Pharmacologic management of periodontal diseases using systemically administered agents.

Since the establishment of bacteria-laden plaque as a causative agent in gingivitis, the search for specific bacteria that induce different types of periodontitis has generated extensive research. In contrast to many other microbial-induced disorders, the specific periodontal pathogen(s) has not been identified to date. Therefore, the search for an effective systemic agent to prevent the loss of attachment through the selective reduction of known periodontal pathogens has remained elusive. It is not surprising then that antibiotics are not used solely to manage periodontal diseases but rather as an adjunct to the mechanical débridement of root surfaces in select periodontal diseases. Further, the sole use of antibiotics in patients with adult periodontitis (or those who exhibit signs of inflammation but are periodontally stable) has shown little benefit and only increases the chance of microbial resistance to antibiotics. Despite these limitations, considerable progress in antibiotic therapy has delivered regimens that enhance the effectiveness of conventional therapy. In contrast to traditional antimicrobial therapy, new treatment modalities have begun to focus on modulating the responses of host cells to bacteria rather than modulating only the bacteria. Current drugs used to regulate host cells inhibit the cyclooxygenase pathway, reduce the activity of metalloproteinases, or inactivate bone resorptive cells (see Table 1). Although these drugs offer great potential to modulate a variety of mammalian cells, a notable and consequential limitation of these agents is a lack of specificity. Inflammation, bone metabolism, and connective tissue metabolism are two-edged swords; all are necessary for the homeostasis of the tissue, but some or all may also be involved in the pathologic destruction of that same tissue. Hence, drugs that inhibit destruction of the connective tissue in one site of the periodontium also interfere with wound healing at another. As a result of these limitations, the efficacy, safety, and cost-effectiveness of the long-term use of these agents is unknown. Preliminary results of treatment with these drugs are promising, and future generations of host-modulating drugs will provide clinicians with additional agents to help improve the success rate of periodontal treatment for patients. Antibiotics remain an important adjunctive therapy in the treatment of periodontal diseases, and the use of host modulating drugs as supplemental agents in the management of periodontal diseases continues to grow. As more knowledge is gained about the causes of periodontal diseases, new drugs that are potent, effective, site specific, and safe can be delivered at optimal times by simply having the patient take a few tablets. Considering the dramatic progress in the past decade in understanding the cause and pharmacologic management of periodontal diseases, the twenty-first century holds great promise for the development of magic bullets.

Dose-dependent pain-facilitatory and -inhibitory actions of neurotensin are revealed by SR 48692, a nonpeptide neurotensin antagonist: influence on the antinociceptive effect of morphine.

Neurotensin has bipolar (facilitatory and inhibitory) effects on pain modulation that may physiologically exist in homeostasis. Facilitation predominates at low (picomolar) doses of neurotensin injected into the rostroventral medial medulla (RVM), whereas higher doses (nanomolar) produce antinociception. SR 48692, a neurotensin receptor antagonist, discriminates between receptors mediating these responses. Consistent with its promotion of pain facilitation, the minimal antinociceptive responses to a 30-pmol dose of neurotensin microinjected into the RVM were markedly enhanced by prior injection of SR 48692 into the site (detected using the tail-flick test in awake rats). SR 48692 had a triphasic effect on the antinociception from a 10-nmol dose of neurotensin. Antinociception was attenuated by femtomolar doses, attenuation was reversed by low picomolar doses (corresponded to those blocking the pain-facilitatory effect of neurotensin) and the response was again blocked, but incompletely, by higher doses. The existence of multiple neurotensin receptor subtypes may explain these data. Physiologically, pain facilitation appears to be a prominent role for neurotensin because the microinjection of SR 48692 alone causes some antinociception. Furthermore, pain-facilitatory (i.e., antianalgesic) neurotensin mechanisms dominate in the pharmacology of opioids; the response to morphine administered either into the PAG or systemically was potentiated only by the RVM or systemic injection of SR 48692. On the other hand, reversal of the enhancement of antinociception occurred under certain circumstances with SR 48692, particularly after its systemic administration.

