Selective extraction of small and large molecules from the cerebrospinal fluid by Purkinje neurons.

Cerebellar Purkinje neurons accumulated propidium iodide, granular blue, and horseradish peroxidase conjugated to wheat germ agglutinin but not unconjugated horseradish peroxidase, bisbenzimide, or Evans blue when these compounds were infused into the lateral cerebral ventricles of awake, unrestrained rats. Accumulation of propidium iodide by Purkinje neurons of the vermis was associated with a reproducible behavioral abnormality characterized by truncal tremor, ataxia, and nystagmus. Both the accumulation of propidium iodide in Purkinje cells and the behavioral abnormality were prevented by prior intracerebroventricular administration of ouabain or colchicine, drugs that block neuronal transport processes. The ability of cerebellar Purkinje neurons to extract small and large molecules from the cerebrospinal fluid has important implications for their physiology and pathology.

The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells.

Human multiple myeloma (MM) is a presently incurable hematological malignancy, and novel biologically based therapies are urgently needed. Proteasome inhibitors represent a novel potential anticancer therapy. In this study, we demonstrate that the proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis of human MM cell lines and freshly isolated patient MM cells; inhibits mitogen-activated protein kinase growth signaling in MM cells; induces apoptosis despite induction of p21 and p27 in both p53 wild-type and p53 mutant MM cells; overcomes drug resistance; adds to the anti-MM activity of dexamethasone; and overcomes the resistance to apoptosis in MM cells conferred by interleukin-6. PS-341 also inhibits the paracrine growth of human MM cells by decreasing their adherence to bone marrow stromal cells (BMSCs) and related nuclear factor kappaB-dependent induction of interleukin-6 secretion in BMSCs, as well as inhibiting proliferation and growth signaling of residual adherent MM cells. These data, therefore, demonstrate that PS-341 both acts directly on MM cells and alters cellular interactions and cytokine secretion in the BM millieu to inhibit tumor cell growth, induce apoptosis, and overcome drug resistance. Given the acceptable animal and human toxicity profile of PS-341, these studies provide the framework for clinical evaluation of PS-341 to improve outcome for patients with this universally fatal hematological malignancy.

Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: implications for systemic nuclear factor-kappaB inhibition.

Inducible activation of nuclear factor-kappaB (NF-kappaB) inhibits the apoptotic response to chemotherapy and irradiation. Activation of NF-kappaB via phosphorylation of an inhibitor protein IkappaB leads to degradation of IkappaB through the ubiquitin-proteasome pathway. We hypothesized that inactivation of proteasome function will inhibit inducible NF-kappaB activation, thereby increasing levels of apoptosis in response to chemotherapy and enhancing antitumor effects. To assess the effects of proteasome inhibition on chemotherapy response, human colorectal cancer cells were pretreated with the dipeptide boronic acid analogue PS-341 (1 microM) prior to exposure to SN-38, the active metabolite of the topoisomerase I inhibitor, CPT-11. Inducible activation of NF-kappaB and growth response were evaluated in vitro and in vivo. Effects on p53, p21, p27 and apoptosis were determined. Pretreatment with PS-341 inhibited activation of NF-kappaB induced by SN-38 and resulted in a significantly higher level of growth inhibition (64-75%) compared with treatment with PS-341 alone (20-30%) or SN-38 alone (24-47%; P < 0.002). Combination therapy resulted in a 94% decrease in tumor size compared with the control group and significantly improved tumoricidal response to treatment compared with all treatment groups (P = 0.02). The level of apoptosis was 80-90% in the treatment group that received combination treatment compared with treatment with single agent alone (10%). Proteasome inhibition blocks chemotherapy-induced NF-kappaB activation, leading to a dramatic augmentation of chemosensitivity and enhanced apoptosis. Combining proteasome inhibition with chemotherapy has significant potential to overcome the high incidence of chemotherapy resistance. Clinical studies are currently in development to evaluate the role of proteasome inhibition as an important adjuvant to systemic chemotherapy.

