Discovery of 2,4-diamino-5-cyanopyrimidine derivatives as protein kinase C theta inhibitors with mitigated time-dependent drug-drug interactions.

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and has therapeutic potential for T cell-mediated diseases such as transplant rejection and rheumatoid arthritis. PKCθ inhibitors have emerged as effective immunomodulative agents for the prevention of transplant rejection. We previously reported that the 2,4-diamino-5-cyanopyrimidine derivative 2 was a potent PKCθ inhibitor; however, it exhibited CYP3A4 time-dependent inhibition (TDI). Here, we report the structural modification of compound 2 into 34 focusing on mitigating CYP3A4 TDI. Compound 34 exhibited potent in vitro activity with mitigated CYP3A4 TDI and efficacy in vivo transplant model.

Validated HPLC method for simultaneous quantification of mutant IDH1/2 inhibitors (enasidenib, ivosidenib and vorasidenib) in mouse plasma: Application to a pharmacokinetic study.

Isocitrate dehydrogenase (IDH) inhibitors comprise a novel class of anticancer drugs, which are approved to treat acute myeloid leukemia patients having mutations on IDH1/2. We report the development and validation of a high-performance liquid chromatography (HPLC) method for the simultaneous quantitation of IDH inhibitors, namely enasidenib (EDB), ivosidenib (IDB) and vorasidenib (VDB), in mouse plasma as per the US Food and Drug Administration regulatory guidelines. The method involves extraction of EDB, IDB and VDB along with internal standard (IS; phenacetin) from mouse plasma (100 µl) using a simple protein precipitation process. The chromatographic analysis was performed on an HPLC system using a gradient mobile phase (comprising 10 mm ammonium acetate and acetonitrile in a flow-gradient) and an X-Terra Phenyl column. The UV detection wave length was set at λmax 265 nm. EDB, IDB, VDB and the IS eluted at 7.36, 8.60, 9.50 and 5.12 min, respectively, with a total run time of 10 min. The calibration curve was linear over a concentration range of 0.20-12.5 µg/ml for EDB and 0.50-12.5 µg/ml for IDB and VDB (r2  = ≥0.998 for all of the analytes). Validation results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

A phase I, open-label, two-stage study to investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of the oral AKT inhibitor GSK2141795 in patients with solid tumors.

Background We sought to determine the recommended phase II dose (RP2D) and schedule of GSK2141795, an oral pan-AKT kinase inhibitor. Patients and Methods Patients with solid tumors were enrolled in the dose-escalation phase. Pharmacokinetic (PK) analysis after a single dose (Cycle 0) informed dose escalation using accelerated dose titration. Once one grade 2 toxicity or dose-limiting toxicity was observed in Cycle 1, the accelerated dose titration was terminated and a 3 + 3 dose escalation was started. Continuous daily dosing was evaluated along with two intermittent regimens (7 days on/7 days off and 3 times per week). In the expansion phase at RP2D, patients with endometrial or prostate cancer, as well as those with select tumor types with a PIK3CA mutation, AKT mutation or PTEN loss, were enrolled. Patients were evaluated for adverse events (AEs), PK parameters, blood glucose and insulin levels, and tumor response. Results The RP2D of GSK2141795 for once-daily dosing is 75 mg. The most common (>10%) treatment-related AEs included diarrhea, fatigue, vomiting, and decreased appetite. Most AEs were low grade. The frequency of hyperglycemia increased with dose; however, at the RP2D, grade 3 hyperglycemia was only reported in 4% of patients and no grade 4 events were observed. PK characteristics were favorable, with a prolonged half-life and low peak-to-trough ratio. There were two partial responses at the RP2D in patients with either a PIK3CA mutation or PTEN loss. Conclusion GSK2141795 was safe and well-tolerated, with clinical activity seen as monotherapy at the RP2D of 75 mg daily. NCT00920257.

SAR refinement of antileishmanial N(2),N(4)-disubstituted quinazoline-2,4-diamines.

Visceral leishmaniasis is a neglected parasitic disease that has a high fatality rate in the absence of treatment. New drugs that are inexpensive, orally active, and effective could be useful tools in the fight against this disease. We previously showed that N(2),N(4)-disubstituted quinazoline-2,4-diamines displayed low- to sub-micromolar potency against intracellular Leishmania, and lead compound N(4)-(furan-2-ylmethyl)-N(2)-isopropyl-7-methylquinazoline-2,4-diamine (4) exhibited modest efficacy in an acute murine model of visceral leishmaniasis. In the present work, thirty-one N(2),N(4)-disubstituted quinazoline-2,4-diamines that had not previously been examined for their antileishmanial activity were evaluated for their potency and selectivity against Leishmania donovani, the causative parasite of visceral leishmaniasis. Quinazoline-2,4-diamines with aromatic substituents at both N(2) and N(4) exhibited potent in vitro antileishmanial activity but relatively low selectivity, while compounds substituted with small alkyl groups at either N(2) or N(4) generally showed lower antileishmanial potency but were less toxic to a murine macrophage cell line. Based on their in vitro antileishmanial potency, N(4)-benzyl-N(2)-(4-chlorobenzyl)quinazoline-2,4-diamine (15) and N(2)-benzyl-N(4)-isopropylquinazoline-2,4-diamine (40) were selected for in vivo evaluation of their pharmacokinetic and antileishmanial properties. While 15 displayed a longer plasma half-life and a greater area under the curve than 40, both compounds showed low efficacy in an acute murine visceral leishmaniasis model. Although the present study did not identify new quinazoline-2,4-diamines with promising in vivo efficacy, the reduced in vitro toxicity of derivatives bearing small alkyl groups at either N(2) or N(4) may provide clues for the design of safe and effective antileishmanial quinazolines.