Biochemical and pharmacological characterization of multiple beta-endorphinergic antinociceptive systems in the rat periaqueductal gray.

Based on the differential abilities of the opioid antagonists naltrexone and D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP) to antagonize the antinociceptive action of beta-endorphin and morphine in the rat periaqueducatal gray (PAG), three pharmacologically distinct mechanisms were determined to mediate the antinociceptive effect of beta-endorphin. Two of these mechanisms are unique to beta-endorphin, possess a high affinity for CTP and can be discriminated based on their differential sensitivity to naltrexone. The third mechanism displays characteristics common to that activated by morphine. The results of radioligand binding studies were consistent with these observations. [125I]-beta-Endorphin labeled a population of sites in the PAG which (compared to those labeled by [3H]morphine) displayed a significantly higher affinity for CTP. In addition, a naltrexone-insensitive binding component was identified in the [125I]-beta-endorphin, but not [3H]morphine assays. Furthermore, comparable competitor affinities were determined across assays, suggesting an interaction of the radioligands with common PAG sites. A naltrexone-insensitive component to beta-endorphin antinociception also was identified in studies which evaluated the ability of the antagonist to shift the beta-endorphin dose-response curve. Interestingly, the ability of low doses of CTP and naltrexone to inhibit increasing doses of beta-endorphin was described by a U-shaped dose effect curve. The response to low and high, but not intermediate, doses of beta-endorphin were antagonized by picomole doses of both antagonists. As there was no evidence for allosteric interactions between [125I]-beta-endorphin binding sites in the PAG, it appears that beta-endorphin also may activate pain facilitory mechanisms which counterbalance its overall antinociceptive effect.

Spinal nonadrenergic imidazoline receptors do not mediate the antinociceptive action of intrathecal clonidine in the rat.

The intrathecal administration of clonidine to rats results in profound antinociception which is thought to be mediated through an interaction of the agonist with spinal alpha-2 adrenergic receptors. However, clonidine has been shown to also interact with nonadrenergic imidazoline receptors. Consequently, this study was undertaken to determine if nonadrenergic imidazoline receptors are present in the rat spinal cord, and the extent to which they are involved in the antinociceptive action of spinally administered clonidine. By using the tail-flick test, the antinociceptive action of spinally administered clonidine was found to be blocked completely by the intrathecal administration of the imidazoline idazoxan. Similarly, yohimbine (a nonimidazoline alpha-2 adrenergic antagonist) also blocked completely the antinociceptive action of clonidine. Results of radioligand binding studies demonstrated that norepinephrine did not interact with approximately 20% of all specific spinal sites labeled by 4 nM [3H]clonidine, indicating the presence of nonadrenergic spinal sites. Affinity data obtained from competition binding assays demonstrated that the spinal nonadrenergic sites labeled by [3H]clonidine possess little affinity for yohimbine. Therefore, nonadrenergic imidazoline receptors are not involved in the antinociceptive action of spinally administered clonidine.

Opioid effects on spinal [3H]5-hydroxytryptamine release are not related to their antinociceptive action.

Several opioid compounds were evaluated for an ability to modulate the K(+)-stimulated release of [3H]serotonin ([3H]5-hydroxytryptamine, [3H]5-HT) from rat spinal cord synaptosomal and tissue slice preparations. Selective kappa-opioid receptor agonists depressed K(+)-stimulated release of the radiolabelled transmitter from both tissue preparations, an effect which was reversed by norbinaltorphimine. Conversely, the selective mu- and delta-opioid receptor agonists [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO) and [D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, enhanced the K(+)-stimulated release of [3H]5-HT. This effect was only seen using the tissue slice preparation. When used at concentrations near its reported Kd for mu-opioid receptors, the selective mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) blocked the action of DAMGO, but had no effect on the action of DPDPE. However, higher concentrations of CTOP, as well as all effective concentrations of selective delta-opioid receptor antagonists, blocked the action of both DAMGO and DPDPE. All agonist effects on spinal 5-HT release, regardless of the tissue preparation, were only seen at high (microM) concentrations. Moreover, effects of the opioid agonists were not consistent with the reported involvement of spinal 5-HT neurotransmission in the mediation of their antinociceptive action. Thus, the ability of opioids to modulate spinal 5-HT release appears to be of minimal physiological significance.

