A new perspective for schizophrenia: TAAR1 agonists reveal antipsychotic- and antidepressant-like activity, improve cognition and control body weight.

Schizophrenia is a chronic, severe and highly complex mental illness. Current treatments manage the positive symptoms, yet have minimal effects on the negative and cognitive symptoms, two prominent features of the disease with critical impact on the long-term morbidity. In addition, antipsychotic treatments trigger serious side effects that precipitate treatment discontinuation. Here, we show that activation of the trace amine-associated receptor 1 (TAAR1), a modulator of monoaminergic neurotransmission, represents a novel therapeutic option. In rodents, activation of TAAR1 by two novel and pharmacologically distinct compounds, the full agonist RO5256390 and the partial agonist RO5263397, blocks psychostimulant-induced hyperactivity and produces a brain activation pattern reminiscent of the antipsychotic drug olanzapine, suggesting antipsychotic-like properties. TAAR1 agonists do not induce catalepsy or weight gain; RO5263397 even reduced haloperidol-induced catalepsy and prevented olanzapine from increasing body weight and fat accumulation. Finally, TAAR1 activation promotes vigilance in rats and shows pro-cognitive and antidepressant-like properties in rodent and primate models. These data suggest that TAAR1 agonists may provide a novel and differentiated treatment of schizophrenia as compared with current medication standards: TAAR1 agonists may improve not only the positive symptoms but also the negative symptoms and cognitive deficits, without causing adverse effects such as motor impairments or weight gain.

PSICalc: a novel approach to identifying and ranking critical non-proximal interdependencies within the overall protein structure.

Motivation: AlphaFold has been a major advance in predicting protein structure, but still leaves the problem of determining which sub-molecular components of a protein are essential for it to carry out its function within the cell. Direct coupling analysis predicts two- and three-amino acid contacts, but there may be essential interdependencies that are not proximal within the 3D structure. The problem to be addressed is to design a computational method that locates and ranks essential non-proximal interdependencies within a protein involving five or more amino acids, using large, multiple sequence alignments (MSAs) for both globular and intrinsically unstructured proteins. Results: We developed PSICalc (Protein Subdomain Interdependency Calculator), a laptop-friendly, pattern-discovery, bioinformatics software tool that analyzes large MSAs for both structured and unstructured proteins, locates both proximal and non-proximal inter-dependent sites, and clusters them into pairwise (second order), third-order and higher-order clusters using a k-modes approach, and provides ranked results within minutes. To aid in visualizing these interdependencies, we developed a graphical user interface that displays these subdomain relationships as a polytree graph. To demonstrate, we provide examples of both proximal and non-proximal interdependencies documented for eukaryotic topoisomerase II including between the unstructured C-terminal domain and the N-terminal domain. Availability and implementation: https://github.com/jdeweeselab/psicalc-package. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Relationship between modulation of natural killer cell activity and antitumor activity of bropirimine when used in combination with various types of chemotherapeutic drugs.

Bropirimine (ABPP), a pyrimidinone, is currently under clinical trial for its antitumor potential. Bropirimine alone was marginally active against some experimental tumors such as B16 melanoma but was ineffective against others such as P388 or L1210 leukemia. However, it produced statistically significant synergistic activity against P388 leukemia when used in combination with cyclophosphamide (CY). The aim of this investigation was to determine whether the synergism could be achieved with different types of cytotoxic drugs. Actinomycin D (act D), adriamycin, 5-azacytidine, cisplatin, melphalan, mitomycin C, and vincristine were selected. Using an experimental protocol identical to that of CY and bropirimine combination therapy, and using a more or less equally effective dosage of the drug for the initial reduction of tumor burden (i.e., around 100% increase of life span), cisplatin and bropirimine also produced a statistically significant synergism over the treatment with cisplatin alone. The combination of bropirimine with either adriamycin, mitomycin, or vincristine was beneficial but the effect was not as consistent or as striking as that seen with the CY and bropirimine combination. It is clear, however, that the combination of act D and bropirimine was not synergistic under the experimental conditions. Since the antitumor activity of pyrimidinone has been reported to be mediated in part by its stimulation of natural killer cell activity, the effect of these cytotoxic drugs on the immunomodulatory activity of bropirimine was investigated. Like CY, cisplatin did not alter the augmentation of natural killer cell activity by bropirimine. However, adriamycin, mitomycin, or vincristine showed a marked inhibition (25-50%) of the augmentation. Act D completely inhibited the immunomodulating activity of bropirimine 4 days after drug administration and continued to show marked inhibition 18 days later. This may partially explain the reasons for lack of synergism between act D and bropirimine. A prolonged immunosuppressive effect exhibited by act D and the degree of tumor repopulation during this period could render bropirimine ineffective. In addition to the magnitude of initial tumor burden reduction by the chemotherapeutic drugs, the present results indicate that the immunosuppressive property of these drugs may also affect the outcome of chemoimmunotherapy.

