GnRH analogues--agonists and antagonists

GnRH analogues have achieved widespread clinical use for the control of reproduction in animals. Over 2000 analogues of GnRH have been developed and tested over the last 30 years. Paradoxical anti-fertility effects are seen when the more potent agonists are delivered continuously to animals. The evaluation of agonist potency depends largely on the model used and wide varying potencies are reported for the same agonist. The design of analogues has centered on improving the receptor-binding and subsequent activation for agonists. Antagonists have been produced with strong receptor binding but without activation. Deslorelin is classified as a superagonist, with a potency perhaps 100 times that of GnRH. The interactions between agonist potency, dose and duration of treatment largely determine whether pro- or anti-fertility effects are induced. Due to the peptide nature of the synthetic analogues oral administration and potential gastrointestinal enzymatic degradation poor bioavailability results necessitating a parenteral delivery system. Some GnRH antagonists have been associated with significant histamine release, inhibiting their widespread use. More recently, antagonists have been developed that avoid this side effect without compromising potency. However the GnRH antagonist development has lagged behind that of the agonists, in part related to their high cost of production. In conclusion, GnRH agonists have achieved widespread clinical use in animals for controlling reproduction in either pro- or anti-fertility roles, yet antagonist development has been slower.     

Using gonadotropin-releasing hormone (GnRH) and GnRH analogs to modulate testis function and enhance the productivity of domestic animals

Gonadotropin releasing hormone (GnRH) controls the activity of the gonadotrope cells of the pituitary gland and, as a consequence, is a critical component of the endocrine cascade that determines the growth, development, and functional activity of testicular tissue. The use of GnRH and GnRH analogs is common in domestic animal production systems. Although GnRH and GnRH analogs are most commonly used to control the fertility and reproductive events in female animals, GnRH agonists and antagonists are increasingly used to modulate the fertility, behavior, and productivity of male animals as well. This review will focus on recent advances in this use of GnRH agonists and antagonists.     

Regulation of primate testicular function by GnRH analogues

The potential of GnRH analogues for regulating testicular function is reviewed. Our experiments showed that constant infusion of GnRH agonists effectively suppressed testicular function in monkeys. In men, however, spermatogenesis could not be suppressed to achieve azoospermia uniformly. GnRH antagonists, although at much higher dosages than agonists, caused a more rapid and uniform inhibition of testis function. Spermatogenesis was reversibly disrupted at the spermatogonial level. Concomitant testosterone supplementation, used to maintain libido and potency, attenuated the antitesticular effects of GnRH analogues. In monkeys testosterone appears to stimulate spermatogenesis directly on the testicular level, while evidence has been obtained that in rats testosterone can also stimulate the release and synthesis of FSH under antagonist mediated blockage of pituitary GnRH receptors. When extrapolating to human studies special care has to be exerted in the selection of testosterone substitution regimens. Although the agonistic and antagonistic analogues of GnRH ultimately exert their antireproductive effects via inhibition of gonadotropin secretion the antagonists may have the greater potential for male fertility regulation due to quicker pituitary and testicular suppression.     

A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists. Identification of D-Lys(6) in the ligand and extracellular loop two of the receptor as determinants

Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indicated that the chicken GnRH receptor has a distinct pharmacology, and certain antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnRH receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. The chicken GnRH receptor had high affinity for mammalian GnRH (K(i) 4.1 +/- 1.2 nM), similar to the human receptor (K(i) 4.8 +/- 1.2 nM). But, in contrast to the human receptor, it also had high affinity for chicken GnRH ([Gln(8)]GnRH) and GnRH II ([His(5),Trp(7),Tyr(8)]GnRH) (K(i) 5.3 +/- 0.5 and 0.6 +/- 0.01 nM). Three mammalian receptor antagonists were also pure antagonists in the chicken GnRH receptor. Another three, characterized by D-Lys(6) or D-isopropyl-Lys(6) moieties, functioned as pure antagonists in the human receptor but were full or partial agonists in the chicken receptor. This suggests that the Lys side chain interacts with functional groups of the chicken GnRH receptor to stabilize it in the active conformation and that these groups are not available in the activated human GnRH receptor. Substitution of the human receptor extracellular loop two with the chicken extracellular loop two identified this domain as capable of conferring agonist activity to mammalian antagonists. Although functioning of antagonists as agonists has been shown to be species-dependent for several GPCRs, the dependence of this on an extracellular domain has not been described.     

