographic signs of PCO disease, have an infertility due to any of the following factors: tubal factor, mild endometriosis, male factor or unexplained factor; no more than three previous assisted reproduction attempts; presence of both ovaries and a normal uterine cavity; a BMI up to 28 kg/m 2 , no history of either OHSS or poor response to gonadotropin therapy, no azoospermia or clinical signs of infection detected in a semen analysis within the previous 12 months.

Study results
Table 2: Results of study 8237 (Bergh) in Controlled ovarian hyperstimulation prior to ART
Primary Endpoint GONAL-f 75 IU Metrodin HP 75 IU p-value
Oocytes retrieved (mean ± SD) 12.2 ± 5.5 7.6 ± 4.4 <0.001

Two hundred and thirty seven patients were recruited in the study and received the down regulation, 119 of them were randomized in the GONAL-f group and 118 in the Metrodin HP group. These patients were included in the analysis of demography and in the analysis of safety.

Two hundred and thirty-five patients received stimulation, 119 of them were in the GONAL-f group and 116 in the Metrodin HP group.

Two hundred and twenty- one patients received hCG and so were included in the analysis of efficacy. One hundred and nineteen (100 %) of them were in the GONAL-f treatment group and 102 (87 %) were in the Metrodin HP treatment group.

The mean number of oocytes recovered (primary endpoint) was higher in the r-hFSH compared to the u-hFSH- HP group (12.2 vs. 7.6 respectively; p < 0.001). On the other hand, the number of FSH treatment days (11.0 vs. 13.5) and the number of 75 IU ampoules (21.9 vs. 31.9) used were significantly less (p < 0.001) in the r-hFSH group than in the u-hFSH-HP group. The mean numbers of embryos obtained were 8.1 vs. 4.7 (p < 0. 001) in favor of the r-hFSH group.

The clinical pregnancy rates per started cycle and per embryo transfer were 45 and 35%, and 48 and 46%, respectively in the r-hFSH and u-hFSH-HP groups (not significant).

Table 3: Results on stimulation and gamete characteristics (Study 8237)
Parameter GONAL-f u-hFSH HP p-value
N patients randomized 119 116
N of patients receiving hCG 119 102
N of days FSH treatment 11.0 ± 1.6 13.5 ± 3.7 <0.001
N of ampoules FSH (75 IU equiv.) 21.9 ± 5.1 31.9 ± 13.4 <0.001
Follicles >10 mm day of hCG 12.7 ± 4.9 8.4 ± 4.2 0.002
E2 (nmol/l) day of hCG 6.55 ± 5.75 3.95 ± 3.90 <0.001
Oocytes retrieved 12.2 ± 5.5 7.6 ± 4.4 <0.001
N Oocyte nuclear maturity N (%) 634 (83) 323 (79) ns
N of cleaved embryos on day 2 8.1 ± 4.2 4.7 ± 3.5 <0.001
N of embryos cryopreserved 3.2 ± 3.0 1.7 ± 2.5 <0.001
N of Patients with embryo transfer (%) 111/119 (93.3) 89/116 (76.7)
Table 4: Results of study 8407 (Frydman) in Controlled ovarian hyperstimulation prior to ART
Primary Endpoint GONAL-f 75 IU Metrodin HP 75 IU p-value
Oocytes retrieved
(mean ± SD)
11.0 ± 5.9 8.8 ± 4.8 0.044

Two hundred and seventy-eight (278) patients were enrolled in this study: 139 were randomized in the GONAL-f group and 139 in the Metrodin HP group. The efficacy analysis of this study was performed on all randomized patients who received hCG (246): 130 in the GONAL-f group and 116 in the Metrodin HP group, apart from the pregnancy criteria which were analysed successively in the Starting FSH Patient population, the population of patients where oocyte recovery was carried out and the population of patients having had embryo transfer.

The mean number of oocytes (primary endpoint) retrieved was higher in the r-hFSH group (11.0 ± 5.9 vs. 8.8 ± 4.8; p = 0.044). There were also significant differences between the two groups in several of the secondary efficacy parameters, mainly fewer days of FSH stimulation were required with r-hFSH to reach the criteria for triggering follicle maturation than with u-hFSH HP (11.7 ± 1.9 versus 14.5 ± 3.3 respectively; p < 0.001). Correspondingly, the total dose of FSH required to reach these criteria was lower for r-hFSH than u-hFSH HP (27.6 ± 10.2 x 75 IU ampoules, compared with 40.7 ± 13.6; p < 0. 001). Only 56.2% of patients in the r-hFSH group required an increase in dosage after the first 6 days of treatment, compared with 85.3% of those receiving u-hFSH HP (p = 0.001).