Mechanism of antitumor action of pyrimidinones in the treatment of B16 melanoma and P388 leukemia.

This study was undertaken in an attempt to understand the mechanism of antitumor action of pyrimidinones alone and in combination with cyclophosphamide (CY). Pyrimidinones such as 2-amino-5-bromo-6-(3-fluorophenyl)-4(3H)pyrimidinone (ABMFPP) were relatively nontoxic toward murine L1210 leukemia cell growth in vitro with the concentration of drug required for a 50% inhibition of cell growth being greater than 50 micrograms/ml. In contrast, ABMFPP showed anti-B16 melanoma activity in vivo which was sensitive to X-irradiation of the hosts. These results collectively suggest that pyrimidinones may act differently from conventional cytotoxic antitumor agents. Multiple i.p. injections of ABMFPP (125 mg/kg/injection) significantly augmented the cytotoxicity of both natural killer cells and macrophages in peritoneal exudates. The augmentation of both effector cell populations was delayed, but was more pronounced when animals received a dose of CY (100 mg/kg) prior to ABMFPP injections. The combination of CY and ABMFPP also showed a synergistic anti-P388 leukemia effect which appeared to be related to the initial reduction of the tumor burden by CY and the marked augmentation of the cytotoxicity of both natural killer cells and macrophages by ABMFPP. The antitumor activity of ABMFPP against B16 melanoma was almost completely eliminated when animals received a dose of 400 rads X-irradiation 5 days prior to tumor inoculation or a dose of 200 rads X-irradiation followed by several injections of anti-asialo monosialoganglioside antibody. The administration of anti-asialo monosialoganglioside alone also markedly reduced the anti-B16 melanoma activity of ABMFPP. The magnitude of reduction of the antitumor effect of ABMFPP by radiation and/or anti-asialo monosialoganglioside antibody directly correlated with the inhibition of the ABMFPP-mediated augmentation of immune responses. These results strongly suggest that the antitumor effect of ABMFPP alone or in combination with CY is at least in part mediated through its augmentation of natural killer cell and/or macrophage activities.

Chemoimmunotherapy of B 16 melanoma and P388 leukemia with cyclophosphamide and pyrimidinones.

Since increasing evidence indicates that combination modality of cancer treatment is preferable, and a series of 5-halo-6- phenylpyrimidinones has been found to induce interferon production and to stimulate a variety of immune responses, several were tested alone or in combination with cyclophosphamide (CY) against B 16 melanoma and P388 leukemia. Thus far, 2-amino-5-bromo-6-(3-fluorophenyl)-4(3H)pyrimidinone ( ABMFPP ) and its sister compound 2-amino-5-bromo-6-(2-fluorophenyl)-4(3H)pyrimidinone ( ABOFPP ) were found to be superior to other pyrimidinones including 2-amino-5-bromo-6-(6-phenyl)-4-pyrimidinone which is currently under clinical investigation. Neither ABMFPP nor ABOFPP alone had any significant activity against P388 leukemia. However, a marked synergistic effect was observed when a single i.p. injection of CY at 24 hr after tumor inoculation (10(6) cells/mouse) was followed by multiple i.p. injections of either ABMFPP or ABOFPP . For instance, the increase of life span was about 180% when animals received both CY (150 mg/kg) and ABMFPP (125 mg/kg/injection) as compared to 100% increased life span when animals received CY alone, and 0% increased life span when animals received ABMFPP alone. Also, 80% of the animals were long-term survivors (greater than 30 days) when animals received the combination therapy as compared to 20% survivors when animals received CY alone. The synergistic effect exhibited by ABMFPP or ABOFPP correlated positively to the initial reduction of tumor burden by CY. The optimal gap between CY and pyrimidinone administration was one day. The best therapeutic response was observed when pyrimidinone was given every 4 days for a total of 7 injections; however, other schedules and dosing frequencies also gave significant responses. The synergistic effect was also observed with B 16 melanoma when animals received the combination therapy. The significance of these findings, in terms of theoretical consideration as well as drug development, is discussed.