New GnRH analogs in canine reproduction

Gonadotropin-releasing hormone (GnRH) analogs, which include agonists and antagonists, have been produced by amino acid substitutions of the native GnRH molecule to create greater potency and longer duration of effectiveness. The aim of this article was to review the pharmacological effects and the existing clinical literature of new GnRH analogs, namely agonists released from long-term formulations and third generation antagonists, in domestic dogs. Long-term administration of agonists functions through desensitization and down-regulation of GnRH pituitary receptors inhibiting gonadotropin production and release after an initial stimulation. Conversely, GnRH antagonists bind to gonadotrope GnRH receptors and compete successfully with endogenous GnRH for occupancy, thereby inhibiting the pituitary-gonadal axis immediately. There is a promising place for both agonists and antagonists in future canine reproduction. They can be used in the control of estrous cycle, hormone-dependent diseases as well as in contraception. Some information on the effectiveness and safety of these new analogs in canine reproduction is already available, yet further work is needed before they could be widely recommended. The increase in gonadotropins and gonadal steroids following administration of agonists might have adverse effects when used on hormone-dependent diseases. This initial "flare" should also be carefully managed in anestrous and prepubertal bitches. At present, the main application of antagonists seems limited to situations where an acute endocrine, inhibitory effect is required, e.g. proestrus or pregnancy termination. Future commercial availability of long-acting, single-dose antagonists could go far towards controlling pet population.     

Long-term suppression of pituitary-gonadal function with three-month depot of leuprorelin acetate in a girl with central precocious puberty

OBJECTIVE: The efficacy of a 3-month depot preparation of the GnRH agonist leuprorelin acetate in central precocious puberty was studied. METHODS: Treatment with a 3-month depot of leuprorelin acetate was performed subcutaneously in a 7.3-year-old girl with central precocious puberty. RESULTS: During treatment the hormonal suppression was constant and complete as demonstrated by suppressed GnRH stimulation tests and prepubertal estradiol plasma levels. The size and volume of the uterus and ovaries returned to the normal range. The rate of bone maturation was significantly reduced with a ratio deltaBA/deltaCA of 0.58 for 3 treatment years. Thus, the effects of treatment were comparable to those reported for treatment with 1-month depot of GnRH agonists. CONCLUSION: Three-month depots have the advantage of a prolonged injection interval which is more convenient for the patients and reduces costs by necessitating fewer visits to the physician and being approximately 10% cheaper than the 1-month depot. We suggest that comparative and randomized studies be performed to make 3-month depots of GnRH agonists available for routine use in children with central precocious puberty.     

Male contraceptive technology for nonhuman male mammals

Contraceptive techniques applied to males have potential to mitigate diverse instances of overpopulation in human and animal populations. Different situations involving different species dictate that there is no ideal male contraceptive, and emphasizes the value of varying approaches to reducing male fertility. A majority of work in this field has focused on non-surgically destroying the testes or obstructing the epididymis, and suppressing gonadotropin secretion or inducing immune responses to reproductive hormones or sperm-specific antigens. Injection of tissue-destructive agents into the testes or epididymides can be very effective, but often is associated with unacceptable inflammatory responses and sometimes pain. Hormonal vaccines are often not efficacious and provide only short-term contraception, although GnRH vaccines may be an exception to this generality. Finally, there are no clearly effective contraceptive vaccines based on sperm antigens. Although several techniques have been developed to the point of commercialization, none has yet been widely deployed other than surgical castration.     

Role, mechanism of action and application of gonadoliberins in reproductive processes