Intravenous RMP-7 selectively increases uptake of carboplatin into rat brain tumors.

Rats implanted with RG-2 gliomas were administered i.v. RMP-7 and [14C]carboplatin. Changes in the permeability of the blood-brain barrier to carboplatin were determined using quantitative autoradiography. i.v. infusions of RMP-7 induced an increase in the permeability of the vascular barrier within the tumor to carboplatin. Additionally, permeability of brain tissue proximal to, but clearly outside the tumor mass, was also increased. Progressively less uptake of [14C]carboplatin was observed as distance from the tumor border increased. The increases in permeability induced by RMP-7 occurred in a dose-related fashion. No increase in carboplatin level was observed in several nonbrain tissues, including sciatic nerve, retina, heart, lung, liver, kidney, and spleen. Finally, the permeabilizing effects of RMP-7 were shown to occur independent of histaminergic or hypotensive mechanisms. These data provide additional insight into the permeabilizing effects and mechanism of RMP-7 and offer additional support for the therapeutic utility of this novel compound as an adjunctive treatment for human gliomas.

Proteasome inhibitors: a novel class of potent and effective antitumor agents.

The ubiquitin-proteasome pathway plays a critical role in the regulated degradation of proteins involved in cell cycle control and tumor growth. Dysregulating the degradation of such proteins should have profound effects on tumor growth and cause cells to undergo apoptosis. To test this hypothesis, we developed a novel series of proteasome inhibitors, exemplified by PS-341, which we describe here. As determined by the National Cancer Institute in vitro screen, PS-341 has substantial cytotoxicity against a broad range of human tumor cells, including prostate cancer cell lines. The PC-3 prostate cell line was, therefore, chosen to further examine the antitumor activity of PS-341. In vitro, PS-341 elicits proteasome inhibition, leading to an increase in the intracellular levels of specific proteins, including the cyclin-dependent kinase inhibitor, p21. Moreover, exposure of such cells to PS-341 caused them to accumulate in the G2-M phase of the cell cycle and subsequently undergo apoptosis, as indicated by nuclear condensation and poly(ADP-ribose) polymerase cleavage. Following weekly i.v. treatment of PS-341 to mice bearing the PC-3 tumor, a significant decrease (60%) in tumor burden was observed in vivo. Direct injection of PS-341 into the tumor also caused a substantial (70%) decrease in tumor volume with 40% of the drug-treated mice having no detectable tumors at the end of the study. Studies also revealed that i.v. administration of PS-341 resulted in a rapid and widespread distribution of PS-341, with highest levels identified in the liver and gastrointestinal tract and lowest levels in the skin and muscle. Modest levels were found in the prostate, whereas there was no apparent penetration of the central nervous system. An assay to follow the biological activity of the PS-341 was established and used to determine temporal drug activity as well as its ability to penetrate tissues. As such, PS-341 was shown to penetrate PC-3 tumors and inhibit intracellular proteasome activity 1.0 h after i.v. dosing. These data illustrate that PS-341 not only reaches its biological target but has a direct effect on its biochemical target, the proteasome. Importantly, the data show that inhibition of this target site by PS-341 results in reduced tumor growth in murine tumor models. Together, the results highlight that the proteasome is a novel biochemical target and that inhibitors such as PS-341 represent a unique class of antitumor agents. PS-341 is currently under clinical evaluation for advanced cancers.

Postischemic (6-Hour) treatment with recombinant human tissue plasminogen activator and proteasome inhibitor PS-519 reduces infarction in a rat model of embolic focal cerebral ischemia.