Mechanism of interaction of CC-1065 (NSC 298223) with DNA.

CC-1065 (NSC 298223), a potent new antitumor antibiotic produced by Streptomyces zelensis, interacts strongly with double-stranded DNA and appears to exert its cytotoxic effects through disruption of DNA synthesis. We undertook this study to elucidate the sites and mechanisms of CC-1065 interaction with DNA. The binding of CC-1065 to synthetic and native DNA was examined by differential circular dichroism or by Sephadex chromatography with photometric detection. The binding of CC-1065 with calf thymus DNA was rapid, being complete within 2 hr, and saturated at 1 drug per 7 to 11 base pairs. The interaction of CC-1065 with synthetic DNA polymers indicated a specificity for adenine- and thymine-rich sites. Agarose gel electrophoresis of CC-1065-treated supercoiled DNA showed that CC-1065 did not intercalate. Site exclusion studies using substitutions in the DNA grooves showed CC-1065 to bind primarily in the minor groove. CC-1065 did not cause DNA breaks; it inhibited susceptibility of DNA to nuclease S1 digestion. It raised the thermal melting temperature of DNA, and it inhibited the ethidium-induced unwinding of DNA. Thus, in contrast to many antitumor agents, CC-1065 stabilized the DNA helix. DNA helix overstabilization may be relevant to the mechanism of action of CC-1065.

Synthetic matrix metalloproteinase inhibitors and tissue inhibitor of metalloproteinase (TIMP)-2, but not TIMP-1, inhibit shedding of tumor necrosis factor-alpha receptors in a human colon adenocarcinoma (Colo 205) cell line.

The solubilization of plasma membrane receptors through proteolytic cleavage of the ligand binding domain at the cell surface is an important mechanism for regulating cytokine function and receptor signaling. The inhibition of the shedding of a variety of receptors by synthetic inhibitors of the matrix metalloproteinases (MMPs) implicates metalloproteinases in this regulatory event. We examined the effects of two naturally occurring tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, and several synthetic MMP inhibitors (MMPIs) on the shedding of both tumor necrosis factor alpha receptor type I (TNFalpha-RI; Mr 55,000) and TNFalpha-RII (Mr 75,000) by the Colo 205 human colon adenocarcinoma cell line. Culture of Colo 205 cells for 48 h resulted in the shedding of both TNFalpha-RI and TNFalpha-RII, as determined by ELISA. The shedding of TNFalpha receptors was not affected by TIMP-1 or protease inhibitors aprotinin, pepstatin, or leupeptin but was inhibited in a dose-dependent manner by the following synthetic MMPIs: batimastat and marimastat (BB-94 and BB-2516, respectively, British Biotech, Inc.); CT1418 (Celltech Therapeutics); CGS27023A (Novartis Pharmaceuticals); and RO31-9790 (Roche), with IC50s ranging from 3.2 to 38.0 microM. Similarly, TIMP-2 from two different sources reproducibly inhibited the shedding of both TNFalpha-RI and TNFalpha-RII in a dose-dependent manner (IC50 = 286 +/- 33 nM for TNFalpha-RI shedding and 462 +/- 52 nM for shedding of TNFalpha-RII). The inhibition of TNFalpha-RI shedding was confirmed in the SW626 human ovarian adenocarcinoma cell line. The synthetic MMPIs and TIMP-2, but not TIMP-1, also caused a dose-dependent increase in the number of TNFalpha receptors retained on the surface of Colo 205 cells, as determined by flow cytometry. Inhibition of TNFalpha receptor shedding with TIMP-2 occurs at molar concentrations 10-100 times less than those required with low molecular weight, synthetic MMPIs but at concentrations greater than those required to inhibit collagen degradation. Modulation of TNFalpha receptor shedding by TIMP-2 could have important implications for the pleiotropic effects of TNFalpha in both normal and malignant cells and for the pharmacological activity of synthetic MMPIs.

Cytotoxicity and antitumor activity of carzelesin, a prodrug cyclopropylpyrroloindole analogue.