Repetitive exposure to the hot-plate test produces stress induced analgesia and alters beta-endorphin neuronal transmission within the periaqueductal gray of the rat.

Repetitive exposure of rats to a hot plate induced a novel non-opioid form of stress induced analgesia. The exposure caused a persistent 1.5-2 s increase in tail flick latency which was not attenuated by systemic naltrexone, but was completely inhibited by systemic MK-801. Concomitantly, alterations occurred in the ability to pharmacologically distinguish multiple beta-endorphin receptors in the periaqueductal gray. Thus, in response to different forms of stress, different pathways may be activated by beta-endorphin, resulting in stress induced analgesias with varied pharmacological characteristics (e.g., opioid and non-opioid).

5-HT3 receptors which modulate [3H]5-HT release in the guinea pig hypothalamus are not autoreceptors.

The 5-HT3 agonist 2-methyl-5-HT had previously been shown to enhance the electrically evoked release of [3H]5-HT from preloaded slices of the guinea pig brain. In the present study, 2-methyl-5-HT (1 microM) was also found to increase the K+ evoked release of [3H]5-HT from preloaded slices of the guinea pig hypothalamus and this effect was blocked by the selective 5-HT3 antagonist ondansetron. In the presence of tetrodotoxin, the enhancement of the K(+)-evoked release of [3H]5-HT by 2-methyl-5-HT in hypothalamus slices was blocked, thus suggesting that the 5-HT3 receptors mediating this effect are not located directly on 5-HT terminals. In agreement with this, 2-methyl-5-HT did not alter the K(+)-evoked release of [3H]5-HT in a synaptosomal preparation of the same brain structure, even at a concentration 10-fold greater than that used in the slices. Taken together, these data indicate that these facilitatory 5-HT3 receptors are not located on 5-HT terminals in the guinea pig hypothalamus and therefore are not autoreceptors.

Antinociception from the administration of beta-endorphin into the periaqueductal gray of rat is enhanced while that of morphine is inhibited by barbiturate anesthesia.

Pentobarbital anesthesia causes about a 10-fold increase in the antinociceptive potency of beta-endorphin microinjected into the periaqueductal gray (PAG) region of the rat brain. The antinociceptive response to PAG morphine was markedly attenuated during anesthesia, but returned as the rats regained consciousness. As they recovered from anesthesia, muscular rigidity and body stiffness (catalepsy) also occurred in the pentobarbital treated animals receiving morphine. These results are consistent with the activation of separate and distinct descending pain inhibitory neuronal systems by these two opioid agonists, and the differential modulation of the systems by pentobarbital. They also suggest that the mechanism underlying muscular responses to morphine is sensitive to pentobarbital, and is not shared by beta-endorphin.

Opioid receptors mediating antinociception from beta-endorphin and morphine in the periaqueductal gray.

beta-Endorphin and morphine produce an increase in the latency of the tail-flick reflex when administered into the PAG of awake rats. The antinociceptive effect of both opioid agonists was blocked by the sequential local injection of either CTP (D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2), a selective mu opioid receptor antagonist, naltrexone, or beta-endorphin (1-27), a putative epsilon opioid receptor antagonist, with minimal selectivity. When either CTP or naltrexone was used as the antagonist, the dose-inhibition curves generated for beta-endorphin and morphine were not parallel, suggesting the involvement of separate and distinct receptors. Also, synergism occurred when a dose of morphine producing submaximum antinociception was administered simultaneously with either a submaximal or ineffective dose of beta-endorphin. Inhibition of the antinociceptive response to beta-endorphin by mu antagonists and the non-selective antagonism of both beta-endorphin and morphine by beta-endorphin (1-27) suggested that epsilon opioid receptors were not involved. Additionally, a mu/delta opioid receptor complex was not involved, since ICI 174,864 (Allyl2-Tyr-Aib-Aib-Phe-Leu-OH), a selective delta opioid receptor antagonist, did not alter the response to beta-endorphin. Thus, although additional characterization is required, beta-endorphin and morphine appear to act (at least in part) through different opioid receptors, demonstrable using selected mu opioid receptor antagonists.