BACKGROUND AND PURPOSE: The proteasome inhibitor PS-519 blocks activation of nuclear factor-kappaB, a major mediator of inflammation. We tested the hypothesis that combination treatment of recombinant human tissue plasminogen activator (rhtPA) and PS-519 extends the therapeutic window for treatment of stroke with rhtPA without increasing incidence of hemorrhagic transformation. METHODS: The middle cerebral artery (MCA) of male Wistar rats (n=56) was occluded by an embolus. After embolization, animals were randomly divided into the following groups: PS-519 treatment groups: PS-519 was given at 2, 4, or 6 hours after MCA occlusion; rhtPA treatment groups: rhtPA was given at 2 or 4 hours after MCA occlusion; combination treatment groups: PS-519 and rhtPA were given at 2, 4, or 6 hours after MCA occlusion; control group: the same volume of saline was given at 2 hours after MCA occlusion. RESULTS: Administration of PS-519 alone at 2 or 4 hours, but not 6 hours, significantly (P<0.05) reduced infarct volume and improved neurological recovery compared with the control group. Administration of rhtPA alone at 2 hours, but not 4 hours, significantly (P<0.05) reduced infarct volume and improved neurological recovery compared with the control group. Furthermore, combination treatment with rhtPA and PS-519 even at 6 hours significantly (P<0.05) reduced infarct volume, improved neurological recovery, and did not increase the incidence of hemorrhagic transformation compared with the control group or the group treated with PS-519 alone. CONCLUSIONS: Our data suggest that combination treatment with PS-519 and rhtPA extends the neuroprotective effect to at least 6 hours after embolization.

Postischemic administration of AK275, a calpain inhibitor, provides substantial protection against focal ischemic brain damage.

Experiments were conducted to determine whether a potent, reversible calpain inhibitor could reduce the cortical ischemic brain damage associated with focal ischemia in the rat. AK275 (Z-Leu-Abu-CONH-CH2CH3), the active isomer of the diastereomeric mixture, CX275, was employed in conjunction with a novel method of perfusing drug directly onto the infarcted cortical surface. This protocol reduced or eliminated numerous, nonspecific pharmacokinetic, hemodynamic, and other potentially confounding variables that might complicate interpretation of any drug effect. Focal ischemia was induced using a variation of the middle cerebral artery occlusion method. These studies demonstrated a reliable and robust neuroprotective effect of AK275 over the concentration range of 10 to 200 microM (perfused supracortically at 4 microliters/h for 21 h). Moreover, a 75% reduction in infarct volume was observed when initiation of drug treatment was delayed for 3 h postocclusion. Our data further support an important role of calpain in ischemia-induced neuropathology and suggest that calpain inhibitors may provide a unique and potentially powerful means of treating stroke and other ischemic brain incidents.

Two distinct monoclonal antibodies raised against mouse beta nerve growth factor. Generation of bi-specific anti-nerve growth factor anti-horseradish peroxidase antibodies for use in a homogeneous enzyme immunoassay.

Two hybridomas producing monoclonal antibodies against mouse beta nerve growth factor (NGF) were obtained from the fusion of hyperimmune splenocytes from rats immunized with polymerized beta-NGF and Sp2/0.Ag mouse myeloma cells. The monoclonal antibodies coded IgG 24 and 30 produced and secreted by the hybrid cells are both of the IgG2a subclass. Both monoclonal antibodies are capable of recognizing native NGF coated on microassay plates as well as the denatured factor on Western blots. However, only IgG 30 has been found to block NGF-induced process outgrowth from the rat pheochromocytoma cell line (PC12) as well as NGF-induced increase in choline acetyltransferase activity in rat primary septal cell cultures. In addition, only IgG 30 was able to detect immunocytochemically NGF-immunoreactive sites in fixed tissue. And, finally, IgG 24 could not compete for IgG 30 binding to immobilized native NGF. Consequently, it appears that these antibodies are recognizing different epitopes on the NGF molecule. Neither monoclonal antibody displayed any crossreactivity with serum albumin, aprotinin, epidermal growth factor or insulin. A hybrid-hybridoma producing bi-specific anti-NGF anti-horseradish peroxidase (HRP) monoclonal antibodies was generated from the fusion of an azaguanine resistant anti-HRP hybridoma, coded RAP2.Ag and the anti-NGF IgG 30 hybridoma treated with emetine. The potential merits of using these bi-specific antibodies in combination with their mono-specific anti-NGF parent in a homogeneous sandwich immunoassay for the quantitation of NGF are discussed.

