Improved efficacy, tolerance, safety, and abuse liability profile of the combination of CR4056 and morphine over morphine alone in rodent models.

BACKGROUND AND PURPOSE: Prolonged use of opioids causes analgesic tolerance and adverse effects including constipation and dependence. Compounds targeting imidazoline I2 receptors are known to potentiate opioid analgesia in rodents. We investigated whether combination with the I2 receptor ligand CR4056 could improve efficacy and safety of morphine and explored the mechanisms of the CR4056-opioid interaction. EXPERIMENTAL APPROACH: We used the complete Freund's adjuvant (CFA) model in rats to study the effects of treatments on hyperalgesia, morphine tolerance and microglia activation as measured by immunofluorescence. Opioid-induced adverse effects were assessed in rodent models of morphine-induced constipation, sedation (open field, sedation rating scale, and rotarod), physical dependence (naloxone-induced withdrawal), and abuse (conditioned place preference-associated reward). Chemiluminescence assays tested CR4056 as allosteric modulator of µ-opioid receptors. KEY RESULTS: CR4056 (ED50 = 4.88 mg·kg-1 ) and morphine (ED50 = 2.07 mg·kg-1 ) synergized in reducing CFA-induced hyperalgesia (ED50 = 0.52 mg·kg-1 ; 1:1 combination). Consistently, low doses of CR4056 (1 mg·kg-1 ) spared one third of the cumulative morphine dose administered during 4 days and prevented/reversed the development of tolerance to morphine anti-hyperalgesia. These opioid-sparing effects were associated with decreased activation of microglia, independent of CR4056 interactions on µ-opioid receptors. Importantly, the low doses of CR4056 and morphine that synergize in analgesia did not induce constipation, sedation, physical dependence, or place preference. CONCLUSION AND IMPLICATIONS: We showed selective synergism between CR4056 and morphine as analgesics. Their combination showed an improved safety and abuse liability profile over morphine alone. CR4056 could be developed as an opioid-sparing drug in multimodal analgesia.

CR4056, a powerful analgesic imidazoline-2 receptor ligand, inhibits the inflammation-induced PKCε phosphorylation and membrane translocation in sensory neurons.

BACKGROUND AND PURPOSE: CR4056 is a first-in-class imidazoline-2 (I2 ) receptor ligand characterized by potent analgesic activity in different experimental animal models of pain. In a recent phase II clinical trial, CR4056 effectively reduced pain in patients with knee osteoarthritis. In the present study, we investigated the effects of CR4056 on PKCε translocation in vitro and on PKCε activation in vivo in dorsal root ganglia (DRG) neurons. EXPERIMENTAL APPROACH: Effects of CR4056 on bradykinin-induced PKCε translocation were studied in rat sensory neurons by immunocytochemistry. PKCε activation was investigated by immunohistochemistry analysis of DRG from complete Freund's adjuvant-treated animals developing local hyperalgesia. The analgesic activity of CR4056 was tested on the same animals. KEY RESULTS: CR4056 inhibited PKCε translocation with very rapid and long-lasting activity. CR4056 decreased hyperalgesia and phospho-PKCε immunoreactivity in the DRG neurons innervating the inflamed paw. The effect of CR4056 on PKCε translocation was blocked by pertussis toxin, implying that the intracellular pathways involved Gi proteins. The inhibition of PKCε translocation by CR4056 was independent of the α2 -adrenoeceptor and, surprisingly, was also independent of idazoxan-sensitive I2 binding sites. The I2 agonist 2BFI had no effect alone but potentiated the activity of low concentrations of CR4056. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that CR4056 shares the ability to inhibit PKCε translocation with other analgesics. Whether the inhibition of PKCε involves binding to specific subtype(s) of I2 receptors should be further investigated. If so, this would be a new mode of action of a highly specific I2 receptor ligand.

Post-thaw Addition of Caffeine and/or Pentoxifylline Affect Differently Motility of Horse and Donkey-Cryopreserved Spermatozoa.

