Development of analgesic-antiinflammatory drugs acting on pain sensory nerve endings for neurogenic inflammation, neuropathic pain and rheumatoid arthritis

It is a surprising fact, that neither analgetic target molecules or drugs with alternative mechanism of action have been identified during the last one hundred years of pharmaceutical research since the discovery of the widely used analgesics and antiinflammatory drugs of the late 19^th century. Opioid receptor agonists, paracetamol and cyclooxygenase (COX) inhibitors cover the entire scope of analgetics listed in pharmaceutical textbooks even today in contrast to the breakthrough in all areas of drug discovery.

Focus has been laid on increased specificity (e.g. selective COX-2 inhibitors), as well as potency (e.g. synthetic morphine derivatives), etc. Peripheral pain-sensory neurons have come into the focus of the research of János Szolcsányi and his group decades ago because of the fact that they may provide simple, specific targets potentially free of side effects on the central nervous system (CNS). Because neuropeptides released from the activated sensory nerve endings during pain sensation have been found to increase local blood flow, and cause plasma extravasation as well as induce accumulation of inflammatory cells the phenomenon has been called neurogenic inflammation. This set of pathophysiological changes initiated by sensory nerve endings have been suggested to play a significant role in the pathogenesis of numerous "idiopathic", "autoimmune" disorders like bronchial asthma, rheumatoid arthritis, allergic rhinitis, conjuctivitis, dermatitis and psoriasis.

Activation of the so called polymodal nociceptors in the sensory nerve endings elicits pain particularly during inflammatory processes in parallel with the release of proinflammatory neuropeptides. This sensory neuropeptide-induced "neurogenic" inflammatory process is suggested to accompany all inflammatory diseases when pain sensation is a cardinal symptom. Pain induces neurogenic inflammation, which in turn induces additional pain, generating a vicious circle. Among these neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) have been found the most important to elicit local neurogenic inflammation, vasodilatation and plasma extravasation).

Neurogenic inflammation is suggested to participate in all inflammatory processes to a variable extent, where nociception or pain sensation is present. This inflammatory response is resistant to COX inhibitors no matter of specificity e.g. non-steroidal antiinflammatory agents or selective COX-1/2 inhibitors. Application of opioid agonsits in clinical practice is limited by serious side-effects. It was described by this group, that a third potent neuropeptide is also released from the sensory nerve endings during painful conditions in addition to tachykinins and CGRP, identified as somatostatin. This endogenous anti-secretory and inhibitory hormone was found to be a potent anti-inflammatory and analgesic agent, but its numerous endocrine effects represent a serious limitation to its routine clinical application. Elucidation of the molecular pharmacology of TT-232 action has put the molecule into the front of drug discovery of neurogenic inflammation after it became clear, that this somatostatin-related action is mediated via SSTR4 receptors, the subtype of somatostatin receptors this drug was shown to inhibit with high specificity.

Rheumatoid arthritis affects about 0.5-1% of the population in the developed world. The therapeutic effect of native somatostatin on rheumatoid arthritis has been confirmed in the clinical setting, too.

Development of TT-232 and follow-up compounds

Our recent approach with a stable peptide somatostatin 4 receptor agonist, TT-232 opened another target on capsaicin-sensitive nociceptors. This compound induced in μg/kg doses or in nanomolar concentrations in vitro a marked inhibition of these capsaicin-sensitive nerve endings producing in this way analgesic and antiinflammatory effects. Sensory neuropeptide substance P released from these nerve endings induces neurogenic inflammation, which cannot be inhibited by COXinhibitors, but could be almost completely abolished by TT-232 pre-treatment. It was particularly striking that the compound markedly inhibited also the cartilage and bone destruction in the chronic arthritis model evoked in the rat by Freund’s complete adjuvant (Arthritis and Rheumatism 2004). Non-peptide TT-232 analogs might produce a breakthrough in this field because of their possible oral application.

General formula of compounds with TT-232 comparable biological effect in substance P release study.

Small molecule follow-up compounds of TT232 were designed from an in-house kinase-inhibitory molecular library, with the help of artificial intelligence softwares on the basis of chemical similarity with neurogenic or non-neurogenic anti-inflammatory as well as analgesic molecules. In this computed modelling we have used biological data on non-peptide somatostatin agonists to selected molecules to be screened in an in vitro substance P release assay and subsequently in animal models, in vivo. One molecule family (compounds VI87, VI460, VI4053, VI4055) of general formula I had significant biological effect comparable to TT-232 in a substance P release study.

Compounds of general formula I were originally developed in-house as kinase inhibitors, acting at the substrate binding-site. Although a complete peptidomimetic molecule library of 1600 very similar molecules were tested, only this compound family, with 13-14 Ao distance of the aromatic rings was found as active pp60c-src enzyme inhibitors. We have used this active (significant substance P release inhibitor) compound family I from our small molecular kinase inhibitory library as a starting point for generating non-peptide TT232 follow up molecules. In order to enhance biological availability we tried to replace the linker, connecting the aromatic rings, with more stabile and soluble moieties. L1, L3: -CH=, -C=N-, imidazole, pyrazole, pyrrole, pyridine, pyrimidine, pyrazolo-pyrimidines, pirrolo-pyrmidines, purines, any substitution on the above listed heteroaryls is also allowed L2:-NH-, cyclopentanone, cyclohexanone, pyran-4-one, N-methylpiperidi-4-one. X, Y: -OH, -O-alkyl, -O- [(CH)2]1,2-O-, -alkyl, halogene.

Inhibition of electrical field stimulation induced substance P release by TT-232 and phenylamino-oxoacetic derivatives (* shows the significant anti-inflammatory effect)

As a result of our approach we managed to optimise the structure in three iteration cycle and we found patent free drug-like small molecules (VI11498, VI11527, VI11530 and VI11562) of general formula II, having as good or better biological in vitro activity as the original TT-232 heptapeptide. The very promising biological activity and moreover the novelty of compounds made us to file a patent application in 2004. Starting out with compound family (II) we developed further two different patentable compound classes with the representative, pharmacologically active members VI16665 and VI17299.