Severo Salvadori

Opioid tolerance is a limiting factor in chronic pain. Delta opioid peptide (DOP)(δ) receptor antagonism has been shown to reduce tolerance. Here, the common clinical mu opioid peptide (MOP)(µ) receptor agonist fentanyl has been linked to the DOP antagonist Dmt-Tic (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydrisoquinoline-3-carboxylic acid) to create new bivalent compounds. Binding affinities of bivalents(#9, #10, #11, #12 and #13) were measured in Chinese hamster ovary (CHO) cells expressing recombinant human MOP, DOP, Kappa opioid peptide (KOP)(κ) and nociceptin/orphanin FQ opioid peptide (NOP) receptors. Functional studies, measuring GTPγ[(35)S] or β-arrestin recruitment, were performed in membranes or whole cells respectively expressing MOP and DOP. The new bivalents bound to MOP (pKi : #9:7.31; #10:7.58; #11:7.91; #12:7.94; #13:8.03) and DOP (#9:8.03; #10:8.16; #11:8.17; #12:9.67; #13:9.71). In GTPγ[(35)S] functional assays, compounds #9(pEC50:6.74; intrinsic activity:0.05) #10(7.13;0.34) and #11(7.52;0.27) showed weak partial agonist activity at MOP. Compounds #12 and #13, with longer linkers, showed no functional activity at MOP. In antagonist assays at MOP, compounds #9 (pKb:6.87), #10(7.55) #11(7.81) #12(6.91) and #13(7.05) all reversed the effects of fentanyl. At DOP, all compounds showed antagonist affinity (#9:6.85; #10:8.06; #11:8.11; #12:9.42; #13:9.00), reversing the effects of DPDPE ([D-Pen(2,5)]enkephalin). In β-arrestin assays, compared with fentanyl (with response at maximum concentration (RMC):13.62), all compounds showed reduced ability to activate β-arrestin (#9 RMC:1.58; #10:2.72; #11:2.40; #12:1.29; #13:1.58). Compared with fentanyl, the intrinsic activity was: #9:0.12; #10:0.20; #11:0.18; #12:0.09 and #13:0.12. The addition of a linker between fentanyl and Dmt-Tic did not alter the ability to bind to MOP and DOP, however a substantial loss in MOP functional activity was apparent. This highlights the difficulty in multifunctional opioid development.

The new chemokine Prokineticin 2 (PROK2) and its receptors (PKR₁and PKR₂) have a role in inflammatory pain and immunomodulation. Here we identified PROK2 as a critical mediator of neuropathic pain in the chronic constriction injury (CCI) of the sciatic nerve in mice and demonstrated that blocking the prokineticin receptors with two PKR₁-preferring antagonists (PC1 and PC7) reduces pain and nerve damage. PROK2 mRNA expression was upregulated in the injured nerve since day 3 post injury (dpi) and in the ipsilateral DRG since 6 dpi. PROK2 protein overexpression was evident in Schwann Cells, infiltrating macrophages and axons in the peripheral nerve and in the neuronal bodies and some satellite cells in the DRG. Therapeutic treatment of neuropathic mice with the PKR-antagonist, PC1, impaired the PROK2 upregulation and signalling. This fact, besides alleviating pain, brought down the burden of proinflammatory cytokines in the damaged nerve and prompted an anti-inflammatory repair program. Such a treatment also reduced intraneural oedema and axon degeneration as demonstrated by the physiological skin innervation and thickness conserved in CCI-PC1 mice. These findings suggest that PROK2 plays a crucial role in neuropathic pain and might represent a novel target of treatment for this disease.

Objectives It is recommended that the Jevtana (current parenteral cabazitaxel formulation) final infusion solution should be used within 8 h when stored at ambient temperature or within 24 h if refrigerated. We determined the physical and chemical stability of cabazitaxel and docetaxel over prolonged periods after dilution in infusion solutions from their Jevtana and Hospira, respectively, parenteral formulations. Methods The stability of these antineoplastic drugs was determined after (i) reconstitution of the injection concentrate and (ii) further dilution in 0.9% NaCl solution contained in PVC-free infusion bags. Chemical stability was determined using both high-performance liquid chromatography (HPLC) with ultraviolet detection and high-resolution (HR)-HPLC–mass spectrometry (MS) techniques. Physical stability was determined by visual inspection. Results The stability tests revealed that reconstituted cabazitaxel solutions (premix solutions) stored at 4°C were physicochemically stable (at a level of ≥95% cabazitaxel) for a minimum of 4 weeks. Diluted infusion solutions in PVC-free infusion bags (docetaxel concentration 0.30 mg/mL; cabazitaxel concentration 0.15 mg/mL) were physicochemically stable (at a level of ≥95% cabazitaxel or docetaxel) for a minimum of 4 weeks, independently of storage temperature (4°C or 25°C). Diluted cabazitaxel infusion solutions appeared stable (at a level of ≥95% cabazitaxel) for a minimum of 4 weeks when stored in the presence of saturated oxygen at 25°C. Conclusions Cabazitaxel and docetaxel are characterised by high stability in customary infusion fluids for at least 4 weeks.

Background and Purpose: An innovative chemical approach, named peptide welding technology (PWT), allows the synthesis of multibranched peptides with extraordinary high yield, purity and reproducibility. With this approach, three different tetrabranched derivatives of nociceptin/orphanin FQ (N/OFQ) have been synthesized and named PWT1‐N/OFQ, PWT2‐N/OFQ and PWT3‐N/OFQ. In the present study we investigated the in vitro and in vivo pharmacological profile of PWT N/OFQ derivatives and compared their actions with those of the naturally occurring peptide.Experimental Approach: The following in vitro assays were used: receptor and [³⁵S]‐GTPγS binding, calcium mobilization in cells expressing the human N/OFQ peptide (NOP) receptor, or classical opioid receptors and chimeric G proteins, electrically stimulated mouse vas deferens bioassay. In vivo experiments were performed; locomotor activity was measured in normal mice and in animals with the NOP receptor gene knocked out [NOP(−/−)].Key Results: In vitro PWT derivatives of N/OFQ behaved as high affinity potent and rather selective full agonists at human recombinant and animal native NOP receptors. In vivo PWT derivatives mimicked the inhibitory effects exerted by the natural peptide on locomotor activity showing 40‐fold higher potency and extremely longer lasting action. The effects of PWT2‐N/OFQ were no longer evident in NOP(−/−) mice.Conclusions and Implications: The results showed that the PWT can be successfully applied to the peptide sequence of N/OFQ to generate tetrabranched derivatives characterized by a pharmacological profile similar to the native peptide and associated with a higher potency and marked prolongation of action in vivo.

The synthesis of non natural amino acid 2-amino-3,3,4-trimethyl-pentanoic acid (Ipv) ready for solid phase peptide synthesis has been developed. Copper (I) chloride Michael addition, followed by a Curtius rearrangement are the key steps for the Ipv synthesis. The racemic valine/leucine chimeric amino acid was then successfully inserted in position 5 of neuropeptide S (NPS) and the diastereomeric mixture separated by reverse phase HPLC. The two diastereomeric NPS derivatives were tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPS receptor where they behaved as partial agonist and pure antagonist.