To increase sperm motility, several molecules have been tested in mammals. Methylxanthines have shown effects on sperm motility, capacitation, and on in vitro fertilization processes. The aim of the study was to evaluate if the post-thaw addition of caffeine and/or pentoxifylline changes motility parameters of cryopreserved stallion and donkey spermatozoa. Straws derived from 14 horses and 7 donkeys were thawed and diluted in a milk-based extender to obtain the following final concentrations: CTR (control, no additives), CAF 5 (5 mM caffeine), CAF 10 (10 mM caffeine), PTX 5 (5 mM pentoxifylline), PTX 10 (10 mM pentoxifylline), CAF-PTX (5 mM caffeine and 5 mM pentoxifylline). Samples were evaluated immediately post-thaw and after 60 and 120 minutes of incubation at 37°C. In horses, overall total motility was significantly lower in CTR than in to CAF5, CAF-PTX, PTX5, PTX10, whereas progressive motility increased only in CAF5 and PTX5 (P < .05). No differences between control and treatments were seen for donkey semen. In CTR, during the first hour of incubation horses' sperm cells showed a larger decrease than donkeys' ones in all parameters (P < .05), except for lateral sperm head displacement. Thus, post-thaw motility and velocity decreased more sharply in horses than in donkeys. Caffeine and pentoxifylline-added post-thaw were able to increase the proportion of motile spermatozoa only for stallions and not for donkeys. Whether the improvement in post-thaw motility of equine spermatozoa may have an effect on in vivo or in vitro pregnancy rates remains to be determined.

Development of subnanomolar-affinity serotonin 5-HT4 receptor ligands based on quinoline structures.

Two small series of quinoline derivatives were designed starting from previously published quinoline derivatives 7a and b in order to obtain information about their interaction with the 5-HT4R binding site. Initially, the structure of 7a and b was modified by replacing their basic moiety with that of partial agonist 4 (ML10302) or with that of reference ligand 6 (RS-67-333). Then, the aromatic moieties of 4-quinolinecarboxylates 7a, d-f, and h-k or 4-quinolinecarboxamides 7b, c, and g were modified into those of 2-quinolinecarboxamides 9a-e. Very interestingly, this structure-affinity relationship study led to the discovery of 7h-j as novel 5-HT4R ligands showing K i values in the subnanomolar range. The structures of all these compounds contain the N-butyl-4-piperidinylmethyl substituent, which appear to behave as an optimized basic moiety in the interaction of these 4-quinolinecarboxylates with the 5-HT4R binding site. However, this basic moiety was ineffective in providing 5-HT4R affinity in the corresponding 4-quinolinecarboxamide 7g, but it did in 2-quinolinecarboxamide ligands 9c-e. Thus, a subtle interrelationship of several structural parameters appeared to play a major role in the interaction of the ligands with the 5-HT4R binding site. They include the kind of basic moiety, the position of the carbonyl linking group with respect to the aromatic moiety and its orientation, which could be affected by the presence of the intramolecular H-bond as in compounds 9c-e.

Variables affecting semen quality and its relation to fertility in the dog: A retrospective study.

Although semen analysis is routinely used to evaluate the male reproductive potential in the canine species, only a few authors have attempted to relate semen characteristics to donor traits, such as age or weight, and fertility. This study evaluated the effects of dogs' age (young: 0-24 months, adult: 25-84 months, old: >84 months), size (small: <15 kg, medium: 16-40 kg, large: >40 kg) and reason for semen collection (evaluation, artificial insemination, cryopreservation or research purposes) on the seminal characteristics and related the main seminal characteristics with fresh AI results. Overall, 251 semen examinations were performed from 140 dogs presented between 2000 and 2015 at the University of Pisa Veterinary Teaching Hospital. Conventional semen parameters (semen volume, sperm concentration and total number, subjective motility and sperm morphology) were evaluated. Overall, 227 (90.4%) samples contained spermatozoa. Azoospermic samples resulted from complete (n = 13, 9 dogs), or incomplete ejaculations (n = 11, 8 dogs), based on seminal alkaline phosphatase concentration and/or ancillary examinations. Reason for semen collection had a significant effect on semen volume, sperm total number and sperm concentration. The proportion of normal spermatozoa was lower in dogs presented for semen evaluation (P < 0.05). Dog size affected ejaculate volume and total sperm number, which was lower in small dogs compared to medium (P < 0.01 and P < 0.05, respectively) and large-sized dogs (P < 0.01). Age had no influence on volume, sperm number or motility. The proportion of normal spermatozoa was higher in young animals than in old ones (P < 0.05). There was a significantly higher proportion of midpiece defects in old dogs compared to young ones (P < 0.01). Total sperm number, motility and proportion of morphologically normal spermatozoa in the semen used in AIs resulting in a pregnancy were 627.6 × 106, 83.9% and 64.9%, respectively, all significantly higher than in the unsuccessful AIs, where they were 389.4 × 106, 66.5% and 42% (P < 0.05). In conclusion, age, size and reason for semen collection may affect main sperm parameters and must be taken into consideration during the interpretation of a routine semen evaluation. In turn, altered semen parameters may affect fertility after artificial insemination.

