Synthesis of potent antagonists of receptors for growth hormone-releasing hormone with antitumor and anti-inflammatory activity

The syntheses and biological evaluation of GHRH antagonists of AVR series with high anticancer and anti-inflammatory activities are described. Compared to our previously reported GHRH antagonist 602 of MIAMI series, AVR analogs contain additional modifications at positions 0, 6, 8, 10, 11, 12, 20, 21, 29 and 30, which induce greater antitumor activities. Five of nineteen tested AVR analogs presented binding affinities to the membrane GHRH receptors on human pituitary, 2-4-fold better than MIA-602. The antineoplastic properties of these analogs were evaluated in vitro using proliferation assays and in vivo in nude mice xenografted with various human cancer cell lines including lung (NSCLC-ADC HCC827 and NSCLC H460), gastric (NCI-N87), pancreatic (PANC-1 and CFPAC-1), colorectal (HT-29), breast (MX-1), glioblastoma (U87), ovarian (SK-OV-3 and OVCAR-3) and prostatic (PC3) cancers. In vitro AVR analogs showed inhibition of cell viability equal to or greater than MIA-602. After subcutaneous administration at 5 μg/day doses, some AVR antagonists demonstrated better inhibition of tumor growth in nude mice bearing various human cancers, with analog AVR-353 inducing stronger suppression than MIA-602 in lung, gastric, pancreatic and colorectal cancers and AVR-352 in ovarian cancers and glioblastoma. Both antagonists induced greater inhibition of GH release than MIA-602 in vitro in cultured rat pituitary cells and in vivo in rats. AVR-352 also demonstrated stronger anti-inflammatory effects in lung granulomas from mice with lung inflammation. Our studies demonstrate the merit of further investigation of AVR GHRH antagonists and support their potential use for clinical therapy of human cancers and other diseases.

in the field, had decided to become involved in the synthesis and evaluation of GHRH antagonists for possible uses in therapy of cancer [6][7][8].
We and others incorporated pentafluoro-Phe at different positions into several GHRH analogs [14,21]. Acylation of GHRH antagonists with octanoic acid or 12-aminododecanoic acid improved the anti-proliferative effects of these antagonists [8,14]. Our work between 1994 and 2017 resulted in several series of potent GHRH antagonists intended for cancer therapy [6][7][8][14][15][16][17][18]. Antagonists of MIA series, after subcutaneous administration in microgram doses suppressed tumor growth of diverse human cancer lines xenografted into nude mice [14]. Antagonists MIA-602 and MIA-690 were among the most potent anti-tumor analogs and also displayed anti-inflammatory activities [22][23][24]. Thus, GHRH analogs of the Miami series powerfully hinder tumor growth and inflammatory activities, but have only a weak endocrine GH inhibitory activity [14]. GHRH antagonists of Miami class, inhibited tumor growth in vivo in nude mice of some 16 types of solid human cancers represented by nearly 50 human cancer lines. In this work we describe the design and syntheses of new class of GHRH antagonists [25] with greater tumor inhibitory potency and augmented suppressive activity on the release of GH.