Evaluation of the interactions of serotonergic and adrenergic drugs with mu, delta, and kappa opioid binding sites.

Several serotonergic and adrenergic agents were tested for an ability to interact with mu, delta, and kappa opioid binding sites. Spiroxatrine interacted nearly equipotently with all three opioid subtypes, yielding Ki values near 110 nM. A number of other serotonergic and adrenergic agents interacted with affinities in the 1-50 microM range. Most of the other compounds tested in this study were found to compete for opioid binding to some degree, though not achieving a 50% inhibition of binding at concentrations up to 100 microM. If this interaction between monoaminergic agents and opioid receptors is found to have functional significance, it must be considered in the interpretation of results from studies using these agents to evaluate the contribution of monoaminergic systems to opioid-mediated events.

Complement C5a receptor assay for high throughput screening.

The complement C5a receptor on U937 cells, a human histiocytic lymphoma cell line, stimulated with dibutyryl-cAMP have been stabilized for at least 3 months at a dilute, ready to use concentration. [125I]-Bolton Hunter labeled C5a, (recombinant, human) has been prepared by reverse phase HPLC to 2200 Ci/mmol. Using a filtration binding assay the Kd from receptor saturation analysis is 10-40 pM and there are 50,000-100,000 receptor sites per cell. These reagents have permitted the development of a reliable, reproducible and convenient drug screening assay, in kit format, for compounds acting at the C5a receptor.

Properties of the interaction between ketamine and opiate binding sites in vivo and in vitro.

Analgesia induced by ketamine appears to be partially mediated by opiate mechanisms. Not only is its action attenuated by the narcotic antagonist naloxone, but the drug has a weak affinity for, and interacts stereoselectively at, opiate receptors. It also produces a classical narcotic action on the guinea-pig ileum. The present study showed that analgesic doses of the drug in rats yielded concentrations sufficient to interact effectively at opiate binding sites in vivo. A dose-dependent (80-120 mg/kg i.p.) inhibition of the binding of [3H]naloxone was observed in both brain and spinal cord. All regions of the brain (except the cerebellum) were affected, but the reduction was significant in the cortex, hippocampus, thalamus and striatum. Thus, a component of ketamine-induced analgesia could be related to a functional interaction with opiate receptors. Additionally, ketamine may be similar to morphine in its preference for the mu, rather than the delta sub-type of opiate receptors, and thus may promote mu-mediated pharmacological effects. For example, in vitro studies of radioligand binding showed that ketamine and morphine were four times more effective in inhibiting the binding of [3H]dihydromorphine than that of [3H] [D-Ala2, D-Leu5] enkephalin. On the other hand, ketamine also effectively interacted at a component of the sigma opiate/phencyclidine binding sites that appears to be relatively insensitive to morphine. This component may be involved in dysphoria induced by ketamine.

[3H]spiroxatrine labels a serotonin1A-like site in the rat hippocampus.

[3H]Spiroxatrine was examined as a potential ligand for the labeling of 5-HT1A sites in the rat hippocampus. Analysis of the binding of [3H]spiroxatrine in the absence and presence of varying concentrations of three monoamine neurotransmitters revealed that serotonin (5-HT) had high affinity (IC50 = 20.7 nM for the [3H]spiroxatrine binding sites, consistent with the labeling of 5-HT1 sites, while dopamine and norepinephrine had very low affinity (IC50 = 57600 nM and greater than 10(-4) M respectively). Saturation studies of the binding of [3H]spiroxatrine revealed a single population of sites with a Kd = 2.21 nM. Further pharmacologic characterization with the 5-HT1A ligands 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, and WB4101 and the butyrophenone compounds spiperone and haloperidol gave results that were consistent with [3H]spiroxatrine labeling 5-HT1A sites. This ligand produced stable, reproducible binding with a good ratio of specific to nonspecific binding. The binding of [3H]spiroxatrine was sensitive to GTP, suggesting that this ligand may act as an agonist. This was supported by the finding that spiroxatrine inhibits forskolin-stimulated adenylate cyclase activity (a proposed 5-HT1A receptor model) in the rat hippocampus. Since [3H]spiroxatrine is structurally distinct from other currently available radioligands for the 5-HT1A site, it should provide new information about the properties of this putative serotonergic receptor.

