Description
Nafarelin is a gonadorelin analogue. It produces an initial phase of stimulation followed by down-regulation of gonadotrophin-releasing hormone receptors, thus reducing the release of FSH and LH, leading to inhibition of androgen and oestrogen production.
Chemical Properties
Nafarelin is a synthetic analogue of gonadotrophin-releasing hormone (GnRH), which is about 200 times more potent than the native hormone and is more resistant to proteolysis Chrisp and Goa (1990), Parker and Schimmer (2001). Like GnRH, it binds with high affinity to the GnRH receptor on anterior pituitary cells, where it acts as an agonist.
Originator
Nafarelin Acetate,Bachem AG
Manufacturing Process
In the reaction vessel of a Beckman 990 Peptide Synthesizer was placed 0.8 g
(0.8 mmol) of benzhydrylamino-polystyrene-divinylbenzene resin (Lab
Systems, Inc.) as described by Rivaille, supra. Amino acids were added
sequentially to this resin by means of the usual methods of Boc-strategy of
peptide synthesis on above copolymer.
The resin was coupled sequentially with a 2.5 molar excess of each protected amino acid and DCC. Thus, the resin was treated during successive coupling
cycles with 0.433 g Boc-Gly-OH, 0.432 g Boc-Pro-OH, 0.857 g Boc-Arg(Tosyl)-
OH, 0.462 g Boc-Leu-OH, 0,504 g Boc-3-(2-naphthyl)-D-alanine and 0.272 g
1-hydroxybenzotriazole, 0.724 g N-Boc, O-2-bromobenzoyloxycarbonyl-Ltyrosine, 0.59 g Boc-Ser(Benzyl)-OH, 0.608 g Boc-Trp-OH, 0.654 g BocHis(Tosyl)-OH and 0.524 g pyroglutamic acid. A coupling cycle for one amino
acid and completeness of the reaction is checked by the ninhydrin method of
E. Kaiser, et al., Anal. Biochem., 34, 595 (1970).
The resin was removed from the reaction vessel, washed with CH2Cl2, and
dried in vacuo to yield 2.0 g of protected polypeptide resin.
The polypeptide product was simultaneously removed from the resin and
completely deprotected by treatment with anhydrous liquid HF. A mixture of
2.0 g of protected polypeptide resin and 2 mL of anisole (scavenger) in a KelF reaction vessel was treated with 20 mL of redistilled (from CoF3) anhydrous
liquid HF at 0°C for 30 minutes. The HF was evaporated under vacuum and
the residue of (pyro)-Glu-His-Trp-Ser-Tyr-3-(2-naphthyl)-D-alanyl-Leu-ArgPro-Gly-NH2,as its HF salt, was washed with ether. The residue was then
extracted with glacial acetic acid. The acetic acid extract was lyophilized to
yield 0.8 g of crude material. The crude polypeptide was loaded on a 4x40
cm. Amberlite XAD-4 column (polystyrene-4% divinylbenzene copolymer) and
eluted with a concave gradient from water (0.5 L) to ethanol (1 L). The tubes
containing fractions from effluent volume 690 mL to 1,470 mL were pooled
and stripped to dryness to yield 490 mg of partially purified polypeptide.
A 150 mg sample of the partially purified product was subjected to partition
chromatography on a 3 times 50 cm. column of Sephadex G-25 using the
solvent system 1-butanol/toluene/acetic acid/water containing 1.5% pyridine
in the ratios 10:15:12:18. The pure fractions were pooled on the basis of thin
layer chromatography (silica gel; BuOH/H2O/HOAc/EtOAc; 1:1:1:1) and HPLC
(5 micron, reverse phase, octadecylsilyl packing; 40% 0.03 M NH4OAc/60%
acetonitrile). The desired product came off the column in fractions from
effluent volume 1,000 mL to 1,400 mL (Rf 0.1). The pure fractions were
pooled, stripped to dryness, taken up in H2O, and lyophilized to yield 57 mg of
pure pyro-glutamyl-histidyl-tryptophylseryl-tyrosyl-3-(2-naphthyl)-D-alanylleucyl-arginylprolyl-glycinamide, as its acetic acid addition salt, [α]D25-27.4°
(c 0.9, HOAc), m.p. 185°-193°C (dec.).
Brand name
Synarel (Searle).
Therapeutic Function
Gonadotropic
Biological Functions
Acutely, Nafarelin stimulates the secretion of LH and FSH from the anterior pituitary, but prolonged, continuous exposure leads to inhibition of secretion by desensitization of the pituitary gonadotropes Vickery (1985). The initial stimulation of gonadotrophin secretion results in an increase in serum concentrations of estradiol in females and testosterone in males. These then decrease to very low concentrations equivalent to those found in menopausal women and castrated men respectively.
Clinical Use
Nafarelin has potential wide therapeutic applications in the treatment of endometriosis, uterine leiomyoma (fibroids), malignant neoplasms, and in assisted reproduction protocols. However, its use is currently restricted to the treatment of endometriosis and in assisted reproduction protocols.
Side effects
Nafarelin does not have the same androgenic adverse effects as danazol, but it can cause menopausal symptoms. The most commonly reported symptoms are hot flushes, vaginal dryness, altered libido and headaches. Treatment for 6 months results in a loss of bone density. The vertebral trabecular bone density falls by nearly 9% and may not return to normal after treatment. Nafarelin also has a similar effect to oestrogen deficiency on blood lipids e.g. concentrations of total cholesterol and triglycerides may increase.
Synthesis
i. 1 mmol from preparation A was placed in the reaction vessel of a 5 L Vega 296 automatic solid phase peptide synthesizer.
ii. Following amino acids were added to the Preparation A resin:
Nα-Boc-Pro 2 equiv.
Nα-Boc-Arg 2 equiv.
Nα-Boc-Leu 2 equiv.
Nα-Boc-D-NaI 1.5 equiv.
Nα-Boc-Tyr 1.5 quiv
Nα-Boc-Ser(tBu) 2 equiv.
Nα-Boc-Trp 1.75 equiv.
Nα-Boc-His(tos) 1.75 equiv.
(pyro)Glu 2.5 equiv.
iii. Crude peptide was dissolved in 2M acetic acid and converted to acetate salt by passage through a column of AG3-X4A resin.
iv. Acetate was dissolved in minimal amount of methanol.
v. It was then reprecipitated using acetone.
vi. It was then purified using HPLC to get the Nafarelin.