peptide synthesis and purification
AVR GHRH antagonists were prepared by solid-phase methodology using Fmoc synthesis strategy [26]. The antagonists with C-terminal amide such as Har-NH 2 or Ada-NH 2 were synthesized using Rink amide MBHA resin; and the antagonists with C-terminal Har-NHCH 3 or Ada-NHCH 3 were synthesized on Methyl Indole AM resin.
For the synthesis, the Fmoc group was eluted from the Rink amino MBHA resin with 20% piperidine in dimethyl-formamide for 20 min. The side chains of Fmoc-amino acids were protected with acid labile groups such as β-tert-butyl ester for Asp;tert-butyl (But) for Ser, Thr and Tyr; Nω-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Arg and D-Arg; Nδ-tert-butoxycarbonyl (Boc) for Orn; Nγ-trityl for Asn and Nδtrityl for Gln. Dat was unprotected. The coupling of Fmoc amino acid was achieved with 3 equivalents of Fmoc amino acid, HBtU [2-(1Hbenzotriazole-1-Yl)-1,1,3,3-tetramethyluronium hexa-fluorophosphate] and HOBt  dissolved in DMF, followed by addition of 6 equivalents of N,N-diisopropylethylamine (DIPEA) and stirred for 5 min to go into a complete solution. The mixture was immediately added to Fmoc-deblocked resin and shaken for 2 h. After finishing the peptide synthesis, the final de-protection and cleavage of the peptides from the resin were performed by treatment with a mixed reagent and scavengers containing TFA/thioanisole /1,2-ethanedithiol / phenol / H 2 O (95 : 1.5 : 1 : 1 : 1.5 by volume) at room temperature for 3 h. All amino acid derivatives, resins, and reagents were obtained from Chem-Impex Int'l INC (Wooddale IL), Sigma-Aldrich, (Billerica, MA), or Novabiochem (Burlington MA). The scale of synthesis was around 0.5 mmol for most of our AVR compounds. The scale for purification was 200 mg for crude products; the average yield was around 30 % with > 95 % purity.
Purification of the crude peptides was performed on a Beckman Gold The HPLC analyses of crude and purified peptides were carried out on an Agilent 1290 Infinity High Performance Liquid Chromatography unit (Agilent, Santa Clara, CA) equipped with Discovery HS C18 column (2.1 × 50 mm, 120 Å pore size, 3 μm particle size, Supelco Bellefonte, PA). An isocratic and/or gradient elution was used from 40 to 80 % B in 10 min with a solvent system consisting of solvents A and B, described above, with a flow rate of 0.5 ml/min. The peaks were monitored at 220 and 280 nm. The peptides were judged to be substantially (>95 %) pure by analytical HPLC. Molecular masses were determined by an Agilent 6210 time-of-flight Mass Spectrometer in conjunction with 1200 CapLC (Agilent,Santa Clara, CA). Peptides were eluted on an Agilent Zorbax C18 column (0.5 × 150 mm, 300 Å pore size, 5 μm particle size) with a solvent system consisting of solvent A (0.1 % formic acid) and solvent B (0.1 % formic acid in 90 % aqueous acetonitrile) in a linear gradient mode of 35-85 % solvent A: solvent B, at a flow rate of 15 μl/min in 30 min. TOF settings were as follows: capillary voltage:4000 V, drying gas flow: 7 l/ min, drying gas temperature: 300 • C, nebulizer gas: 30 psi, fragmentor voltage: 350 V.

Animals
Male rats (Wistar, obtained from Charles River Laboratory) weighing ~200 g were used for the in vivo and in vitro endocrine tests; Athymic (Ncr nu/nu) nude mice, 5 to 6-week-old, obtained from Envigo Labs (Tampa, FL) were used in oncologic studies; 6-week-old C57BI/6 male mice (Jackson Laboratory, Bar Harbor, ME) were used in lung granulomatous study. All animals were housed in Laminar airflow cabinets under pathogen-free conditions [27]. The experiments were conducted in accordance with the principles and procedures of the NIH Guide for the Care and Use of Laboratory Animals. The protocol of the animal experiments was reviewed and approved by the Institutional Animal Care and Use Committee of the Miami VA Medical Center.

Cell lines
Various human cancer cell lines including HCC827 human lung cancer NSCLC-ADC, H460 human lung cancer LCLC, CFPAC-1 and PANC-1 human pancreatic cancer, NCI-N87 human stomach cancer, HT-29 colon cancer, MX-1 breast cancer OVCAR-3 and SK-OV-3 ovarian, U87 glioblastoma and PC-3 prostate cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA) and maintained in culture in as instructed by the manufacturer.

Evaluation of receptor binding affinities in vitro
Preparation of human pituitary membrane fractions and receptor binding assay were performed as previously described [26]. Human pituitaries were purchased from the National Hormone and Peptide Program. In the competitive binding analyses, 125 I-labeled [His 1 , Nle 27 ]-hGHRH(1-32)-NH 2 (0.2 nM) was displaced by GHRH antagonists at 10 − 6 -10 − 12 M. The final binding affinities were expressed as IC 50 value and were calculated by using the LIGAND PC computerized curve-fitting program of Munson and Rodbard [28] as modified by McPherson [29].