Reproductive parameters of donkey jacks undergoing puberty.

In male donkeys, puberty and the related events have been poorly characterized. The aim of this study was to evaluate the age at which male donkeys reach puberty, and characterize age associated changes in testicular size, testicular blood flow, serum testosterone concentration and semen quality. Every two months, starting at 6 months and finishing at 24 months of age, five male donkeys born in May to July were subjected to B-mode ultrasound examination to assess testicular size and scrotum content and blood serum sampling for testosterone concentration. From the age of 8 months, pulsed Doppler was employed to evaluate blood flow in the testicular artery. Testosterone serum concentration was evaluated via RIA. From the age of 12 months, monthly semen collections were attempted and semen was evaluated for sperm number, motility and morphology. Onset of puberty was defined as the first ejaculate containing ≥50 × 106 spermatozoa with ≥10% total motility. One of the donkeys was excluded from the statistical analyses due to a hydrocele presented during the study. Testes width was affected by age (P < 0.0001) and after an initial plateau increased linearly from 10 months of age. Pulsatility and resistivity indexes were also affected by age (P < 0.01), being significantly higher at 14 months than at 24 months. Testosterone serum concentration was affected by age (P < 0.0001) and was significantly lower at 6 months (0.1 ng/ml) compared to 22-24 months (≥0.8 ng/ml). Spermatozoa appeared in the ejaculate at a mean age of 18.7 months and puberty was attained between 19 and 20 months of age (mean: 19.5 months), between January and February. In conclusion, late spring born Amiata donkey colts reached puberty at 19-20 months of age. Puberty was accompanied by changes in testicular size, testicular blood flow and serum testosterone concentration.

Multivalent ligands for the serotonin 5-HT4 receptor.

5-HT4 receptors are known to form constitutive dimers in membranes. To explore whether multivalency can enhance ligand interactions and/or efficacy in 5-HT4 receptors, the structure of the partial agonist ML10302 was modified with oligo(ethylene glycol) chains, thus generating, by a gradual approach, short and long tethered bivalent or tetravalent ligands and the corresponding spanner-linked monovalent controls. Both bivalent and tetravalent ligands displayed a 10-20-fold increase in binding affinity compared to appropriate controls, but no multivalent ligand showed greater binding energy than ML10302 itself. Furthermore, the direct assessment of receptor-Gs interaction and studies of cAMP signalling indicated that multivalency does not enhance the efficacy of ML10302.

Phenylindenone isomers as divergent modulators of p38α MAP kinase.

Two new fluorophenylindenone derivatives were designed as potential p38α MAPK modulators by preserving the key interactions of the vicinal pyridine/fluorophenyl pharmacophore with the enzyme protein. Interestingly, these two fluorophenylindenone isomers showed divergent activities, with compound 6 behaving as an inhibitor and 5 as a putative activator. These results were rationalized by docking studies and molecular dynamics simulations in terms of stabilization of DFG loop, by compound 5 in a conformation more accessible to phosphorylation.

Biological profile and bioavailability of imidazoline compounds on morphine tolerance modulation.

Tolerance to opioid administration represents a serious medical alert in different chronic conditions. This study compares the effects of the imidazoline compounds 1, 2, and 3 on morphine tolerance in an animal model of inflammatory pain in the rat. 1, 2, and 3 have been selected in that, although bearing a common scaffold, preferentially bind to α2-adrenoceptors, imidazoline I2 receptors, or both systems, respectively. Such compounds have been tested in vivo by measuring the paw withdrawal threshold to mechanical pressure after complete Freund's adjuvant injection. To determine the ligand levels in rat plasma, an HPLC-mass spectrometry method has been developed. All the compounds significantly reduced the induction of morphine tolerance, showing different potency and duration of action. Indeed, the selective imidazoline I2 receptor interaction (2) restored the analgesic response by maintaining the same time-dependent profile observed after a single morphine administration. Differently, the selective α2C-adrenoceptor activation (1) or the combination between α2C-adrenoceptor activation and imidazoline I2 receptor engagement (3) promoted a change in the temporal profile of morphine analgesia by maintaining a mild but long lasting analgesic effect. Interestingly, the kinetics of compounds in rat plasma supported the pharmacodynamic data. Therefore, this study highlights that both peculiar biological profile and bioavailability of such ligands complement each other to modulate the reduction of morphine tolerance. Based on these observations, 1-3 can be considered useful leads in the design of new drugs able to turn off the undesired tolerance induced by opioids.

