Neurotensin Analogues Containing Cyclic Surrogates of Tyrosine at Position 11 Improve NTS2 Selectivity Leading to Analgesia without Hypotension and Hypothermia.

Neurotensin (NT) exerts its analgesic effects through activation of the G protein-coupled receptors NTS1 and NTS2. This opioid-independent antinociception represents a potential alternative for pain management. While activation of NTS1 also induces a drop in blood pressure and body temperature, NTS2 appears to be an analgesic target free of these adverse effects. Here, we report modifications of NT at Tyr11 to increase selectivity toward NTS2, complemented by modifications at the N-terminus to impair proteolytic degradation of the biologically active NT(8-13) sequence. Replacement of Tyr11 by either 6-OH-Tic or 7-OH-Tic resulted in a significant loss of binding affinity to NTS1 and subsequent NTS2 selectivity. Incorporation of the unnatural amino acid ß3hLys at position 8 increased the half-life to over 24 h in plasma. Simultaneous integration of both ß3hLys8 and 6-OH-Tic11 into NT(8-13) produced a potent and NTS2-selective analogue with strong analgesic action after intrathecal delivery in the rat formalin-induced pain model with an ED50 of 1.4 nmol. Additionally, intravenous administration of this NT analogue did not produce persistent hypotension or hypothermia. These results demonstrate that NT analogues harboring unnatural amino acids at positions 8 and 11 can enhance crucial pharmacokinetic and pharmacodynamic features for NT(8-13) analogues, i.e., proteolytic stability, NTS2 selectivity, and improved analgesic/adverse effect ratio.

The neurotensin-1 receptor agonist PD149163 inhibits conditioned avoidance responding without producing catalepsy in rats.

Agonists for neurotensin (NT)-1 receptors have produced antipsychotic-like effects in many animals, including reversal of prepulse inhibition deficits and psychostimulant-induced increases in spontaneous activity. The present study sought to provide a basic assessment of the putative antipsychotic effects of PD149163 in rats using a two way conditioned avoidance response task, which is highly validated for screening antipsychotic drugs, and an inclined grid assessment, which is used to assess extrapyramidal side effect liability. PD149163 (0.0625-8.0 mg/kg) significantly suppressed conditioned avoidance responding (CAR) following administration of a 1.0 or 8.0 mg/kg dose. PD149163 failed to significantly increase catalepsy scores. The typical antipsychotic drug haloperidol (0.01-1.0 mg/kg) significantly suppressed CAR at a 0.1, 0.3, and 1.0 mg/kg dose, and a significant increase in catalepsy scores was found at the 1.0 mg/kg dose. The atypical antipsychotic drug clozapine (2.5-10.0 mg/kg) also produced a significant inhibition of CAR, which occurred following administration of a 10.0 mg/kg dose. Clozapine failed to significantly increase catalepsy scores. Finally, D-amphetamine (1.0 mg/kg), serving as a negative control, failed to suppress CAR or increase catalepsy scores. These data further suggest that PD149163 may have atypical antipsychotic-like properties.

Design and in vitro evaluation of branched peptide conjugates: turning nonspecific cytotoxic drugs into tumor-selective agents.

The use of peptide receptors as targets for tumor-selective therapies was envisaged years ago with the findings that receptors for different endogenous regulatory peptides are overexpressed in several primary and metastatic human tumors, and can be used as tumor antigens. Branched peptides can retain or even increase, through multivalent binding, the biological activity of a peptide and are very resistant to proteolysis, thus having a markedly higher in vivo activity compared with the corresponding monomeric peptides. Oligo-branched peptides, containing the human regulatory peptide neurotensin (NT) sequence, have been used as tumor-specific targeting agents. These peptides are able to selectively and specifically deliver effector units, for cell imaging or killing, to tumor cells that overexpress NT receptors. Results obtained with branched NT conjugated to different functional units for tumor imaging and therapy indicate that branched peptides are promising novel multifunctional targeting molecules. This study is focused on the role of the releasing pattern of drug-conjugated branched NT peptides. We present results obtained with oligo-branched neurotensin peptides conjugated to 6-mercaptopurin (6-MP), combretastain A-4 (CA4) and monastrol (MON). Drugs were conjugated to oligo-branched neurotensin through different linkers, and the mode-of-release, together with cytotoxicity, was studied in different human cancer cell lines. The results show that branched peptides are very promising pharmacodelivery options. Among our drug-armed branched peptides, NT4-CA4 was identified as a candidate for further development and evaluation in preclinical pharmacokinetic and pharmacodynamic studies. This peptide-drug exhibits significant activity against pancreas and prostate human cancer cells. Consequently, this derivative is of considerable interest due to the high mortality rates of pancreas neuroendocrine tumors and the high incidence of prostate cancer.

Antinociceptive, hypothermic, hypotensive, and reinforcing effects of a novel neurotensin receptor agonist, NT69L, in rhesus monkeys.