Cell proliferation assay
Human cancer cells were seeded into 96-wellplates and exposed to 1 μM, 2 μM, and 5 μM of a selected AVR class of GHRH antagonists in quintuplicate wells, each for 70-72 h. The GHRH antagonist MIA-602 was used for comparison of activity. At the end of treatment, cell proliferation was measured using CellTiter 96 aqueous one solution kit (Promega, Madison WI), as described previously [27]. Relative inhibitory potency of AVR-class of GHRH antagonists in comparison to MIA-602 was calculated based on the inhibition of cell viability after cancer cells were treated with the antagonists at a concentration of 5 μM for 72 h.

Oncologic study in vivo
As previously described [27], athymic (Ncr nu/nu) nude mice were xenografted subcutaneously (s.c) with approximately 3 mm 3 pieces of tumor tissue derived from donor animals. When tumors reached a mean volume of ≈40− 50 mm 3 , the animals were randomly assigned into groups (6-10 mice per group) and treated s.c. with GHRH antagonists: MIA-602, and selected AVR antagonists at doses of 5 μg/day (prepared in a vehicle solution,10 % propylene glycol containing 0.1 % DMSO) or otherwise indicated. Controls were treated with vehicle solution. Tumor size was measured once a week with a micro-caliper. Tumor volume was calculated as previously described [30]. At the end of the experiment, (after 4-7 weeks of treatment) mice were anesthetized and sacrificed. Tumor growth rate "Vn/V0", tumor volume at the termination of treatment / tumor volume at beginning of the treatment, was calculated.

In vivo assessment of the antagonistic activity of GHRH analogs on GH release
The potency and duration of inhibitory effects of GHRH antagonists were tested in male rats using 8 rats per group [14]. Twenty min after anesthesia, GHRH antagonists MIA-602, AVR-352 and AVR-353, at dose of 5 μg/kg were injected into the jugular vein of the rats, followed by an iv injection of 3 μg/kg GHRH (1-29)NH 2 5 min later. Controls received vehicle solution (10 % propylene glycol containing 0.1 % DMSO) before GHRH stimulus. Serum GH levels before the administration of the antagonists and 5, 15 and 30 min following the injection of GHRH (1-29) NH 2 were measured by ELISA assay kit (ALPCO Diagnostics, Mill Valley, CA).

In vitro assessment of the antagonistic activity of GHRH analogs on GH release
Rats were decapitated and pituitaries were collected in prewarmed HBSS (Hanks' Balanced Salt Solution). Tissue was cut into small pieces and digested in HBSS medium containing 3 % BSA, 50 μg/mL gentamycin and 0.5 % collagenase) and seeded onto poly-D-lysine-coated 24well plates using 7 pituitaries/plate. Cells were allowed to recover for 4 days. Growth medium was then replaced with serum-free DMEM for 4 h and 20 nM of selected GHRH analogs were added in DMEM containing 0.1 % BSA for 30 min. Cells were then incubated in medium containing 20 nM concentration of the analog and 1 nM GHRH(1-29)NH 2 for 30 min. The medium from this step was collected and centrifuged at 800 g for 3 min. GH concentration was determined by using Spin Bio Growth hormone (rat) EIA KIT (Cayman chemical, Ann Arbor, MI).
The mice were sacrificed on day 21 days after challenges and the lungs were harvested for protein analyses.

Protein isolation and Western blot analyses
Lung tissues retrieved from mice with granulomatous lung reaction, or tumors from mice carrying xenografted human lung cancer HCC827 after treatment with antagonists were lysed in lysis buffer (Cell Signaling Technology, Beverly, MA) with protease inhibitor cocktail (Cell Signaling Technology, Beverly, MA). Samples were sonicated and centrifuged at 10,000 g for 5 min 3 times. The supernatant was collected, and protein concentration was determined by BCA protein assay using a kit (Cell Signaling Technology).

Statistical analysis
Statistical analyses of the results of oncological studies were performed by t-test or one-way ANOVA, followed by Tukey's test, using the computer software Sigma Stat (Jandel, San Rafael, CA). Differences were considered significant when p < 0.05. GH data was analyzed similarly.