The initial pregnancy rate (excluding biochemical) per started cycle was 32/139 (23.0%) for r-hFSH and 38/139 (27.3%) for u-hFSH HP (not significant).

Table 5: Results on stimulation and gamete characteristics (Study 8407)
Parameter Gonal-F u-hFSH HP p-value
No. of patients starting FSH 139 139
No. of patients receiving hCG 130 116
No. of days FSH treatment 11.7 ± 1.9 14.5 ± 3.3 <0.001
No. of ampoules FSH (75 IU equiv.) 27.6 ± 10.2 40.7 ± 13.6 <0.001
Follicles > 12 mm day of hCG 12.1 ± 5.2 10.5 ± 4.6 0.004
Oocytes retrieved 11.0 ± 5.9 8.8 ± 4.8 0.044
Oocyte nuclear maturity B MII No. 8.1 ± 4.4 6.3 ± 3.5 0.001
No. of embryos obtained 5.0 ± 3.7 3.5 ± 2.9 <0.001
No. of patients with embryo transfers/hCG received 116 (89%) 98 (84%)
No. of embryos transferred/cryopreserved 3.5 ± 2.8 2.6 ± 2.2 0.009
No. ongoing clinical pregnancies/cycle started 25 (18%) 25 (18%) NS
% multiple pregnancies 9 7 NS

Both studies confirm that r-hFSH is more effective than u-hFSH HP in inducing ovulation in women undergoing assisted reproductive treatment. Patients given the recombinant product required fewer days of treatment and a lower total dose of FSH to reach the criteria for hCG administration than those receiving u-hFSH HP.

Ovulation induction

Study demographics and trial design
Table 6: Summary of patient demographics for clinical trials in OI
Study # Trial design Dosage, route of
administration and
duration
Study
subjects
(n=number
randomized)
Mean age (SD) Gender
Study 5642 Phase III, open label, randomized, comparative, parallel group, multicenter 75 IU as starting dose on day 3-5. Dose adjustments every 7 days by dose of 37.5 IU every 7 days not before than day 14 222 29.3 +/- 3.2 (r-hFSH)

29.1+ 3.6 (Metrodin HP )
female
Study 5727 Phase III, open label, randomized, comparative, parallel group, multicenter 75 IU as starting dose on day 3-5. Dose adjustments every 7 days by dose of 37.5 IU every 7 days not before than day 14 232 29.3 +/- 3.7 (r-hFSH)29.3 +/- 3.7 (r-hFSH)

30.0 + 3.4 (Metrodin HP )
female

Both studies recruited the same population: women between 18 and 39 years old; ovulatory dysfunction: either anovulation or oligoovulation who had failed to ovulate or conceive when treated for at least 3 months with clomiphene citrate; BMI less than 35.

Study results
Table 7: Results of study 5642 in OI
Primary Endpoints Associated value and
statistical significance for
GONAL-f at specific
dosages
Associated value for Metrodin
(u-HP)
Cumulative ovulation rate 88% (P=0.071) 95%

The primary efficacy parameter was the ovulation rate. Two hundred and twenty-two patients entered into the first cycle of treatment, of whom 110 received GONAL-f and 112 received Metrodin HP. In study GF 5642, the cumulative ovulation rate was 84% and 91% in the GONAL-f and Metrodin HP treatment groups respectively. This difference was not statistically significant. The 95% confidence interval of the difference in the cumulative ovulation rate between the Metrodin HP and GONAL-f treatment groups was [-1.3%; 16.17%] which is less than the 20% difference defined as the limit of the clinically acceptable difference between the two treatment groups. Seventy-five patients delivered at least one baby during this study, 31 (28%) in the GONAL- f treatment group and 44 (39%) in the Metrodin HP treatment group. No statistically significant difference was recorded between the two treatment groups. The overall multiple pregnancy rate was 6% and 14% in the GONAL-f and Metrodin HP treatment group, respectively.