The cyclopropylpyrroloindole analogues are DNA minor-groove binders containing a cyclopropyl group, which mediates N3-adenine covalent adduct formation in a sequence-selective fashion. Carzelesin (U-80244) is a cyclopropylpyrroloindole prodrug containing a relatively nonreactive chloromethyl precursor to the cyclopropyl function. Activation of carzelesin requires two steps, (a) hydrolysis of a phenylurethane substituent to form U-76073, followed by (b) ring closure to form the cyclopropyl-containing DNA-reactive U-76074. The formation of the DNA-reactive U-76074, via U-76073, from carzelesin was shown to proceed very slowly in phosphate-buffered saline (t1/2 greater than 24 h) but to occur rapidly in plasma from mouse, rat, dog, and human (initial t1/2 values ranging from 18 min for mouse to 52 min for rat) and in cell culture medium (t1/2 approximately 40 min). Although carzelesin was less potent in terms of in vitro cytotoxicity and in vivo optimal dosage and showed low affinity for binding to DNA, it was therapeutically more efficacious against mouse L1210 leukemia than was U-76074 or adozelesin (U-73975), another cyclopropylpyrroloindole analogue which is currently in phase I clinical trials. Carzelesin also proved to be more efficacious than U-76074 or adozelesin against mouse pancreatic ductal 02 adenocarcinoma, a system reported to be resistant to every agent tested. Carzelesin was highly effective against this tumor and produced 97% tumor growth inhibition. In addition, i.v. administered carzelesin showed significant activity (National Cancer Institute criteria) against i.v. or s.c. implanted Lewis lung carcinoma, i.p. or s.c. implanted B16 melanoma, s.c. implanted colon 38 carcinoma, and five s.c. implanted human tumor xenografts, including clear cell Caki-1 carcinoma, colon CX-1 adenocarcinoma, lung LX-1 tumor, ovarian 2780 carcinoma, and prostatic DU-145 carcinoma. Carzelesin treatment produced 100% complete remissions (no palpable tumor mass at the termination of the experiment) in mice bearing early-stage human ovarian 2780. Pharmacologically, carzelesin proved to be relatively schedule and route independent and was highly active against i.p. implanted L1210 leukemia, regardless of whether the analogue was given i.v., i.p., s.c., or p.o. These results, collectively, suggest that carzelesin is absorbed and distributed well. Both carzelesin and adozelesin caused marked tumor shrinkage in mice bearing human lung LX-1 or advanced-stage human ovarian 2780 carcinoma; however, tumor regrowth occurred shortly after the treatment with adozelesin was stopped. Little or no apparent tumor regrowth occurred after treatment with carzelesin.(ABSTRACT TRUNCATED AT 400 WORDS)

Methamphetamine-induced neurotoxicity alters locomotor activity, stereotypic behavior, and stimulated dopamine release in the rat.

The neurochemical evidence of methamphetamine (MA)-induced toxicity to dopaminergic nerve terminals is well documented; however, the functional consequences are not clearly defined. The present study was designed to investigate whether MA-induced dopamine depletions affect locomotor activity, stereotypic behavior, and/or extracellular dopamine concentrations in the neostriatum. Male rats were treated with a neurotoxic regimen of MA (10 mg/kg, i.p., every 2 hr for four injections) or vehicle and tested for functional effects 1 week later. Animals that had received the neurotoxic regimen of MA showed a reduction in both caudate nucleus and nucleus accumbens dopamine contents of 56 and 30%, respectively. Furthermore, MA-treated rats exhibited a significant attenuation in spontaneous activity, as well as a significant diminution in MA (low dose)-stimulated locomotor activity as compared to vehicle-treated rats. However, there were no differences in the MA (low dose)-induced increases in extracellular dopamine concentrations in the caudate nucleus or the nucleus accumbens core of either group. Interestingly, the acute administration of higher doses of MA elicited a significantly augmented stereotypic response and a significantly attenuated increase in the extracellular concentration of dopamine in the caudate nucleus of rats treated with a neurotoxic regimen of MA as compared to vehicle-treated animals. These data indicate that MA-induced neurotoxicity results in abnormal dopamine-mediated behaviors, as well as a brain region-specific impairment in stimulated dopamine release.

Synthesis of a series of stromelysin-selective thiadiazole urea matrix metalloproteinase inhibitors.