Design and synthesis of new hGHRH antagonists
We have synthesized 115 GHRH antagonists of AVR series using Fmoc-chemistry method instead of Boc chemistry used for GHRH antagonists of MIA series [26]. This AVR series of antagonists contains modification at positions 0, 6,8,9,10,11,12,20,21,29 and 30 of the structures of GHRH antagonist MIA-602. Table 1 shows the key amino acid replacements in nineteen AVR antagonists which were selected for testing of antitumor activities in comparison to MIA-602. These AVR compounds have 5FPhAC-Ada at N-terminal; 5FPhe or Cpa at position 6, Asn or Ala at position 8, Arg or Har at position 9, Tyr (Me) or 5FPhe at position 10, Arg or His in position 11 and 20, Lys or Orn at position 12 and 21 and Har-NH 2 or Har-NHCH 3 at position 29; or modified C-terminal-NH 2 with Aoc-NHCH 3 or Ada-NH 2 , Ada-NHCH 3 as an additional extension at position 30.

Binding affinities of new hGHRH antagonists
The nineteen AVR and two MIA GHRH antagonists MIA-602 and Table 1 Structures of AVR antagonists tested in vivo. 1

Inhibition of cell proliferation in vitro
The  Table 2 summarizes the inhibitory potency of AVR-compounds relative to MIA-602 in different cell lines, based on the inhibitory effects on cell viability exposed to 5μM of antagonists. Antagonists AVR-235, AVR-333, AVR-352, AVR-353 and AVR-354 showed greater inhibitory effects on the cancer cell lines tested than MIA-602.

Oncological studies in vivo
The compounds which displayed high inhibitory potencies in vitro, were further tested in vivo for their suppressive effects on the growth of human cancers xenografted into nude mice. Based on the results in Tables 2 and 3, we selected the AVR analogs with higher binding affinity to GHRH receptors on human anterior pituitary cells for further testing for anti-tumor and anti-inflammatory activity in vivo. Table 4 shows a comparison of inhibition of the growth of tumors after therapy with GHRH antagonist MIA-602 and new GHRH antagonists of AVR class. Various human cancers were tested including lung (HCC827 and H460); pancreatic (PANC-1 and CFPAC-1), gastric (NCI-N87), colorectal (HT-29), breast (MX-1), ovarian (SK-OV-3 and OVCAR-3), prostatic (PC-3) cancers and glioblastoma (U87). Table 4 (Table 4 and Fig. 2A-D). These results support the merit  (Table 4).

Inhibition of GH release in vitro and in vivo after administration of GHRH antagonists of AVR class
The GHRH antagonists which displayed high inhibitory potency in oncologic experiments, were further tested for their effects on GH release in vitro using primary cultures of rat pituitaries. Fig. 3A shows that GH-release induced by 1 nM GHRH was partially inhibited by 20 nM MIA-602 by 22 %, AVR-235 by 40 %, AVR-352 by 28 % and AVR-353 by 35 % respectively. The results demonstrated that these AVR GHRH antagonists exhibit inherent inhibitory activity on GH release from the pituitary. Fig. 3B shows that intravenous administration of 5 μg/kg of AVR-352 or AVR-353 into rats caused a significant reduction (28.0 %, 32.0 % respectively) in the serum concentration of GH compared to the GH levels in the animals given vehicle control. The inhibition of GH release induced by AVR compounds was greater than that by MIA-602 (22.7 %) at the same doses.

The mechanism mediating the enhanced anticancer effects of GHRH antagonists
Our previous studies have revealed that antagonist MIA-602 inhibited tumor progression of lung cancer HCC827 by modulating the  [27]. The proteins were extracted from the tumor tissues retrieved from mice xenografted with lung cancer HCC827 treated with MIA-602 or AVR-353 at dose of 5 μg (Table 2 and Fig. 2A) and analyzed by Western blot analysis. As shown in Fig. 4, treatment with AVR-353 significantly down-regulated the expression of GHRH-R, SV1, pSTAT3/STAT3, PAK1, pCREB/CREB; cyclin D1, cyclin D2, cyclin-dependent kinases (CDKs; CDK4, and CDK6); and upregulated the expression of p27 kip1 (the conditional inhibitor for CDK4/6). GHRH antagonist AVR-353 displayed similar but greater effects in comparison to MIA-602.