Gonadoliberin (gonadotropin releasing hormone, GnRH) plays a central role in the regulation of reproductive functions as it regulates the release of both luteinizing hormone (LH) and follicle stimulating hormone (FSH). The isolation and structure determination of GnRH opened the possibility of its use for influencing reproductive processes. This possibility initiated a rapid development in the design of potent and long-acting GnRH agonists and antagonists. The most important structural modifications of GnRH leading to superagonists are the D-amino acid substitutions in position 6 combined with Pro9-ethylamide or azaGly10 at the C-terminus. We have synthesized several superagonists of GnRH according to these substitution principles. Furthermore, our L-isoaspartyl modification in position 6, as a new approach to GnRH agonist design, also resulted in superactive analogs. The recently discovered sequences of non-mammalian GnRH-s opened new routes for us to synthesize species specific GnRH agonists. All three groups of the above mentioned GnRH analogs have been successfully used for the treatment of sexual disorders of different animals (cattle, pigs, rabbits, etc.). Ovulation synchronization and a 30% increase in the fertility rate could be achieved by using GnRH agonists in cattle breeding. Analogs derived from species specific sequences could be applied for the induced artificial propagation of fish even out of the spawning season. It is known that superactive GnRH analogs can suppress the growth of certain hormone-dependent tumours. In vitro and in vivo tests of our analogs showed promising antitumour activity in breast cancer which might be explained by the mechanism of desensitization. Almost a hundred antagonist analogs of GnRH have been developed in our laboratory. The most effective ones contain 4 or 5 D-amino acids, and one of them is even orally active. The inhibition of ovulation can also be achieved by the administration of GnRH superagonists. This phenomenon might also be explained by the desensitization of LH-release. Radioactive analogs specifically labeled with tritium in different amino acid residues have been synthesized and used for studying tissue distribution and biodegradation of gonadoliberins. Analogs containing a photoreactive group have been prepared and applied for the trials of GnRH receptor isolation.     

Receptor-mediated uptake of GnRH agonist and antagonists by cultured gonadotropes: evidence for differential intracellular routing

The binding and uptake of the GnRH agonist D-Lys6-GnRH and of the antagonists [N-Ac-D-(pyro)-Cl-Phe1,2-D-Trp3-Lys6-D-Ala10]-GnRH and D-p-Glu1-D-Phe2-D-Trp3-D-Lys6-GnRH by dispersed pituitary gonadotropes was studied with electron microscopy. The peptides were coupled to colloidal gold markers with a diameter of 6 or 20 nm which were incubated separately or together for time periods between 15 and 180 min. Both antagonists could be found after 45 and 180 min at 37 degrees C in lysosomes as well as at the plasma membrane of gonadotropes. Co-incubation of both antagonists or of agonist and either antagonist resulted in uptake of the conjugates into separate lysosomes as well as mixed together into the same lysosome. Localization of the antagonists in structures associated with the Golgi apparatus was not observed at the time points studied. The results show that both GnRH agonist- and antagonist-conjugates are biologically active and that they are internalized by the gonadotropes via receptor mediated endocytosis. The failure to detect antagonist conjugates in the Golgi apparatus may indicate that passage through this organelle requires activation of the receptors by agonists and that the uptake of antagonist into lysosomes due to normal membrane protein turnover.     

Two mutations in extracellular loop 2 of the human GnRH receptor convert an antagonist to an agonist

GnRH regulates the reproductive system through cognate G protein-coupled receptors in vertebrates. Certain GnRH analogs that are antagonists at mammalian receptors behave as agonists at Xenopus laevis and chicken receptors. This phenomenon provides the opportunity to elucidate interactions and the mechanism underlying receptor activation. A D-Lys(iPr) in position 6 of the mammalian GnRH receptor antagonist is required for this agonist activity (inositol phosphate production) in the chicken and X. laevis GnRH receptors. Chimeric receptors, in which extracellular loop domains of the human GnRH receptor were substituted with the equivalent domains of the X. laevis GnRH receptor, identified extracellular loop 2 as the determinant for agonist activity of one of the mammalian antagonists: antagonist 135-18. Site-directed mutagenesis of nine nonconserved residues in the C-terminal domain of extracellular loop 2 of the human GnRH receptor showed that a minimum of two mutations (Val(5.24(197))Ala and Trp(5.32(205))His) is needed in this region for agonist activity of antagonist 135-18. Agonist activity of antagonist 135-18 was markedly decreased by low pH (<7.0) compared with GnRH agonists. These findings indicate that D-Lys(iPr)(6) forms a charge-supported hydrogen bond with His(5.32(205)) to stabilize the receptor in the active conformation. This discovery highlights the importance of EL-2 in ligand binding and receptor activation in G protein-coupled receptors.     

GnRHs and GnRH receptors

GnRH is the pivotal hypothalamic hormone regulating reproduction. Over 20 forms of the decapeptide have been identified in which the NH2- and COOH-terminal sequences, which are essential for receptor binding and activation, are conserved. In mammals, there are two forms, GnRH I which regulates gonadotropin and GnRH II which appears to be a neuromodulator and stimulates sexual behaviour. GnRHs also occur in reproductive tissues and tumours in which a paracrine/autocrine role is postulated. GnRH agonists and antagonists are now extensively used to treat hormone-dependent diseases, in assisted conception and have promise as novel contraceptives. Non-peptide orally-active GnRH antagonists have been recently developed and may increase the flexibility and range of utility. As with GnRH, GnRH receptors have undergone co-ordinated gene duplications such that cognate receptor subtypes for respective ligands exist in most vertebrates. Interestingly, in man and some other mammals (e.g. chimp, sheep and bovine) the Type II GnRH receptor has been silenced. However, GnRH I and GnRH II still appear to have distinct roles in signalling differentially through the Type I receptor (ligand-selective-signalling) to have different downstream effects. The ligand-receptor interactions and receptor conformational changes involved in receptor activation have been partly delineated. Together, these findings are setting the scene for generating novel selective GnRH analogues with potential for wider and more specific application.     