The synthesis and enzyme inhibition data for a series of thiadiazole urea matrix metalloproteinase (MMP) inhibitors are described. A broad screening effort was utilized to identify several thiadiazoles which were weak inhibitors of stromelysin. Optimization of the thiadiazole leads to include an alpha-amino acid side chain with variable terminal amide substituents provided a series of ureas which were moderately effective stromelysin inhibitors, with Ki's between 0.3 and 1.0 microM. The most effective analogues utilized an L-phenylalanine as the amino acid component. In particular, unsubstituted 46 had a Ki of 710 nM, while the p-fluoro analogue 52 displayed increased potency (100 nM). Stromelysin inhibition was further improved using a pentafluorophenylalanine substituent which resulted in 70, a 14 nM inhibitor. While gelatinase inhibition was generally poor, the use of 1-(2-pyridyl)piperazine as the amide component usually provided for enhanced activity, with 71 inhibiting gelatinase with a Ki of 770 nM. The combination of this heterocycle with a p-fluorophenylalanine substituent provided the only analogue, 69, with collagenase activity (13 microM). The SAR for analogues described within this series can be rationalized through consideration of the X-ray structure recently attained for70 complexed to stromelysin. Uniquely, this structure showed the inhibitor to be completely orientated on the left side of the enzyme cleft. These results suggest that thiadiazole urea heterocycles which incorporate a substituted phenylalanine can provide selective inhibitors of stromelysin. Careful selection of the amide substituent can also provide for analogues with modest gelatinase inhibition.

Methamphetamine enhances the cleavage of the cytoskeletal protein tau in the rat brain.

The view that methamphetamine is neurotoxic to dopaminergic and serotonergic axon terminals has been based largely on biochemical and histological studies. In the present study, methamphetamine-induced structural damage to axons was quantified using a sensitive sandwich enzyme-linked immunosorbent assay developed for the detection of the cleaved form of the cytoskeletal protein tau. The administration of a monoamine-depleting regimen of methamphetamine (4 x 10 mg/kg, i.p. every 2 hours for a total of four injections) produced a time-dependent increase in the concentration of cleaved tau in the striatum. Maximal concentrations of cleaved tau were detected 3 days following methamphetamine administration. Cleaved tau concentrations also were significantly elevated in the dorsal hippocampus and, to a lesser extent, in the prefrontal cortex of methamphetamine-treated rats. Maintenance of rats in a cold (4 degrees C) environment not only prevented the methamphetamine-induced depletion of striatal dopamine and serotonin but also prevented the methamphetamine-induced increase in striatal cleaved tau concentrations. The novel findings from this study are supportive of the view that methamphetamine produces acute structural damage to neurons that may lead to the long-term neurotoxic effects of repeated, high-dose administration of the drug and that cleaved tau reliably quantifies the time-dependent neurotoxic effects of methamphetamine.

Mechanism of action of didemnin B, a depsipeptide from the sea.

With the brush border membrane vesicles prepared from the rat kidney cortex, didemnin B and its parent compound, didemnin A function neither as a K+-specific ionophore nor as an ionophore for Na+ ions while other depsipeptide antibiotics such as valinomycin and gramicidin promote transmembrane movement of K+ and Na+ ions, respectively. Didemnin B inhibits protein synthesis and DNA synthesis much more than RNA synthesis and is in general more potent than didemnin A. Time course studies reveal that the action of didemnin B is rapid and cannot be reversed after 2 h in contact with the cells. The inhibition of protein synthesis is almost superimposable to that of L1210 cells growth. DNA synthesis is also markedly inhibited. These results collectively suggest that didemnin B acts differently, at least in part, from other depsipeptide antibiotics and its biological effect is primarily mediated through its inhibition of protein synthesis and to a lesser extent its inhibition of DNA synthesis.

Interaction of drugs with calmodulin. Biochemical, pharmacological and clinical implications.