Induction of ovulation with buserelin in jennies: in search of the minimum effective dose.

The aim of this study was to evaluate the minimum dose of buserelin acetate (buserelin) able to induce ovulation between 24 and 48 h from treatment (positive response) in estrous jennies. Jennies were studied during a total of 172 estrous cycles: ovarian activity was routinely monitored by ultrasound; when the dominant follicle reached a diameter of 33 ± 1 mm, estrous jennies were treated by subcutaneous administration of different doses of buserelin, 3.3mg (N = 11), 1.5mg (N = 21), 0.8 mg (N = 12), 0.4 mg (N = 16), 0.2mg (N = 13), 0.1mg (N = 16), 0.04 mg (N = 14), 0.02 mg (N = 16), or employed as controls (N = 53). Single jennies (P = 0.0001) and GnRH dose (P = 0.003) significantly affected ovulation rates. Ovulation rates between 24 and 48 h of each treated group, except for the 0.02 mg group, was higher than in the control group (P < 0.05). The minimum dose of buserelin effective to induce ovulation in estrous jennies was 0.04 mg.

Synthesis and structure-activity relationship studies in serotonin 5-HT4 receptor ligands based on a benzo[de][2,6]naphthridine scaffold.

A small series of serotonin 5-HT4 receptor ligands has been designed from flexible 2-methoxyquinoline compounds 7a,b by applying the conformational constraint approach. Ligands 7a,b and the corresponding conformationally constrained analogues 8a-g were synthesized and their interactions with the 5-HT4 receptor were examined by measuring both binding affinity and the ability to promote or inhibit receptor-G protein coupling. Ester derivative 7a and conformationally constrained compound 8b were demonstrated to be the most interesting compounds showing a nanomolar 5-HT4R affinity similar to that shown by reference ligands cisapride (1) and RS-23,597-190 (4). The result was rationalized by docking studies in term of high similarity in the binding modalities of flexible 7a and conformationally constrained 8b. The intrinsic efficacy of some selected ligands was determined by evaluating the receptor-G protein coupling and the results obtained demonstrated that the nature and the position of substituents play a critical role in the interaction of these ligands with their receptor.

Exploring multitarget interactions to reduce opiate withdrawal syndrome and psychiatric comorbidity.

Opioid addiction is often characterized as a chronic relapsing condition due to the severe somatic and behavioral signs, associated with depressive disorders, triggered by opiate withdrawal. Since prolonged abstinence remains a major challenge, our interest has been addressed to such objective. Exploring multitarget interactions, the present investigation suggests that 3 or its (S)-enantiomer and 4, endowed with effective α2C-AR agonism/α2A-AR antagonism/5-HT1A-R agonism, or 7 and 9-11 producing efficacious α2C-AR agonism/α2A-AR antagonism/I2-IBS interaction might represent novel multifunctional tools potentially useful for reducing withdrawal syndrome and associated depression. Such agents, lacking in sedative side effects due to their α2A-AR antagonism, might afford an improvement over current therapies with clonidine-like drugs.

Fibromyalgia: nosography and therapeutic perspectives.

Fibromyalgia (FM) is an important cause of morbidity and health expenditure. Severe widespread extra-articular chronic pain, along with nonrestorative sleep, dominates the clinical syndrome. The pathogenesis of FM remains unclear. While dysfunction in serotoninergic neurotransmission is believed to play an important role, several neurologic and immuno-endocrine mechanisms may also be relevant. A theory is advanced based on an inherited imbalance in neuro-vegetative systems resulting from increased sympathetic tone because of a metabolic deficiency in the serotoninergic system that, when exposed to a precipitating event, leads to the development of the clinical manifestations of FM. The importance of both nonpharmacological treatments and multimodal medication management is stressed.