Table 8: Results of study 5727 in OI
Primary Endpoints Associated value and statistical
significance for GONAL-f at
specific dosages
Associated value and statistical
significance for Metrodin (u-HP)
Cumulative ovulation rate 88% (ns) 95% (ns)

The second study showed similar results in terms of efficacy endpoint. The patient ovulation rate in patients with known ovulation outcome was 88% in the GONAL-f treatment group and 95% in the Metrodin HP treatment group. This difference is not statistically significant. The one-sided confidence interval for the difference in ovulation rates in the GONAL-f and Metrodin HP treatment groups was (-12.8%, ∞ ), confirming that the ovulation rate for patients treated with GONAL-f is as good as and equivalent to the ovulation rate of those treated with Metrodin HP because the absolute value of the difference in ovulation rates is less than the specified 20%.

Sixty-six patients delivered at least one baby during this study, 34 (29%) in the GONAL-f treatment group and 32 (28%) in the Metrodin HP treatment group. No statistically significant difference was recorded between the two treatment groups. The overall multiple pregnancy rate was 5% and 4% in the GONAL-f and Metrodin HP treatment group, respectively.

Comparative Bioavailability Studies

Study IMP23572

Study Demographics and Design
Table 9: Summary of patient demographics for clinical study 23572
Study Trial design Dosage, route of
administration and
duration
Study
subjects
(n=number
randomized)
Mean age (SD) Gender
Study 23572 Phase I, open-label, cross-over. 2 single injections of 300 IU r-hFSH (either f.d. or liquid) s.c. at 7 days interval 39 18-45 years Female and Male
Study Results

The bioequivalence of the new liquid formulation has been assessed versus a freeze dried formulation. Mean values of Cmax and AUClast were very similar for both formulation and mean values of Tmax were also similar- The 90 % confidence intervals of the ratios of AUClast and Cmax were within the pre-defined range of 0.8 to 1.25.

Table 10: Summary Table of Comparative Bioavailability Parameters
r-hFSH
(Multidose vs Monodose Freeze-dried)
From measured data
Geometric Mean
Arithmetic Mean (CV %)
Parameter r-hFSH Multidose Liquid

(Test)
r-hFSH Monodose Freeze-dried

(Reference)
% Ratio of Geometric Means

(following correction for potency)
90% Confidence Interval

(following correction for potency)
AUCT
(IU·h/L)
487.8
499.9 (22.3)
544.6
552.8 (18.6)
104% 99% – 111%
AUCI
(IU·h/L)
544.6
564.1 (25.8)
614.0
634.0 (24.4)
Not evaluated Not evaluated
CMAX
(IU/L)
6.7
6.9 (25.3)
7.5
7.7 (24.0)
105% 100% - 109%
TMAX
(h)
15 (6 – 48) 12 (4 – 48)
T½
(h)
36.1 (56.8) 37.5 (60.9)

The tolerability of multi-dose liquid GONAL-f was compared to a reference GONAL-f mono-dose freeze-dried formulation in pituitary gonadotrope down-regulated male and pre-menopausal female volunteers. The liquid formulation was well tolerated, and no serious or severe adverse events were reported. Eleven (11) adverse events were reported with the liquid multi-dose formulation (11/41 = 26.8%) versus 15 with the freeze-dried formulation (15/40 = 32.5 %). The most frequently reported adverse event was headache for both formulations.

Detailed Pharmacology

Animal

Pharmacodynamics

To confirm that r-hFSH possessed the well -characterized pharmacodynamic activity of human FSH, both in vivo and in vitro studies were conducted. The in vivo studies, involving rats and monkeys, compared r-hFSH with two u-hFSH products (Metrodin7 and Fertinorm7 HP). The in vitro studies compared r-hFSH with international human pituitary FSH reference standards as well as with the two u-hFSH products.

In vitro

In an in vitro study using calf testes membrane, r-hFSH had the same binding characteristics to the testicular FSH receptor as did reference standards of human pituitary FSH (US NIADDK preparation h-FSH-1-3 and WHO 1st International Standard 83/575) and the two u-hFSH preparations. The binding affinities were very similar for all of the products, and the binding curves were superimposable.

The bioactivity of r-hFSH was indistinguishable from that of the two u-hFSH preparations and from pituitary and urinary FSH reference standards in a bioassay that measures FSH bioactivity according to estradiol production in isolated ovarian granulosa cells.

In vivo

Pharmacological studies in vivo included a comparison of the quantitative dose-response curves of r-hFSH and two clinical preparations of u-hFSH for oocyte formation, and ovarian weight gain (also in comparison with hMG) in young female rats using the Steelman-Pohley ovarian weight gain assay. The activities of the preparations were identical. r-hFSH also produced follicular maturation in mature cynomolgus monkeys similar to that produced with u-FSH.