Calmodulin is a widely distributed, highly active, calcium-binding protein that influences a number of important biological events. Accordingly, agents that inhibit the activity of calmodulin should have profound pharmacological effects. Within the past few years, a number of compounds have been identified that inhibit calmodulin. The most potent of these described so far include certain antipsychotic drugs, smooth muscle relaxants, alpha-adrenergic blocking agents and neuropeptides. Studies of the physicochemical and structural properties of a variety of calmodulin inhibitors have shown that there are ionic and hydrophobic interactions between the drug and calmodulin. From the limited studies conducted so far, we conclude that, for a compound to inhibit calmodulin, it should carry a positive charge at physiological pH, presumably to interact with negative charges on the highly acidic calmodulin, and have hydrophobic groups, presumably to interact with lipophilic regions on calmodulin. But these two factors are not the only ones that are involved in inhibiting calmodulin, for many highly charged and highly hydrophobic agents have relatively little effect on calmodulin activity. The structural relationships between these ionic and hydrophobic regions and other, as yet identified, factors are also important. Many of the biochemical actions of the phenothiazine antipsychotic agents can be explained by the common mechanism of their binding to, and inhibiting, calmodulin. The question of whether these biochemical actions can explain their pharmacological and clinical effects is still unclear. The fundamental role calmodulin plays in biology suggests that this calcium binding protein may provide a new site for the pharmacological manipulation of biological activity. The calmodulin inhibitors described thus far hardly scratch the surface of this fertile area of research.

Alterations in diurnal and nocturnal locomotor activity in rats treated with a monoamine-depleting regimen of methamphetamine or 3,4-methylenedioxymethamphetamine.

RATIONALE: The long-term neurochemical effects produced by the repeated administration of methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) are well documented; however, the functional consequences have not been clearly defined. OBJECTIVE: The present study was designed to investigate whether rats treated with a monoamine-depleting regimen of MA or MDMA exhibit disturbances in locomotor activity during the diurnal and nocturnal cycles. METHODS: Rats were treated with the vehicle or a monoamine-depleting regimen of MA or MDMA (10 mg/kg, IP, every 2 h for four injections on a single day). One week after drug treatment, the rats were placed in residential activity chambers and their locomotor activity was monitored for the next 7-day/night cycles. RESULTS: MA-treated rats exhibited depletions of striatal dopamine and serotonin content of approximately 70%, whereas MDMA-treated rats showed depletions of striatal serotonin content of approximately 50%. Rats treated with MA demonstrated a significant reduction in diurnal, but not nocturnal, locomotor activity, whereas MDMA-treated rats exhibited significant reductions in both diurnal and nocturnal locomotor activity. Analysis of the difference in activity between the nocturnal and diurnal cycles revealed that MA-treated animals exhibited a significantly greater change in activity as compared to that observed in vehicle- and MDMA-treated rats. CONCLUSIONS: Although it is unknown whether the adaptations in locomotor activity observed in MA- and MDMA-treated rats are due to the loss of dopamine and/or serotonin, these data suggest that the administration of a monoamine-depleting regimen of MA or MDMA results in alterations in light-cycle-dependent locomotor activity.

Pharmacological regulation of calmodulin.

A number of psychotropic drugs, particularly the phenothiazines and related antipsychotic compounds, inhibit a variety of calmodulin-dependent enzymes. The mechanism by which these compounds inhibit the activity of calmodulin is through a selective calcium-dependent binding to this protein. With the notable exception of certain stereoisomers, compounds that are clinically effective antipsychotic agents showed the greatest degree of binding to calmodulin. Other classes of pharmacological agents, including aminergic agonists and antagonists, and nonspecific central nervous system depressants and stimulants, showed little or no binding to calmodulin. In fact, the specificity with which antipsychotic drugs bind to calmodulin suggests the possibility of screening for new and clinically more effective antipsychotic agents based on their selective binding to calmodulin. Certain neuropeptides that produce behavioral effects in animals also were found to inhibit the activity of calmodulin, suggesting that there may be endogenous psychotogens or antipsychotic peptides that interact with calmodulin. Although under ordinary conditions the binding of antipsychotics to calmodulin is reversible, the binding of phenothiazine antipsychotics to calmodulin can be made irreversible either photochemically by ultraviolet irradiation, or enzymatically by a hydrogen peroxide-peroxidase system. Such a labeling technique should prove to be a useful tool to study the localization and turnover of calmodulin. These results indicate that several of the diverse biochemical actions of antipsychotic agents can be explained by a common mechanism, namely, by their binding to and inhibition of calmodulin, and raise the possibility that calmodulin may serve as one of the cellular receptors for certain antipsychotic compounds. However, further studies must be completed before we can state with any degree of certainty that these in vitro biochemical findings can explain the pharmacological and clinical actions of the antipsychotics.