2-phenyl-4-piperazinylbenzimidazoles: orally active inhibitors of the gonadotropin releasing hormone (GnRH) receptor

Antagonism of the gonadotropin releasing hormone (GnRH) receptor has shown positive clinical results in numerous reproductive tissue disorders such as endometriosis, prostate cancer and others. Traditional therapy has been limited to peptide agonists and antagonists. Recently, small molecule GnRH antagonists have emerged as potentially new treatments. This article describes the discovery of 2-phenyl-4-piperazinylbenzimidazoles as small molecule GnRH antagonists with nanomolar potency in in vitro binding and functional assays, excellent bioavailability (rat %F>70) and demonstrated oral activity in a rat model having shown significant serum leuteinizing hormone (LH) suppression.     

Use of GnRH agonist implants for long-term suppression of fertility in extensively managed heifers and cows

The ability of gonadotrophin releasing hormone (GnRH) agonist implants to suppress ovarian activity and prevent pregnancies, long-term, was examined in heifers and cows maintained under extensive management. At three cattle stations, heifers (2-year-old) and older cows (3- to 16-year-old) were assigned to a control group that received no treatment, or were treated with high-dose (12 mg, Station A) or low-dose (8 mg, Station B and Station C) GnRH agonist implants. The respective numbers of control and GnRH agonist-treated animals (heifers + cows) at each station were: Station A, 20 and 99; Station B, 19 and 89; Station C, 20 and 76. Animals were maintained with 4% bulls and monitored for pregnancy at 2-monthly intervals for approximately 12 months. Pregnancy rates for control heifers and control cows ranged from 60-90% and 80-100%, respectively, depending on the study site. The respective number of animals (heifers + cows) treated with GnRH agonist that conceived, and days to first conception, were: Station A, 9 (9%) and 336 +/- 3 days; Station B, 8 (10%) and 244 +/- 13 days; Station C, 20 (26%) and 231 +/- 3 days. Treatment with high-dose GnRH agonist prevented pregnancies for longer (approximately 300 days) than treatment with low-dose GnRH agonist (approximately 200 days). In the majority of heifers and cows treated with GnRH agonist, ovarian follicular growth was restricted to early antral follicles (2-4mm). The findings indicate that GnRH agonist implants have considerable potential as a practical technology to suppress ovarian activity and control reproduction in female cattle maintained in extensive rangelands environments. The technology also has broader applications in diverse cattle production systems.     

Luteinizing hormone-releasing hormone analogs: their impact on the control of tumorigenesis

The development of the luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists and the principles of their clinical use were reviewed. In the 28 years that have elapsed since the elucidation of the structure of LH-RH, various applications in gynecology, reproductive medicine, and oncology have been established for LH-RH agonists and antagonists. These clinical applications are based on inhibition of the pituitary and the gonads. The advantage of the LH-RH antagonists is due to the fact that they inhibit the secretion of gonadotropins and sex steroids immediately after the first injection and thus achieve rapid therapeutic effects in contrast to the agonists, which require repeated administration. LH-RH antagonists should find applications in the treatment of benign gynecologic disorders and benign prostatic hypertrophy and in assisted reproduction programs. The primary treatment of advanced androgen-dependent prostate cancer is presently based on the use of depot preparations of LH-RH agonists, but antagonists like Cetrorelix already have been tried successfully. Antagonists of LH-RH might be more efficacious than agonists in treatment of patients with breast cancer as well as ovarian and endometrial cancer. Recently, practical cytotoxic analogs of LH-RH that can be targeted to LH-RH receptors on tumors have been synthesized and successfully tested in experimental cancer models. Targeted cytotoxic LH-RH analogs show a great promise for therapy of prostate, breast, and ovarian cancers.     