Evaluation of the direct actions of drugs with a serotonergic link in spinal analgesia on the release of [3H]serotonin from spinal cord synaptosomes.

Morphine, ketamine, ethylketocyclazocine and quipazine, drugs with an apparent local spinal serotonergic action, which contributes to their analgesic effects, were tested for their ability to alter the release of [3H]serotonin ([3H]5-HT) from a synaptosomal preparation from the spinal cord of the rat. Related compounds including [D-Ala2, D-Leu5]enkephalin (DADLE), n-allylnormetazocine and phencyclidine were also examined. None of the drugs was found to be capable of inducing a direct release of [3H]5-HT or of facilitating potassium-induced release of 5-HT. However, quipazine inhibited the depressant action of exogenous 5-HT on overflow of 3H (mediated through the 5-HT autoreceptor), an action that should facilitate serotonergic neurotransmission. In contrast to the other drugs, DADLE was found to depress K+ stimulated release of 5-HT. The results suggests that the serotonergic mechanism involved in the antinociceptive action of some of these drugs (i.e. ketamine, morphine and ethyl-ketocyclazocine) is not related to direct presynaptic interactions to promote release of 5-HT. On the other hand, a small population of serotonergic nerves critical for analgesia may be involved and are not detected using tissue from the whole spinal cord, However, it seems equally plausible that these drugs may produce their antinociceptive action through interactions with other neurotransmitter systems that in turn interface with the serotonergic nerves, perhaps through interneurons or collateral connections.

Demonstration of an autoreceptor modulating the release of [3H]5-hydroxytryptamine from a synaptosomal-rich spinal cord tissue preparation.

A superfusion system employed to measure the K+-stimulated release of [3H]5-hydroxytryptamine [(3H]5-HT, [3H]serotonin) from a synaptosomal-rich spinal cord tissue preparation was carefully characterized, then used to examine the regulation of spinal 5-HT release. Spinal 5-HT release is apparently modulated by an autoreceptor. Exogenous 5-HT depressed, in a concentration-dependent manner, the K+-stimulated release of [3H]5-HT. Similarly, lysergic acid diethylamide (LSD) produced a concentration-dependent decrease in [3H]5-HT release. Methiothepin and quipazine blocked the inhibition of release induced by exogenous 5-HT. The 5-HT2 receptor antagonists spiperone and ketanserin failed to alter the action of 5-HT at the spinal 5-HT autoreceptor. Spiperone and ketanserin were shown, however, to alter the storage of [3H]5-HT. When used in concentrations greater than 10 nM, the drugs evoked increases in basal [3H]5-HT and [3H]5-hydroxyindoleacetic acid ( [3H]5-HIAA) effluxes which were independent of the presence of calcium ions. A good agreement existed between the potencies of drugs for modifying autoreceptor function and their abilities to compete for high-affinity [3H]5-HT binding in the spinal cord (designated 5-HT1). Furthermore quipazine, in concentrations that preferentially interact with the 5-HT1B subtype, antagonized the actions of exogenous 5-HT on K+-stimulated release. Spiperone, in a concentration that approximated the affinity constant of 5-HT1A sites for the drug, was ineffective in altering the ability of exogenous 5-HT to modulate K+-stimulated [3H]5-HT release. These results suggest that 5-HT1B sites are associated with serotonergic autoreceptor function in the spinal cord.