Pharmacokinetics

In the rat single and repeated dose ADME studies were performed with 125 I-labelled r-hFSH administered subcutaneously (SC) and intravenously (IV). The bioavailability of the SC dose appeared similar to that after IV administration, but elimination was slower. Radioactivity distributed in high concentrations to the thyroid gland, gastrointestinal tract, kidneys and ovaries. Radioactivity distributed to the fetus as to a non-target tissue, with concentrations less than those in maternal plasma, and radioactivity was also found in the milk of lactating female rats. Intact 125 I-r-hFSH was present in plasma for up to 24 hours after dosing but only a series of smaller radioactive products, probably peptide fragments, were found in the urine. The kidneys appeared to be the primary route of excretion. There were no important differences between males and females as well as between non-gravid and gravid females.

In a single dose study in monkeys, r-hFSH was shown to behave similarly to native human urinary FSH (u-hFSH) after IV administration. Both preparations followed a two-compartment model with nearly identical distribution and terminal half-lives (approximately 1.5 and 15 hours, respectively). Their volumes of distribution and total clearance were only slightly different and biologically insignificant. The pharmacokinetic parameters of r-hFSH were also similar when administered as a single dose by the intramuscular (IM) and subcutaneous (SC) routes, and their absolute bioavailability was about 75%.

After repeated IM and SC administration of r-hFSH to monkeys, the pharmacokinetics was similar to that observed after a single dose. Steady state was reached after 2 to 3 days of treatment with an accumulation factor of about two between the first and last dose by both routes of administration.

Monitoring of serum FSH concentrations in the multidose toxicity studies demonstrated extensive exposure in monkeys and dogs, indicating that the studies were a valid test of the effects of r-hFSH. The similar bioavailabilities of r-hFSH observed in the monkey and humans would also indicate that animal results can be extrapolated to humans.

Human

Pharmacodynamics

An evaluation of the dynamics of r-hFSH was performed in 12 pituitary down-regulated healthy female volunteers. The study was divided into two parts. In part I, r-hFSH was administered in a balanced, random order, cross-over sequence as a single-dose of 150 IU on three occasions: IV, IM, and SC, each separated by a one week washout period. In Part II, each subject received a daily SC dose of 150 IU r-hFSH over seven days. The pharmacodynamics of r-hFSH were assessed by measuring daily plasma estradiol concentrations, serum immunoreactive inhibin and follicular growth by ultrasound of the ovaries - before, during, and after the seven days of treatment in Part II of the study. In this study, it was not intended to induce full follicular development and ovulation, but rather to document individual ovarian response to this predetermined fixed dose and duration of FSH treatment.

Mean serum FSH levels reached a steady state after 3-5 days. The first pharmacodynamic marker of ovarian response to FSH was serum inhibin, followed by plasma estradiol, then follicular growth (measured by total volume of follicles > 10 mm diameter). When FSH administration was stopped, inhibin levels dropped while estradiol continued to rise for one day. Follicle size continued to increase over the next four days. When individual responses were analyzed, it was noted the two thirds of the volunteers developed significant follicular growth, inhibin and estradiol secretion. No correlation was found between maximal concentration of r-hFSH and the maximal effects observed. This data indicates that the interindividual variability observed in the ovarian response to FSH therapy is not related to the pharmacokinetics of FSH, but reflects different levels of ovarian sensitivity to FSH.

Pharmacokinetics

In addition to the kinetic evaluation performed in Part I of the study summarized above under Pharmacodynamics , one other kinetic study was performed. This study also used 12 pituitary down-regulated, healthy female volunteers, and was a randomized, cross -over study to compare the pharmacokinetics of 150 IU Metrodin (urofollitropin for injection), 150 IU Fertinorm HP (highly purified Follicle Stimulating Hormone) and 150 IU and 300 IU of r-hFSH given intravenously.

The mean serum FSH concentration-time profiles after a single 150 dose IV of Metrodin, Fertinorm HP, and r-hFSH were superimposable, and the mean profile after a single 300 IU dose of r-hFSH was double that of the 150 IU dose. Total clearance of the preparations was comparable. Based on the immunoassay results, the clearance of u-hFSH was 0.1 L/h while for r-hFSH it was 0.07 L/h, indicating that less than one-fifth of the administered dose was excreted in the urine. Immunoassay data showed that the FSH preparations had similar initial (2 hours) and terminal (17 hours) half-lives. The volumes of distribution at steady state (11 L) were also similar.