Acute, multiple-dose, and genetic toxicology of AR177, an anti-HIV oligonucleotide.

AR177 (Zintevir) is a 17-mer oligonucleotide that has been shown to have anti-HIV activity and to be a potent HIV-1 integrase inhibitor in vitro, and is among the first oligonucleotides to enter human clinical trials. Acute and multiple-dose intravenous toxicity studies were performed in mice, and genetic toxicity studies were performed in vitro and in vivo in order to determine the toxicity profile of AR177. The acute toxicity study in mice showed that AR177 had an LD50 of > or = 1.5 g/kg body weight. The multipledose toxicity study in mice showed that AR177 caused male-specific mortality, and changes in serum chemistry, hematology, and histology at doses of 250 and 600 mg/kg. Clinical chemistry findings included changes in liver function, and decreased erythrocyte values at 250 and 600 mg/kg. Histopathologic findings included vacuolization of reticuloendothelial cells in phagocytic cells in lymphoid tissue, liver, lungs, heart and uterus, and extramedullary hematopoeisis in the spleen. Renal toxicity was exhibited as nephropathy and tubular necrosis in the two high-dose groups of males. A no-effect dose was not established. AR177 did not exhibit genetic toxicity in any of three mutagenic assays. In combination with previously reported toxicity studies of AR177 in monkeys, this study showed that the toxicity of AR177 is species specific.

The reproductive and developmental toxicity of the antifungal drug Nyotran (liposomal nystatin) in rats and rabbits.

Nyotran is a liposomally encapsulated i.v. formulation of the antifungal polyene nystatin. This drug was evaluated in a series of reproductive toxicity studies, according to the guidelines outlined by the International Conference on Harmonization (ICH). A fertility and early embryonic development study (SEG I) and a prenatal and postnatal development (SEG III) study were conducted in rats, and embryo-fetal development (SEG II) studies were conducted in rats and rabbits. Nyotran was administered iv in all studies. In SEG I and SEG III, rats were administered daily doses of 0.5, 1.5, or 3.0 mg/kg Nyotran. In both studies, parental mortality and toxicity in the 3.0 mg/kg dose group necessitated the lowering of the high dose to 2.0 mg/kg/day. Parental toxicity, in the form of decreased body weights, decreased food consumption, and piloerection were also observed at the 1.5 mg/kg/day dose level in the SEG I and SEG III studies. Despite the parentally toxic doses in the SEG I study, there was no effect of Nyotran on F0 male or female fertility or early embryonic development of F1 offspring. In the SEG III study, lactational body weights of the F1 generation were decreased at all Nyotran dose levels. There was no effect on pre-wean developmental landmarks, but post-wean development was affected by Nyotran administration at all dosage levels. Preputional separation was delayed in the 1.5 and 3.0/2.0 mg/kg/day F1 offspring, auditory startle function was decreased in F1 females at all dose levels, and motor activity was decreased in male F1 offspring at all dose levels. However, there were no treatment-related effects on the subsequent mating of the F1 generation and resulting F2 offspring. In SEG II studies, rats and rabbits were also administered 0.5, 1.5, or 3.0 mg/kg/day of Nyotran during gestation. The high dose in these SEG II studies was not lowered, as the maternal animals were able to tolerate the shorter duration of dosing. Maternal effects in rabbits were observed only in the high-dose group and were limited to decreased food consumption and decreased absolute and relative liver weight. Decreased food consumption in high-dose dams and clinical weight loss in some animals at the mid- and high-dose levels evidenced maternal toxicity in rats. Nyotran did not have any effect on Caesarian section parameters in either rats or rabbits and no effect on the incidence of fetal malformations in rabbits. A statistically significant increase in mild hydrocephaly, observed in 4 rat fetuses, was seen at the highest dose level of 3.0 mg/kg/day. The biological significance and relationship to Nyotran treatment of this finding is not clear. This finding may represent a change in the background incidence or a change in the pattern of responsiveness of this strain of rat fetus to the test chemical. Toxicokinetic data were also collected in the SEG II rabbit and rat studies for comparison to human exposures. In both species, systemic exposure to the nystatin at effective antifungal concentrations was demonstrated. The systemic exposures in rats and rabbits were, however, considerably less than have been reported in humans administered clinical doses of 2 or 4 mg/kg/day Nyotran. Thus, humans tolerate higher dosages and systemic exposures of Nyotran relative to rats and rabbits and there is no margin of safety in either dosage level or systemic exposure to drug. Given this lack of a margin of safety and the effects on postnatal development in F1 rats, caution should be exercised when using this drug in females of childbearing potential.

The tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism.

The tetracycline analogs minocycline and doxycycline are inhibitors of metalloproteinases (MMPs) and have been shown to inhibit angiogenesis in vivo. To further study the mechanism of action of these compounds we tested them in an in vitro model of angiogenesis: aortic sprouting in fibrin gels. Angiogenesis was quantitated in this system by a unique application of planar morphometry. Both compounds were found to potently inhibit angiogenesis in this model. To further characterize the activity of these compounds against MMPs, we determined the IC50S of both compounds against representatives of three classes of metalloproteinases: fibroblast collagenase, stromelysin, and gelatinase A. Doxycycline was found to inhibit collagenase, gelatinase A and stromelysin with IC50S of 452 microM, 56 microM and 32 microM, respectively. Minocycline was found to inhibit only stromelysin in the micromolar range with an IC50 of 290 microM. Since these results suggest that these compounds may not have been inhibiting in vitro angiogenesis by an MMP-dependent mechanism, we decided to test the effects of the potent MMP inhibitor BB-94. This compound failed to inhibit aortic sprouting in fibrin gels, thus strongly suggesting that both doxycycline and minocycline act by an MMP-independent mechanism. These results have implications for the mechanism of action of tetracycline analogs, particularly where they are being considered for the treatment of disorders of extracellular matrix degradation including periodontal disease, arthritis, and tumor angiogenesis.

Adozelesin, a potent new alkylating agent: cell-killing kinetics and cell-cycle effects.

Adozelesin (U-73975) was highly cytotoxic to V79 cells in culture and was more cytotoxic than several clinically active antitumor drugs as determined in a human tumor-cloning assay. Phase-specificity studies showed that cells in the M+early G1 phase were most resistant to adozelesin and those in the late G1 + early S phase were most sensitive. Adozelesin transiently slowed cell progression through the S phase and then blocked cells in G2. Some cells escaped the G2 block and either divided or commenced a second round of DNA synthesis (without undergoing cytokinesis) to become tetraploid. Adozelesin inhibited DNA synthesis more than it did RNA or protein synthesis. However, the dose needed for inhibition of DNA synthesis was 10-fold that required for inhibition of L1210 cell growth. The observation that cell growth was inhibited at doses that did not cause significant inhibition of DNA synthesis and that cells were ultimately capable of completing two rounds of DNA synthesis in the presence of the drug suggests that adozelesin did not exert its cytotoxicity by significant inhibition of DNA synthesis. It is likely that adozelesin alkylates DNA at specific sites, which leads to transient inhibition of DNA synthesis and subsequent G2 blockade followed by a succession of events (polyploidy and unbalanced growth) that result in cell death.

Characterization of choline acetyltransferase-sustaining and survival-promoting activities for parasympathetic neurons in pig lung.

A total of 49 tissues from six animal species were screened for substances that would sustain choline acetyltransferase (ChAT) in parasympathetic ciliary neurons in culture. Among these, lungs from several species, particularly pig, contained relatively high concentrations of ChAT-sustaining activity. The bioassay used was one in which embryonic chicken ciliary ganglia were cultured as explants for 3 days with KCl and lung extract. KCl, in the presence of lung extract, sustained high concentrations of choline acetyltransferase activity, but did not maintain enzymatic activity in the absence of lung extract. These effects were dependent on the embryonic age of the ciliary ganglia. Lung extract and KCl would not maintain choline acetyltransferase activity in parasympathetic neurons taken from chickens older than embryonic day 12. In addition to ChAT-sustaining activity, pig lung also contains survival-promoting activity for parasympathetic neurons but not for other neuronal types, including sympathetic or sensory neurons of neural crest or placodal origin. This is a more specific target spectrum than previously reported ciliary survival-promoting factors that have been purified or partially purified and indicates that survival-promoting activities in pig lung are different than those previously observed. Because of the large quantitites that can be obtained, we suggest that pig lung is a preparatively feasible source for possibly novel ciliary neuron ChAT-sustaining and survival-promoting activities for parasympathetic neurons.