Endocrine treatment of prostate cancer

Although androgen deprivation as a treatment for patients with prostate cancer was described more than 60 years ago its optimal use remains controversial. The widespread use of prostate-specific (PSA) assay has lead to earlier diagnosis and earlier detection of recurrent disease. This means that the systemic side effects of androgen deprivation and quality of life have become more important. Debates continue regarding the proper use and timing of endocrine therapy with orchiectomy, oestrogen agonists, gonadotropin hormone-releasing hormone (GnRH) agonists, GnRH antagonists, and androgen antagonists. A critical review of the literature was performed. Data support that androgen deprivation is an effective treatment for patients with advanced prostate cancer. However, although it improves survival, it is not curative, and creates a spectrum of unwanted effects that influence quality of life. Castration remains the frontline treatment for metastatic prostate cancer, where orchiectomy, oestrogen agonists and GnRH agonists produce equivalent clinical responses. Maximum androgen blockade (MAB) is not significantly more effective than single agent GnRH agonist or orchiectomy. Nonsteroidal antiandrogen monotherapy is as effective as castration in treatment of locally advanced prostate cancer offering quality of life benefits. Adjuvant endocrine treatment is able to delay disease progression at any stage. There is, however, controversy of the possible survival benefit of such treatment, including patients having PSA relapse after definitive local treatment for prostate cancer. Neoadjuvant endocrine treatment has its place mainly in the external beam radiotherapy setting. Intermittent androgen blockade is still considered experimental. The decision regarding the type of androgen deprivation should be made individually after informing the patient of all available treatment options, including watchful waiting, and on the basis of potential benefits and adverse effects. Several large studies are under way to investigate the role of adjuvant endocrine treatment in the field of early prostate cancer, intermittent androgen deprivation and endocrine therapy alone compared with endocrine therapy with radiotherapy. The real challenge, however, is to develop better means to avert hormone-refractory prostate cancer and better treatments for patients with hormone-refractory disease when it occurs.     

Functional assays to define agonists and antagonists of the sigma-2 receptor

The sigma-2 receptor has been identified as a biomarker in proliferating tumors. To date there is no well-established functional assay for defining sigma-2 agonists and antagonists. Many sigma-2 ligands with diverse structures have been shown to induce cell death in a variety of cancer cells by triggering caspase-dependent and independent apoptosis. Therefore, in the current study, we used the cell viability assay and the caspase-3 activity assay to determine sigma-2 agonists and antagonists. Three classes of sigma-2 ligands developed in our laboratory were evaluated for their potency to induce cell death in two tumor cell lines, mouse breast cancer cell line EMT-6 and human melanoma cell line MDA-MB-435. The data showed that the EC₅₀ values of the sigma-2 ligands using the cell viability assay ranged from 11.4 μM to >200 μM, which were comparable with the EC₅₀ values obtained using the caspase-3 assay. Based on the cytotoxicity of a sigma-2 ligand relative to that of siramesine, a commonly accepted sigma-2 agonist, we have categorized our sigma-2 ligands into agonists, partial agonists, and antagonists. The establishment of functional assays for defining sigma-2 agonists and antagonists will facilitate functional characterization of sigma-2 receptor ligands and sigma-2 receptors.     

Effects of gonadotropin-releasing hormone agonists on meiotic maturation of follicle-enclosed oocytes in rabbits

The effects of GnRH agonists on in vitro maturation of rabbit follicle-enclosed oocytes were studied. Rabbit preovulatory follicles were cultured with or without hCG (10(2) ng/ml), buserelin (10(2)-10(5) ng/ml), or leuprolide (10(2)-10(5) ng/ml) for 14 hours in vitro. GnRH agonists induced the resumption of meiosis in the follicle-enclosed oocytes in a dose-dependent manner. The percentage of oocytes achieving GVBD following treatment with 10(5) ng/ml buserelin (87.9 +/- 6.3%) or 10(5) ng/ml leuprolide (86.0 +/- 4.1%) did not differ significantly from hCG-treated control (87.3 +/- 3.8%). Mature oocytes initially were detected within 2 hours of GnRH agonist exposure. Concomitant addition of a GnRH antagonist at 10(4) ng/ml significantly blocked the stimulatory effect of GnRH agonist on oocyte maturation. GnRH agonists significantly stimulated both prostaglandin (PG) E2 (PGE2) and PGF2 alpha production by preovulatory follicles (p less than 0.01), but secreted prostanoid levels did not differ significantly among different concentrations of GnRH agonists. Meiotic maturation of follicle-enclosed oocytes following GnRH agonist exposure began 2 hours earlier than production of PGs. PG production stimulated by GnRH agonists was reduced significantly by indomethacin. However, oocyte maturity in the presence of GnRH agonist plus indomethacin did not differ significantly from that of GnRH agonist alone. GnRH agonistic analogues induce the resumption of meiosis in follicle-enclosed oocytes in rabbits by a mechanism other than PG stimulation.     