Comparison of various routes of administration for r-hFSH demonstrated that two thirds of the administered dose was available systemically after IM or SC injection, and the absolute bioavailability was about 70% when assessed by immunoassay. The accumulation factor for repeated SC administration was around three when steady state was reached.

Toxicology

Acute Toxicity

The single dose toxicity studies performed with r-hFSH have been summarized below in Table 11.

Table 11: Acute Toxicity Studies
Species (Strain) #/Sex/Group Route Dose (IU/kg) LD50 (IU/kg)
Rat (Sprague Dawley) 15 SC 1000, 2000, 4000 > 4000
15 IM 1000, 2000, 4000 > 4000
20 IV 0, 1000, 2000, 4000 > 4000
10 PO 10000 -
Dog (Beagle) 1 IV 2000 > 2000
Monkey (cynomolgus) 1 SC 2000, 4000 > 4000
1 IM 2000, 4000 > 4000
1 IV 2000, 4000 > 4000

*1000 IU = 73.3 µg  2000 IU = 146.7 µg  4000 IU = 293.3 µg

There were no signs of local or systemic toxicity in any of the rat studies. In the dog study, there were no overt signs of toxicity following a single intravenous dose of 2000 IU/kg r-hFSH although there was evidence of transient liver toxicity following the administration of the control agent, u-hFSH. In the monkey studies, ovarian and endometrial changes were observed, but these findings are considered to be related to the pharmacological action of the compound.

Repeated Dose Toxicity

A total of seven repeated dose toxicity studies were performed with r-hFSH. These studies have been summarized below in Table 12.

Table 12: Repeated Dose Toxicity Studies with r-hFSH
Species (Strain) #/Sex /Group Route Dose (IU/kg/day) Duration
(weeks)
Recovery
Period
Rat
(Sprague Dawley)
18 SC r-hFSH: 0, 10, 30, 100
u-hFSH: 100
4 4
15 SC r-hFSH: 0, 300, 1000
hMG: 1000
4 2
18 SC r-hFSH: 0, 10, 100, 1000 13 4
Dog
(Beagle)
2 IV r-hFSH: 0, 20, 100
u-hFSH: 20, 100
u-hFSH HP: 20, 100
4 0
Monkey
(Cynomolgus)
3 in the 10 and 30 IU/kg/day groups; 5 in all others IM r-hFSH: 0, 10, 30, 100
u-hFSH: 100
4 4
5 IM r-hFSH: 0, 300, 1000
hMG: 1000
4 2
3 in the 10 and 100 IU/ kg/ day groups; 5 in all others IM r-hFSH: 0, 10, 100, 1000 13 4

* 10 IU = 0.7 µg  20 IU = 1.5 µg  100 IU = 7.3 µg  1000 IU = 73.3 µg

In rats, no treatment related mortalities or clinical signs of intolerance were observed. In the 4 week study at moderate doses, r-hFSH exerted a stimulatory effect on female gonads as evidenced by a number of morphological changes involving the ovaries, genital tract and mammary glands seen mainly in females given 30 and 100 IU/kg/day. These were related to the pharmacological activity of the hormone injected at high doses over a long period of time. In the 4 week study at high doses and in the 13-week study, atrophy of the gonads and secondary sex organs, predominantly in females, were seen. These findings could be related to inhibin production as well as to the inactivation of exogenous FSH by the high levels of antibodies to FSH which were found to have developed in the rats within four weeks of treatment as a consequence of the injection of a foreign protein. This is also in agreement with the fact that serum FSH levels decreased as treatment continued.

In the dog, the serum FSH levels measured during dosing confirmed extensive exposure thus the study was a valid test of the effects of r-hFSH. No mortalities were observed following administration of 20 or 100 IU/kg/day for 4 weeks. Body weight was unaffected by treatment and no signs of systemic toxicity or local reactions at the injection sites were seen. The predominant changes were related to the pharmacological activity of the compound on the reproductive systems of both the male and female animals. At the higher dose of 100 IU/kg/day, slight acute inflammation of the liver was also found in one of the treated females.

In the monkey, as in the dog, the serum FSH levels measured during dosing confirmed extensive exposure despite the development of antibodies to the foreign protein. The general similarity of the toxicological findings, which represent the pharmacological actions of high doses of FSH, in the shorter and longer term experiments demonstrate that some hormonal activity of FSH was still maintained.