Anti-inflammatory effects of AVR class
GHRH antagonists exhibit inhibitory effects on several inflammatory cytokines in the cancer microenvironment [32]. We aimed to test these effects in an in-mouse lung inflammatory model. To achieve this goal, we developed granulomas in the lungs of mice. Mice were randomly grouped and treated with vehicle solution, MIA-602 (5 μg/day), AVR-352 (5 μg/day) or methyl prednisolone (138 μg/day) for three weeks. Fig. 5 shows the expression of IL2, OAS1 (2 ′ -5 ′ -Oligoadenylate Synthetase 1) and ISG15 (Interferon-stimulated gene 15) in the lung tissues retrieved from the animals, analyzed by Western blots. The levels of all three proteins were decreased in comparison with untreated tissues, however only treatment with AVR-352 resulted in significant reductions (55.8 %, 68.7 % and 63.8 % for IL-2, OAS1 and ISG15 respectively, p < 0.05). The results confirmed that AVR-352 has greater anti-inflammatory activity in comparison with MIA-602 or methyl prednisolone.

Structures of best GHRH antagonists of AVR class
The structures of analogs, which displayed highest receptor binding affinities, are listed in Table 4. Analogs AVR-352 and 353 appeared to be the most potent GHRH antagonists based on their inhibitory effects on tumor growth in the in vitro and in vivo tests and had higher antitumor activity than MIA-602.  The data were calculated based on the inhibition of cell proliferation in cancer cell lines treated with antagonists at concentration of 5 μM for 72H. -, not performed, * p < 0.05, ** p < 0.01, ***p < 0.001.
The paper describes the syntheses of a new AVR class of GHRH antagonists, in which further modifications of the structures of MIA-602 and MIA-690 were introduced. Our strategy to induce higher activity included the design and synthesis of three groups of modified AVR compounds as follows: Firstly, we synthesized compounds based on the structure of MIA-602 containing Arg 11, 20 and Lys 12,.21 to replace His 11, 20 Orn 12, 21 ; because His 11, 20 Orn 12, 21 were in the sequence in our previous antagonist JV-1-38 which showed higher inhibitory effects on GH release, but weaker antitumor activity in comparison to MIA-602 or MIA-690 [51]. The analogs with replacements such as AVR-107, AVR-104, AVR-120 displayed weaker antitumor activity in comparison with MIA-602. In the second AVR group we introduced NHCH3 at the C-terminus (analog AVR-201, AVR-234 and AVR-235); interestingly analog AVR-235 containing additional modification with 5FPhAc at the N-terminus and 5FPhe 6 showed higher affinity in the receptor binding assay and promising antitumor activity in stomach cancer (Tables 3 and  4) [45]. In the third group of AVR compounds, we further modified NHCH3 with fatty amino acid Ada at C-terminus, which resulted in a series of analogs (AVR-352, AVR-353 and AVR-354) with both greater antitumor activity on various cancer cells and higher affinities to the pituitary GHRH receptors, as well as inhibitory effects on GH release (Tables 2-4 and Fig. 3). The molecular masses, of analogs as determined by mass spectrometry, matched very well with their molecular weight as calculated from their structures (Table S1, Figs. S1 and S2).
As shown in Table 2, eleven of nineteen tested GHRH antagonists of AVR class, with different combination of modification in the structures, presented a higher binding affinity to GHRH receptor than MIA-602, and nearly all of the AVR compounds had higher binding affinity than MIA-690, which is a potent GHRH antagonist with important antitumor ADC, adenocarcinoma. †Vn, mean tumor size in cubic millimeters at the end of treatment; V0, mean tumor size in cubic millimeters at the beginning of treatment. * p < 0.05, ** P < 0.01, *** P < 0.001.  Table 3. *p < 0.05, **p < 0.01. effects in experimental tumor models [14,27]. The structures of the five most potent AVR analogs listed in Table 5 showed the advantages of the modifications designed. The antitumor effects of these five antagonists: AVR-235, AVR-333, AVR-352, AVR-353 and AVR-354 were evaluated in various human cancer cell lines and compared directly to MIA-602 in the in vitro cell proliferation assay ( Fig. 1 and Table 3). In all cancer cell lines tested these AVR-compounds exerted similar or more potent inhibitory effects to MIA-602. Particularly AVR-352 and AVR-353, presented relatively higher inhibitory action than MIA-602 in all tested human cancer cell lines (Table 3). Greater inhibitory effects on tumor growth in the antineoplastic study