Immunocytochemical use of internally radiolabelled monoclonal antibodies against tyrosine hydroxylase, substance P and enkephalin.

An improved protocol for the internal radiolabelling of monoclonal antibodies with tritiated lysine is described. Hybridoma cell lines producing monoclonal antibodies against the biosynthetic enzyme tyrosine hydroxylase and the neuropeptides, substance P and enkephalin, were employed in this investigation. Immunocytochemical detection of the endogenous antigens with these internally labelled antibodies was performed and when used in immunocytochemistry, the subsequent data were in complete agreement with previous light and electron microscopic studies. These results indicate that the present internal radiolabelling procedure does not alter the ability of the monoclonal antibody to recognise the endogenous antigen. This study, therefore, supports the use of internally labelled antibodies with compatible immunocytochemistry techniques in double staining procedures.

Inhibition of NF-kappa B activation in vitro and in vivo: role of 26S proteasome.

It is becoming increasingly apparent that NF-kappa B plays a critical role in regulating the inflammatory response. Data obtained from studies in our laboratories demonstrate that the proteasome plays an important role in the inflammatory cascade by regulating the activation of NF-kappa B. Indeed, the availability of selective and orally active proteasome inhibitors should prove useful in delineating the roles of the proteasome and NF-kappa B in other pathophysiological conditions such as cancer and heart disease.

Behavioural consequences following infusion of selective neurokinin agonists into the median raphe nucleus of the rat.

The locomotor activity induced after infusion of selective neurokinin (NK) agonists into the median raphé nucleus of rats was investigated. In photocell cages, the NK-2-agonist, GR64349, and the NK-3 agonist, senktide, both increased motor activity in a dose-dependent manner. However, the NK-1 agonist, GR73632, had little effect over a range of doses. In the open field, the motor effect of all three NK agonists was identical to that observed in the photocell cages. In addition, senktide induced straub-tail, hind-limb splaying and various oral movements. Such effects were not noted with the other two agonists. These results suggest that activation of NK-2 or NK-3 receptors by the neurokinins, in the median raphe nucleus of the rat, leads to an increase in locomotor activity.

Substance-P antagonists: effect on spontaneous and drug-induced locomotor activity in the rat.

The substance P (SP) antagonists (D-Pro4, D-Trp7,9, Leu11) SP(4-11), (D-Pro4, D-Trp7,9, Phe11)SP(4-11) and (D-Pro4, D-Trp7,9,10, Leu11) SP (4-11) were infused into the lateral ventricles (i.c.v.) and their effects on spontaneous and drug-induced locomotor activity were investigated. The drug DiMeC7, the stable substance P agonist, was used to stimulate locomotor activity because of its prolonged action. Only (D-Pro4, D-Trp7,9,10) SP (4-11) was found to attenuate the drug-induced increases in motor activity, indicating that it is a substance P antagonist with activity in the CNS.

Responsiveness to dopaminergic agonists after devascularizing lesions of the cortex.

Unilateral devascularizing lesions of neocortex of the rat altered the responsiveness to the direct and indirect dopaminergic agonists, apomorphine and methamphetamine, respectively. These lesions produced subcortical damage manifesting itself as (1) decreased responsiveness to drug-induced locomotor activity, (2) the development of drug-induced rotational activity and (3) loss of body weight. Administration of the monosialoganglioside GM1 (i.c.v.; 5 mg/kg/day; 7 days) reversed these effects when the subjects were tested 10 and 30 days after surgery. The data are interpreted as resulting from damage to dopaminergic systems in the striatum.

Behavioural effects of serotonin agonists and antagonists in the rat and marmoset.

