REG-O3 chimeric peptide combining growth hormone and somatostatin sequences improves joint function and prevents cartilage degradation in rat model of traumatic knee osteoarthritis.

OBJECTIVE: REG-O3 is a 24-aminoacid chimeric peptide combining a sequence derived from growth hormone (GH) and an analog of somatostatin (SST), molecules displaying cartilage repair and anti-inflammatory properties, respectively. This study aimed to investigate the disease-modifying osteoarthritis drug (DMOAD) potential of REG-O3 by analyzing its effect on pain, joint function and structure, upon injection into osteoarthritic rat knee joint. DESIGN: Osteoarthritis was induced in the right knee of mature male Lewis rats (n = 12/group) by surgical transection of the anterior cruciate ligament (ACLT) combined with partial medial meniscectomy (pMMx). Treatments were administered intra-articularly from fourteen days after surgery through three consecutive injections one week apart. The effect of REG-O3, solubilized in a liposomal solution and injected at either 5, 25 or 50 µg/50 µL, was compared to liposomal (LIP), dexamethasone and hyaluronic acid (HA) solutions. The study endpoints were the pain/function measured once a week throughout the entire study, and the joint structure evaluated eight weeks after surgery using OARSI score. RESULTS: ACLT/pMMx surgery induced a significant modification of weight bearing in all groups. When compared to liposomal solution, REG-O3 was able to significantly improve weight bearing as efficiently as dexamethasone and HA. REG-O3 (25 µg) was also able to significantly decrease OARSI histological global score as well as degeneration of both cartilage and matrix while the other treatments did not. CONCLUSION: This study provides evidence of a remarkable protecting effect of REG-O3 on pain/knee joint function and cartilage/matrix degradation in ACLT/pMMx model of rat osteoarthritis. REG-O3 thus displays an interesting profile as a DMOAD.

Low concentration structural dynamics of lanreotide and somatostatin-14.

Lanreotide, a synthetic cyclic octapeptide, analogue of the peptide hormone somatostatin-14 (SST-14), is routinely used as a long-acting medication in the management of neuroendocrine tumors. Despite its therapeutic importance, low concentration structural data is still lacking for lanreotide. In fact, the major part of the previous structural investigations were focused on the remarkable aggregation properties of this peptide, appearing at high concentrations (>5 mM). Here, we have applied three optical spectroscopic techniques, i.e. fluorescence, circular dichroism and Raman scattering, for analyzing the structural dynamics at the concentrations below 5 mM, where lanreotide exists either in a monomer state or at the first stages of aggregation. The obtained data from lanreotide were discussed through their comparison with those collected from SST-14, leading us to the following conclusions: (i) The central D-Trp residue, forming with its adjacent Lys the main receptor interacting part of lanreotide, keeps a constant high rotational freedom whatever the environment (water, water/methanol, methanol). (ii) A solvent-dependent tight ß-turn, belonging to the type-II' family, is revealed in lanreotide. (iii) Raman data analyzed by band decomposition in the amide (I and III) regions allowed estimation of different secondary structural elements within the millimolar range. Interestingly, the applied protocol shows a perfect agreement between the structural features provided by the amide I and amide III Raman markers.

Octreotide used for probing the type-II' ß-turn CD and Raman markers.

Octreotide, a potent somatostatin (SST) analogue, is used as an antiproliferative drug in numerous endocrine tumors. Previous NMR investigations, basically performed in DMSO, had evidenced a type-II' ß-turn structure for this cyclic peptide. However, apart a few incomplete studies by circular dichroism, a systematic analysis of the structural behavior of octreotide in aqueous solution as a function of concentration and ionic strength was still lacking. Here, we report the chemical synthesis and purification of octreotide for optical spectroscopic purposes accompanied by its structural analysis. Furthermore, we have used octreotide as a short size, well-defined model compound for analyzing the CD and Raman markers of a type-II' ß-turn. CD data collected in the 25-250 µM range revealed the general trend of octreotide to undergo a disordered toward ordered structural transition upon increasing concentration. Especially, the ß-turn CD markers could be characterized above 50 µM by a negative band at ~202 nm flanked by a shoulder at ~218 nm. On the basis of Raman spectra recorded as a function of concentration (1-20 mM), we could assign the markers at ~1678 and ~1650 cm(-1) in the amide I region, and at ~1303, ~1288, and ~1251 cm(-1) in the amide III region, to the type-II' ß-turn structure. The stability of the intermolecular antiparallel ß-sheet formed in octreotide could be confirmed by the rigidity of the disulfide bridge which adopts a preferential gauche-gauche-gauche rotamer along the -Cß-S-S-Cß- moiety of the linked cysteines. The present analysis permits a better understanding of the differences between the structural features of SST-14 and its routinely used analogue, octreotide.