in vivo also indicated a higher potency of AVR-compounds than of MIA-602. However, the relative inhibitory potency varies in different human cancer models with other tested compounds (Table 4). Interestingly, AVR-352 and AVR-353 were more potent, or as potent as MIA-602 in the inhibition of tumor growth in all the tested tumor models including lung HCC827 and H460, pancreatic PANC-1 and CFPAC-1, stomach NCI-N87, colorectal HT-29, breast MX-1, glioblastoma U87, ovarian cancer SK-OV-3 and OVCAR-3. Similar results were observed with AVR-235 in lung cancer HCC827 and stomach cancer NCI-N87, AVR-333 in PANC-1, NCI-N87 and U87; and AVR-354 in HT-29, MX-1 and U87 (Table 4). We believe that the impressive antitumor activity seen with these analogs is due in part to the increased binding affinity to GHRH receptors in comparison to MIA-602 (Table 2). GHRH antagonists also have anti-inflammatory effects in mice with pulmonary granulomatous reaction (Fig. 5). These agents significantly reduced the expression of OAS1, ISG15 and IL2, the cytokine, which plays key functions in regulating cell immunity [46]. AVR-352 showed higher anti-inflammatory property than MIA-602 (Fig. 5).
Considerable evidence already exists and continues to be accumulated in support of the concept that the downregulation of GHRH receptors or their blockade by GHRH antagonists, as well as by GHRH agonists plays a major role in the inhibition of tumor growth, [18,27,30,36]. This downregulation appears to be similar to that exerted by LHRH agonists or antagonists on the pituitary gonadal axis [42,47] and which has been used clinically for the past 30 years for therapy of sex hormone dependent tumors. Studies on different classes of GHRH-R antagonists on cancers have demonstrated their abilities to modulate multiple intracellular pathways involved in cellular proliferation, survival, metastasis, apoptosis and inflammation [16][17][18]24,27,[34][35][36][37][38][39]48]. The mechanisms of GHRH-R antagonists identified include the modulation of the expression of effector proteins in the cell cycle to inhibit cell proliferation by blocking the G1 to S transition in the cell cycle, enhancing the expression of E-cadherin and β-catenin leading to less invasive cancers [27]. Other signaling pathways such as Raf/Mek/ERK1/2, PI3K/AKT, EGF/HER, PKC [49][50][51] pathways, and cAMP-CREB pathway [27] were also found to be associated with the inhibitory effects of GHRH antagonists on human cancer cells. These studies extended our knowledge of the possible cross talk between the binding of GHRH antagonists to its receptor and other, not yet explored cell signaling pathways in different cancers. Furthermore, GHRH-R antagonist MIA-602 has been described to modulate multiple inflammation-associated molecules in gastric and lung cancers. In the STAT3/NF-κB signaling pathway, treatment with GHRH antagonist MIA-602 results in downregulation of PAK1, a member of the p21-activated kinase family, and STAT3, a potential effector for G-protein-coupled receptors in regulation of cell progression [36,52]. A recent study [37] also revealed that SV1, splice variant of GHRH-R, is a hypoxia-driven promoter of tumor progression in esophageal squamous cell carcinoma (ESCC). The hypoxia-elevated SV1 activates key glycolytic enzyme, muscle-type phosphofructokinase (PFKM) through nuclear factor-κB (NF-κB) pathway, which enhances glycolytic metabolism and promotes progression of ESCC. The malignant actions induced by SV1-NF-κB-PFKM pathway could be reversed by MIA-602 [37]. Interestingly, the study by Liang et al. [53] revealed that NF-κB subunit p65 transcriptionally activates GHRH-R expression in human ciliary In vivo testing was carried out in rats by intravenous (i.v.) administration of GHRH antagonists MIA-602, AVR-352 or AVR-353 (5μg/kg followed by an iv injection GH-RH (1-29)NH 2 3 μg/kg 5 min later). Controls received vehicle solution. Serum GH levels in animals treated with antagonists were compared at 0, 5 and 15 min following the administration of GHRH. *p < 0.05, **p < 0.01. epithelial cells, indicating a functional role of GHRH-R/JAK2/STAT3 signaling axis in acute anterior uveitis and GHRH antagonist MIA-602 attenuated expression of proinflammatory factors. In recent studies with Covid-19 pandemics, a very important finding made by D. Kotton's group [54], revealed that soon after air sacs in lung are infected with SARS-Cov-2, NF-κB signaling pathway triggers high levels of inflammation. The discovery of NF-kB's role in this deadly cascade makes promising a new therapy based on GHRH antagonists like MIA-602.