The present study was conducted to investigate the effects of various 5-hydroxytryptamine (5-HT) agonists and antagonists on motor behaviour in rats and marmosets. Various motor-based responses were assessed after central or peripheral administration of 5-HT agents to rats and marmosets. Drugs acting as agonists at the 5-HT1A receptor (8-OHDPAT, gepirone, BMY-7378, NAN-190, PAPP (LY165163) and flesinoxan) and 5-HT2/1C receptors (DOI) were found to reverse neuroleptic-induced catalepsy in the rat whereas 5-HT2/1C antagonists (mianserin, ritanserin and ICI-170,809) and the 5-HT1 antagonist ((+/-)pindolol) increased catalepsy. Agonists acting at 5-HT3 receptors (phenylbiguanide and 2-methyl-5-HT) had no effect on catalepsy. The putative 5-HT1A antagonist, (+/-) pindolol, attenuated the reversal of catalepsy by 8-OHDPAT. Although both 8-OHDPAT and BMY-7378 were tested, only the latter was found to reduce apomorphine-induced stereotypy. Bilateral or unilateral infusions of 8-OHDPAT, BMY-7378 or pindolol into the substantia nigra of non-lesioned rats had no effect on spontaneous locomotor or rotational activity, respectively. However, 8-OHDPAT and BMY-7378 were found to increase or decrease motor activity, after injection into the median or dorsal raphe nuclei, respectively. Finally, 8-OHDPAT and BMY-7378 were found to be inactive against MPTP-induced bradykinesia in the marmoset. It is concluded that both 5-HT1A and 5-HT2/1C receptors are involved in the anti-cataleptic effects of 5-HT agents. The 5-HT1A receptors are probably situated within the raphe, whereas the location of the 5-HT2/1C receptors remains undetermined.

Increased sensitivity to the antinociceptive activity of (+/-)-baclofen in an animal model of chronic neuropathic, but not chronic inflammatory hyperalgesia.

Modulation of sensory afferent inputs to the spinal cord by GABA appears to be an important physiological mechanism and may provide an antinociceptive control system. In the present study we have evaluated the antinociceptive activity of the GABAB receptor agonist, (+/-)-baclofen, in rats with unilateral chronic inflammatory or neuropathic hyperalgesia. (+/-)-Baclofen was antinociceptive in untreated control animals and both animal models. In the neuropathic model the sensitivity to (+/-)-baclofen was significantly increased by 3-fold in the ipsilateral limb. By contrast, in animals with chronic inflammation no difference in sensitivity between ipsilateral and contralateral limbs to (+/-)-baclofen was observed. Receptor autoradiographic analysis in spinal cord sections revealed no increase in the density of GABAB receptor binding sites and no change in receptor affinity in the neuropathic model.

Limited neocortical devascularizing lesions causing deficits in memory retention and choline acetyltransferase activity--effects of the monosialoganglioside GM1.

In mature rats with unilateral cortical lesions, choline acetyltransferase activity was found to decrease by 40% in the ipsilateral nucleus basalis magnocellularis, compared with control animals. Intraventricular administration of the monosialoganglioside GM1 (5 mg/kg per day), via minipumps, over a period of 14 days prevented this fall in choline acetyltransferase activity. The activity of this enzyme in the sham operated subjects was not significantly different from that in the unoperated group. This biochemical data is in full agreement with regards to the protective action of monosialoganglioside GM1 on forebrain cholinergic neurons. In this study the behavioral effects of these cortical lesions were investigated for the first time. Results show that these lesions, with their associated retrograde neuronal degenerative effects, altered a variety of sensorimotor and memory-based behaviors. In particular, the limited unilateral devascularization of the neocortex increased baseline locomotor activity above control; affected motor coordination; impaired passive avoidance retention and reacquisition; and decreased the retention of learnt Morris water-maze information. Infusion of the monosialoganglioside into the ventricles, whilst having no effect on the increased motor activity or motor coordination deficit, did enhance the reacquisition of information in both memory-related tasks.

RMP-7, a bradykinin agonist, increases permeability of blood-ocular barriers in the guinea pig.