Vibrational analysis of amino acids and short peptides in aqueous media. V. The effect of the disulfide bridge on the structural features of the peptide hormone somatostatin-14.

To emphasize the role played by the S-S bridge in the structural features of somatostatin-14 (SST-14), newly recorded CD and Raman spectra of this cyclic peptide and its open analogue obtained by Cys-->Ser substitution are presented. CD spectra of both peptides recorded in aqueous solutions in the 100-500 microM concentration range are strikingly similar. They reveal principally that random conformers constitute the major population in both peptides. Consequently, the S-S bridge has no structuring effect at submillimolar concentrations. In methanol, the CD spectrum of somatostatin-14 keeps globally the same spectral shape as that observed in water, whereas its open analogue presents a major population of helical conformers. Raman spectra recorded as a function of peptide concentration (5-20 mM) and also in the presence of 150 mM NaCl provide valuable conformational information. All Raman spectra present a mixture of random and beta-hairpin structures for both cyclic and open peptides. More importantly, the presence or the absence of the disulfide bridge does not seem to influence considerably different populations of secondary structures within this range of concentrations. CD and Raman data obtained in the submillimolar and millimolar ranges of concentrations, respectively, lead us to accept the idea that SST-14 monomers aggregate upon increasing concentration, thus stabilizing beta-hairpin conformations in solution. However, even at high concentrations, random conformers do not disappear. Raman spectra of SST-14 also reveal a concentration effect on the flexibility of the S-S linkage and consequently on that of its cyclic part. In conclusion, although the disulfide linkage does not seem to markedly influence the SST-14 conformational features in aqueous solutions, its presence seems to be necessary to ensure the flexibility of the cyclic part of this peptide and to maintain its closed structure in lower dielectric constant environments.

Local synthesis and dual actions of progesterone in the nervous system: neuroprotection and myelination.

Progesterone (PROG) is synthesized in the brain, spinal cord and peripheral nerves. Its direct precursor pregnenolone is either derived from the circulation or from local de novo synthesis as cytochrome P450scc, which converts cholesterol to pregnenolone, is expressed in the nervous system. Pregnenolone is converted to PROG by 3beta-hydroxysteroid dehydrogenase (3beta-HSD). In situ hybridization studies have shown that this enzyme is expressed throughout the rat brain, spinal cord and dorsal root ganglia (DRG) mainly by neurons. Macroglial cells, including astrocytes, oligodendroglial cells and Schwann cells, also have the capacity to synthesize PROG, but expression and activity of 3beta-HSD in these cells are regulated by cellular interactions. Thus, Schwann cells convert pregnenolone to PROG in response to a neuronal signal. There is now strong evidence that P450scc and 3beta-HSD are expressed in the human nervous system, where PROG synthesis also takes place. Although there are only a few studies addressing the biological significance of PROG synthesis in the brain, the autocrine/paracrine actions of locally synthesized PROG are likely to play an important role in the viability of neurons and in the formation of myelin sheaths. The neuroprotective effects of PROG have recently been documented in a murine model of spinal cord motoneuron degeneration, the Wobbler mouse. The treatment of symptomatic Wobbler mice with PROG for 15 days attenuated the neuropathological changes in spinal motoneurons and had beneficial effects on muscle strength and the survival rate of the animals. PROG may exert its neuroprotective effects by regulating expression of specific genes in neurons and glial cells, which may become hormone-sensitive after injury. The promyelinating effects of PROG were first documented in the mouse sciatic nerve and in co-cultures of sensory neurons and Schwann cells. PROG also promotes myelination in the brain, as shown in vitro in explant cultures of cerebellar slices and in vivo in the cerebellar peduncle of aged rats after toxin-induced demyelination. Local synthesis of PROG in the brain and the neuroprotective and promyelinating effects of this neurosteroid offer interesting therapeutic possibilities for the prevention and treatment of neurodegenerative diseases, for accelerating regenerative processes and for preserving cognitive functions during aging.