No mortalities were observed in any of the monkey studies. Body weight was unaffected by treatment with r-hFSH; the predominant effects were those anticipated from the known pharmacodynamic actions of FSH. Despite the development of anti -FSH antibodies, r-hFSH caused prominent and continued but reversible ovarian stimulation resulting in cysts, some haemorrhagic, endometrial hyperplasia, even some proliferation of mammary glandular cells, and changes in the vagina. Hypertrophy of pituitary acidophilic cells was seen in females receiving r-hFSH at doses of 30 to 1000 IU/kg/day and thymus atrophy was seen at doses of 300 and 1000 IU/kg/day. At a dose of 100 IU/kg/day, r-hFSH caused some stimulation of testicular tubules and a possible increase in spermatogenesis. Slight acute inflammation of the liver was also found in one of the females given r-hFSH at a dose of 100 IU/kg/day.

In the various 4 week experiments, the hormonal actions were largely reversible, but the anatomical changes had progressed so far in the 13 week studies that complete recovery was not possible after the high doses (100 to 1000 IU/kg/day).

In the 4 week studies, it was not possible to determine a definite difference in potency between the SC and IM routes. The 'No observed (pharmacological) effect level' was probably about 10 IU/kg/day in the dog (dosed IV) and was less than 10 IU/kg/day in the monkey (IM) and rat (SC).

No conventional target organ toxicity was found in any species, apart from a possible low incidence of centrilobular inflammation in the liver in dogs at the highest dose of r-hFSH and both of two clinical preparations of u-hFSH, and minor changes in neutrophils, platelets and PTT, also in the same groups. The latter effects cannot be distinguished from normal concomitants of stimulated oestrus.

The findings in these experiments did not show any major difference between recombinant and natural urinary human FSH. Differences from the effects of hMG could be attributed to the LH activity of the latter preparation.

Mutagenicity

r-hFSH did not show any mutagenic activity in the range of mutagenicity studies conducted.

Reproduction and Teratology

Reproductive toxicology studies were conducted to assess the possible effects of r-hFSH on reproduction. Segments I, II, and III were conducted in rats by the SC route as this is the proposed therapeutic route in man. Segment II was also conducted in rabbits as a non-rodent species. The doses of r-hFSH used for all the studies were 5, 40, and 320 IU/kg/day in comparison with hMG at the dose of 320 IU/kg/day. The highest dose of r-hFSH was about 100 times the clinical dose and was expected to produce profound reproductive effects.

Given in a sufficiently high dose, r-hFSH was able to cause death and other forms of fetal effects in the rat and rabbit, but without being a teratogen. It also caused dystocia. Human Menopausal Gonadotropin (standardized to the same follicle stimulating activity but also possessing luteinizing activity) had the same effects. r-hFSH, 5 IU/kg/day SC, had no effect in the rat, and 40 and 320 IU/kg/day had progressively more severe actions. In general terms r-hFSH 320 IU/kg/day had the same activity as hMG 320/kg/day. The retardation of weight gain found in the Segment II test in rats given high doses of r-hFSH and hMG can be attributed to the resorptions produced. The rabbit was more sensitive than the rat, as even 5 IU/kg/day of r-hFSH caused death of almost all embryos in utero.

In the fertility study in the rat, r-hFSH at 40 and 320 IU/kg/day and hMG (320 IU/kg/day) both impaired fertility. As both sexes had been dosed before mating it is not known whether both females and males were affected, although it appears likely that females were affected, judging by the changes observed in the ovaries and the known physiological effects of FSH on follicular development and function. No histological changes were seen in the testes, even at the highest r-hFSH dose of 320 IU/kg/day, in spite of a small decrease in weight.

Other Studies

Although the local tolerance of r-hFSH was assessed in the acute and multidose toxicity studies, in which it was well tolerated on SC, IM and IV injection, a sensitization test in guinea pigs and a local tolerance study in rabbits were also performed.

In guinea pigs, r-hFSH was a sensitizer in the maximization test, but to a lesser degree than u-hFSH. In rabbits, a concentration of 600 IU/mL, administered SC and IM, was well tolerated. The finding of sensitization in guinea pigs is not surprising as both r-hFSH and u-hFSH are foreign proteins to this animal species. The repeated dose toxicity studies clearly demonstrated the formation of antibodies to human FSH when administered to animals. The greater purity of r-hFSH is most likely responsible for the milder response observed. These findings are not considered to be clinically relevant.

Together with the much more extensive evidence from the repeated dose toxicity studies, it can be concluded that r-hFSH is well tolerated at the site of administration.