Specific delta-opioid antagonists exert an agonist-independent inhibitory effect,similar to the agonist,on the release of GnRHIn Vitro

Summary 1. Inin vitro studies with adult male rats we have recently shown that the delta-opioid agonist DTLET inhibits the release of the Gonadotropin-Releasing Hormone (GnRH) from hypothalamic fragments containing the arcuate nucleus and the median eminence. This effect is receptor mediated and eicosanoid dependent (Gerozissiset al., 1993).2. In the present study we report that the delta-opioid antagonists with negative intrinsic activity, Diallyl-G and ICI 174864, applied under the same experimental conditions (30 min static incubations at 37°C, in a potassium rich milieu), in the absence of the agonist DTLET, also exert a similar to the agonist inhibitory effect on the release of GnRH.3. The dose-dependent inhibitory effect of Diallyl-G on GnRH release is reversed by increasing concentrations of DTLET. The mu and delta opioid antagonist, naloxone is without effect in the absence of DTLET. However, naloxone acts as an antagonist on the Diallyl-G-induced inhibition of GnRH release.4. Diallyl-G also inhibits the release of prostaglandin E₂ (PGE₂). In the presence of indomethacin or nordihydroguaiaretic acid, Diallyl-G is ineffective to further inhibit the release of GnRH. These latter observations taken together with the results of eicosanoid estimation suggest that PGE₂ but not leukotrienes participate in the agonist-independent effects of Diallyl-G on GnRH release.5. Therefore these results support the hypothesis that delta-opioid antagonists with negative intrinsic activity exert agonist-independent biological responses similar to those of the agonists.     

The evolution of hormonal therapy for prostatic carcinoma

It is well recognized that testosterone has a number of untoward effects on prostatic carcinoma and that castration is associated with significant tumor shrinkage and resolution of symptoms of advanced prostatic carcinoma. Approaches to hormonal therapy have evolved significantly over the last several decades. Initially castration was utilized, which provided effective reduction of testicular androgens, but with adverse psychological factors. The next approach was utilization of diethylstilbestrol, but with significant cardiovascular toxicity in higher doses. The development of the luteinizing hormone-releasing hormone agonists provided an improvement in pharmacologic castration; however, they are associated with a transient testosterone surge and the potential for exacerbation of clinical manifestations of advanced prostate carcinoma (the so-called "testosterone flare"). Recently, gonadotropin-releasing hormone (GnRH) antagonists have been investigated. Abarelix is a pure GnRH antagonist that blocks the anterior pituitary receptor, resulting in prompt and significant reduction not only of luteinizing hormone but also follicle-stimulating hormone. This results in castrate levels of testosterone while avoiding the testosterone surge.     

Chitosan hydrogels containing liposomes and cubosomes as particulate sustained release vaccine delivery systems

Sustained release depot systems have been widely investigated for their potential to improve the efficacy of subunit vaccines and reduce the requirement for boosting. The present study aimed to further enhance the immunogenicity of a sustained release vaccine by combining a depot formulation with a particulate antigen delivery system. Sustained release of the model subunit antigen, ovalbumin (OVA), was observed in vivo from chitosan thermogel-based formulations containing cationic, nanosized liposomes loaded with OVA and the immunopotentiator, Quil A (QA). Such formulations demonstrated the ability to induce cluster of differentiation (CD)8(+) and CD4(+) T-cell proliferation and interferon (IFN)-γ production, as well as the production of OVA-specific antibody. However, gel-incorporated liposomes showed evidence of instability and similar in vivo immune responses to liposomes in gel formulations were induced by gel-based systems loaded with soluble OVA and QA. The immunogenicity of chitosan thermogels containing cubosomes, a more stable lipidic particulate system, was therefore examined. Similarly, all gel-based formulations produced comparable effector immune responses in experimental mice, irrespective of whether the antigen and immunopotentiator were present in gels within cubosomes or in a soluble form. This work demonstrates the potential for sustained release thermogelling systems and highlights the importance of matching the physicochemical and immunological properties of the particulate system to that of the depot.