PURPOSE: To determine if the bradykinin agonist, RMP-7, could increase the ocular tissue concentration of agents that normally have limited access across the blood-ocular barriers. The extracellular space marker, sucrose, and the anti-viral drug, ganciclovir, were tested. METHODS: Using the perfused eye method in guinea pigs, RMP-7 (1 microgram/kg over 5 minutes) or saline were administered intraarterially into the ocular circulation before either radiolabeled sucrose or ganciclovir (0.4 to 0.6 microCi/ml per minute). At time intervals ranging from 0.25 minute to 10 minutes, perfused eyes were removed, and the radioactivity within various compartments was measured using liquid scintillography. RESULTS: Pretreatment with RMP-7 significantly increased uptake of both sucrose (up to 4.5-fold) and ganciclovir (up to 2-fold) into the guinea pig retina and lens. Smaller and less consistent effects were observed in other eye compartments. CONCLUSIONS: This report demonstrates that RMP-7 enhances the permeability of blood-ocular barriers, and it provides the first pharmacologic evidence for a means to enhance the concentration of ganciclovir into the retina. Thus, these data support the concept that RMP-7 may prove to be a useful adjunct for enhanced delivery of therapeutics to the eye under conditions in which ocular barriers limit treatment.

The proteasome: a new target for novel drug therapies.

The proteasome is an enzyme present in all cells, from yeast to human, and has a central role in the proteolytic degradation of the vast majority of intracellular proteins. Among the key proteins modulated by the proteasome are those involved in controlling inflammatory processes, cell cycle regulation, and gene expression. As such, agents that inhibit the proteasome have been shown to be active in numerous animal models of inflammation and cancer Two proteasome inhibitors are under clinical evaluation. PS-519 is being studied for the treatment of reperfusion injury that occurs following cerebral ischemia and myocardial infarction. The other, PS-341, has recently entered multiple phase 2 clinical trials for the treatment of multiple myeloma, chronic lymphocytic leukemia, and a variety of solid tumors. The proteasome may have an important role in the evolution of HIV-related disorders including AIDS and inflammatory disorders. Therapeutic strategies using proteasome inhibitors for the treatment of these conditions have now entered preclinical development.

Effects of classical and novel agents in a MPTP-induced reversible model of Parkinson's disease.

A modified primate model of Parkinson's disease was developed to assess the effectiveness of various agents that act via dopamine, acetylcholine, serotonin or glutamate systems. Using a MPTP dosing regimen a reversible parkinsonian-like syndrome was produced in the marmoset. An obvious advantage of such a protocol is that it allows multiple drug studies to be undertaken in animals, without the need for prolonged anti-parkinsonian therapy to maintain their health. Results show that dopamine D2 agonists (bromocriptine, quinpirole, N,N-dipropyl,A,5,6-DTN, (+)3PPP and PHNO), anti-muscarinics (atropine, scopolamine and benztropine), in addition to L-DOPA and nomifensine, all reduced the bradykinesia induced by MPTP. The D1 agonist SKF-38393 and the partial dopamine agonist (-)3PPP were both ineffective. Finally, agents with potential therapeutic use in Parkinson's disease were also tested. However, a glutamate antagonist (MK801) and three serotonin antagonists (ritanserin, ketanserin and ICI 170,809) were all unable to alter the MPTP effects, at the doses used in our study.

Diminished nociceptive response in mice following immunization with neurokinins.

Female BALB/C mice were immunized with various neuropeptides and later tested on a hot plate apparatus for responses to nociceptive stimuli. Animals immunized with substance P, neurokinin B (NKB) or fragments of either peptide were found to spend significantly longer periods of time on the hot plate than controls. Subjects treated with substance K (NKA), its N-terminal fragment or neuropeptide Y were not different from controls. Finally, mice injected with histamine or the EXON-4-related peptide were also found to exhibit delayed responses in this test situation. These results suggest roles for SP, NKB, EXON-4-related peptide and histamine in sensory processing and transmission.