Neurotensin (NT) is a tridecapeptide found in the nervous system, as well as elsewhere in the body. It has anatomic and functional relationships to dopaminergic neurons in brain. NT has been implicated in the actions of antipsychotic drugs and psychostimulants, and animal studies suggest that neurotensin directly injected into brain has reinforcing effects. Previously, we showed that one of our brain-penetrating analogs of neurotensin, NT69L (N-methyl-L-Arg, L-Lys, L-Pro, L-neo-Trp, L-tert-Leu, L-Leu), has many pharmacological effects in rats including antinociception, hypothermia, and blockade of the hyperactivity caused by psychostimulants (cocaine, D-amphetamine, and nicotine). Since these studies in rats suggest that this compound may have clinical use in humans, we were interested to know what effects NT69L had in primates. NT69L caused a potent antinociceptive effect against capsaicin (0.1 mg)-induced allodynia in 46 degrees C water in rhesus monkeys, inducing 40% of the maximal possible effect at an intravenous dosage of 0.03 mg/kg; its hypotensive effects precluded evaluation of higher dosages. Core temperature measured by rectal probe was modestly reduced at 0.01 and 0.03 mg/kg. In an intravenous self-administration procedure, NT69L was without reinforcing effects at any dose, including those that caused other pharmacological effects, and did not alter cocaine-maintained behavior when administered as a pretreatment.

[Effect of intracerebral administration of somatostatin and neurotensin on posture and movement disorders]. / Pozo-rukhovi porushennia pry vnytrishnómozkovykh vvedenniakh somatostatyny ta neirotenzynu.

Experiments were performed on male Wistar rats to study posture-movement disorders after intracerebroventricular, intraamygdalar, intrahippocampal and intranigral (pars reticulata) injections of somatostatin and neurotensin. Development of movement disturbances characterized by changes in the posture-movement syndrome was observed after somatostatin and neurotensin administration. Manifestation of movement disorders, their duration and the posture-movement syndrome structure depended on the structure to which neuropeptides were injected as well as on the neuropeptides themselves. Prolonged changes characterized by appearance of the so called "Sphinx" posture in animals developed after somatostatin intranigral administration. The obtained data permit concluding that somatostatin and neurotensin may have pathogenic significance in cataleptic syndrome development.

Neurotensin and neurotensin analogues modify the effects of chronic neuroleptic administration in the rat.

The effects of intracerebroventricular neurotensin and the neurotensin analogues neuromedin N and [D-Trp 11]neurotensin on the behavioural responses to chronic neuroleptic administration were investigated in the rat. Chronic (18 weeks) administration of a low dose (12.5 mg/kg, i.m., every 3 weeks) of fluphenazine decanoate alone failed to elicit the vacuous chewing mouth movements (VCMs) which have previously been reported following higher doses of this drug, but VCMs were seen in neuroleptic-treated animals following the additional administration of neurotensin. A higher dose of fluphenazine (25 mg/kg, i.m., every 3 weeks) greatly increased the VCM response, and this potentiation was suppressed to control levels by [D-Trp11]neurotensin, but unaffected by neuromedin N. These findings suggest that alterations in neurotensin may contribute to the deleterious extrapyramidal effects of long-term neuroleptic administration, and that [D-Trp11]neurotensin may attenuate these effects by blockade of neurotensin receptors within the central nervous system.

Effect of initial blood pressure level on cardiovascular reflexes caused by intraperitoneal neurotensin.

The reflex changes of systemic blood pressure (BP) and heart rate (HR) induced by intraperitoneal (IP) injections of neurotensin-containing solutions (NTCS) were measured in anesthetized guinea pigs whose BP was raised up by intravenous (IV) infusions of either angiotensin II (ATII) or noradrenaline (NA), and compared to those observed in control animals (i.e., saline-infused guinea pigs with low initial BP level). The amplitudes of reflex pressor and tachycardic responses to IP NTCS were either not affected or reduced in animals whose BP was raised with ATII or NA. However, no hypotensive effect was observed, following IP NTCS, in any of the animals tested. The results indicate that high initial BP levels tend to attenuate the reflex increases of BP and HR caused by IP NTCS, the level of inhibition being largely determined by the type of vasopressor agent utilized to raise up the BP. They also suggest that the initial BP level is unlikely to be an important factor in determining whether the activation of capsaicin-sensitive visceral afferents will produce hypotensive or hypertensive effects in this animal model.

Promotion by neurotensin of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The effects of neurotensin on the incidence and histology of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine were investigated in Wistar rats. Rats were given 100 or 200 micrograms per kg of body weight of neurotensin s.c. every other day in depot form after 25 wk of p.o. treatment with the carcinogen. Prolonged alternate-day administration of neurotensin at 200 micrograms per kg of body weight resulted in a significant increase in the incidence of gastric cancers of the glandular stomach by Wk 52. However, it did not influence the histological appearance of the gastric cancers. Furthermore, it caused a significant increase in the labeling indices of the epithelial cells of the antrum and of gastric cancers. In contrast, the administration of neurotensin at 100 micrograms per kg of body weight had a slight, but not significant, influence on the development of gastric cancers. These findings indicate that neurotensin promotes gastric carcinogenesis, and that this effect may be related to its effect in increasing proliferation of epithelial cells in the antral mucosa and in gastric cancers.

Aphagia in the rat following microinjection of neurotensin into the ventral tegmental area.

Neurotensin was microinjected into the lateral cerebral ventricle and the ventral tegmental area of rats which had been deprived of food for 18 hours. Both routes of administration resulted in a significant reduction of food intake compared to vehicle control injections. Additionally, the dose of neurotensin required to produce aphagia following ventral tegmental injection was substantially less than the dose required by the ventricular route. The results are discussed in relation to a possible site and mode of action for this neuropeptide.