The results shown in Fig. 4 reveal that in correlation with the stronger inhibitory activity on tumor growth, AVR-353 displays greater effects in modulating the expression of effector proteins involved in the signaling pathways described in lung cancer HCC827 after treatment with MIA-602 [27]. The fact that GHRH antagonists of AVR class exert higher binding affinity to its receptor and greater inhibition of GH release and tumor growth suggests that these compounds will also display greater activities in modulating the expression of effector proteins involved in multiple signaling pathways in other cancer lines. Peptide analogs can promote and regulate distinct conformational changes in receptors [14,55], GHRH antagonists of AVR class may affect the conformation of GHRH-Rs. Multiple receptor conformations possessing distinct signaling and regulatory properties have been reported in the activation of G-protein-coupled receptor (GPCR, 56). GHRH-R is one of the members of GPCR receptor superfamily [16,17], thus it is important to reveal the possible effects of GHRH antagonists of AVR class on the conformation of GHRH receptors. It is important to study the possible changes in the mechanism of action of AVR-352 and AVR 353 in comparison with MIA-602 in individual human tumor models.
In addition, immunohistochemistry analyses in specimens of tumor samples from animal models and humans revealed the increased levels of GHRH receptors compared to the surrounding normal tissues [36]. Studies on analysis of multiple cohorts of patients with gastric cancer and ESCC [36,37], demonstrated the association between the expression of GHRH receptors in tumors, malignant properties, and poor survival, and suggested that overexpression of GHRH-receptors as an independent predictive factor for patient prognosis. The finding underscores GHRH-R as promising biomarker and therapeutic target for management of human cancers [36,37].
GHRH and its agonistic analogs have been tested in human subjects and showed little or no toxicity and do not cause any significant side effects [30,[57][58][59][60][61][62][63][64]. Khorram et al., described that subcutaneous administration of a synthetic GHRH analog [Norleucine27] GHRH (1-29)-NH2 to elderly subjects for 16 weeks resulted in activation of both immune cells [58] and somatotropic axis [59]; the only adverse side-effect was transient hyperlipidemia, which resolved at end of the study. A long-acting analog of GHRH (1-29)-NH2, CJC-1295, activates the GH/IGF axis while administrated in normal subjects [60]. Tesamorelin, the synthetic analog of GHRH (1-44)-NH2, improves cognitive function in adults with mild cognitive impairment and in healthy older adults [61,62]. In preclinical studies administration of Tesamorelin effectively reduced visceral fat and liver fat and improved liver function in HIV-infected patients [63,64]. Agonist MR-409 synthesized in our laboratory [26] showed feasible and safe in Yorkshire swine model of subacute ischemic cardiomyopathy and significantly reduced infarct size and improved diastolic function [65]. In a recent study the agonist MR-409 ameliorated chronic kidney disease-induced heart failure with preserved ejection fraction in a swine model of chronic kidney disease-induced heart failure [66]. No formal toxicity studies have been performed on AVR antagonists, the investigations have been carried out mainly on antitumor activity of MIA class and AVR class of GHRH antagonists, in those tests, no adverse effects in these antagonists have been recorded in rodents [6,7,14,18,27,67]. The toxicity tests must be done before any clinical studies. However after our extensive studies of GHRH analogs [6][7][8]14,18,27,30,57,67], the side effect/toxicity profile of GHRH analogs is considered as favorable in contrast to chemotherapeutic agents. We expect that the antagonist of AVR class, like MIA-602, would have little or no toxicity [18,67]. We believe that the impressive antitumor and anti-inflammatory activities of GHRH antagonists of AVR class strongly supports their potential use for clinical therapy of human cancer and other diseases. We believe that the impressive antitumor and anti-inflammatory activities of GHRH antagonists of AVR class strongly supports their potential use for clinical therapy of human cancer and other diseases.

Table 5
Key amino acid replacements in AVR-GHRH Antagonists compared to MIA-602.