Rhenium(I) polypyridine diamine complexes as intracellular phosphorogenic sensors: synthesis, characterization, emissive behavior, biological properties, and nitric oxide sensing.

We report the development of a series of rhenium(I) polypyridine complexes appended with an electron-rich diaminoaromatic moiety as phosphorogenic sensors for nitric oxide (NO). The diamine complexes [Re(N^N)(CO)3 (py-DA)][PF6 ] (py-DA=3-(N-(2-amino-5-methoxyphenyl)aminomethyl)pyridine; N^N=1,10-phenanthroline (phen) (1 a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4 -phen) (2 a), 4,7-diphenyl-1,10-phenanthroline (Ph2 -phen) (3 a)) have been synthesized and characterized. In contrast to common rhenium(I) diimines, these diamine complexes were very weakly emissive due to quenching of the triplet metal-to-ligand charge-transfer ((3) MLCT) emission by the diaminoaromatic moiety through photoinduced electron transfer (PET). Upon treatment with NO, the complexes were converted into the triazole derivatives [Re(N^N)(CO)3 (py-triazole)][PF6 ] (py-triazole=3-((6-methoxybenzotriazol-1-yl)methyl)pyridine; N^N=phen (1 b), Me4 -phen (2 b), Ph2 -phen (3 b)), resulting in significant emission enhancement (I/I0 ≈60). The diamine complexes exhibited high reaction selectivity to NO, and their emission intensity was found to be independent on pH. Also, these complexes were effectively internalized by HeLa cells and RAW264.7 macrophages with negligible cytotoxicity. Additionally, the use of complex 3 a as an intracellular phosphorogenic sensor for NO has been demonstrated.

Synthesis and in vivo evaluation of cyclic diaminopropane BACE-1 inhibitors.

The synthesis, evaluation, and structure-activity relationships of a set of related constrained diaminopropane inhibitors of BACE-1 are described. The full in vivo profile of an optimized inhibitor in both normal and P-gp deficient mice is compared with data generated in normal rats.

Design and Optimization of an Acyclic Amine Series of TRPV4 Antagonists by Electronic Modulation of Hydrogen Bond Interactions.

Investigation of TRPV4 as a potential target for the treatment of pulmonary edema associated with heart failure generated a novel series of acyclic amine inhibitors displaying exceptional potency and PK properties. The series arose through a scaffold hopping approach, which relied on use of an internal H-bond to replace a saturated heterocyclic ring. Optimization of the lead through investigation of both aryl regions revealed approaches to increase potency through substituents believed to enhance separate intramolecular and intermolecular H-bond interactions. A proposed internal H-bond between the amine and neighboring benzenesulfonamide was stabilized by electronically modulating the benzenesulfonamide. In the aryl ether moiety, substituents para to the nitrile demonstrated an electronic effect on TRPV4 recognition. Finally, the acyclic amines inactivated CYP3A4 and this liability was addressed by modifications that sterically preclude formation of a putative metabolic intermediate complex to deliver advanced TRPV4 antagonists as leads for discovery of novel medicines.

An intranasal irritation assessment of antibacterial ointment alone or in combination with mupirocin versus Bactroban Nasal in rabbits.

The purpose of this study was to evaluate the potential irritating effects and the systemic exposure level of an antibacterial ointment containing REP8839 as a single agent or in combination with mupirocin versus Bactroban Nasal in rabbits. Additionally, the reversibility of REP8839 effects during a 14-day recovery period was assessed. Five treatment groups of six male and six female New Zealand White rabbits received dose levels of 1%, 2%, and 4% REP8839, 2% Bactroban Nasal, or 2% REP8839/2% mupirocin combination. One additional group of six animals/sex served as the control and received the vehicle, Petrolatum/Softisan 649. The test article or vehicle was administered to all groups via topical administration to the external nares, twice a day (approx. 8h intervals between the doses) for 21 consecutive days, at a dose volume of 100 microL per nare/dose for a total of 400 microL per day (200 microL per nare). Two animals/sex/group were maintained for a 14-day recovery period. The external nares were reflected back and the mucosal lining was evaluated and scored for erythema and edema within 30-60 min following the first dose each day. Blood samples were collected from all animals at designated time points on Day 21 of the study to assess systemic exposure levels. Cross-sectioning of the nasal tract was conducted in all the groups for microscopic evaluation. Mucosal scoring of the nares did not reveal any edema or erythema in any of the dose groups with the antibacterial alone, with the combination product, or with Bactroban Nasal. Mean body weights and food consumption were not adversely impacted by the test articles. Minimal plasma exposure was observed in the rabbits (<5 ng/mL). The REP8839 groups did appear to have dose-responsive exposure (from below the limit of quantitation to 5 ng/mL with 1%, 2%, and 4% REP8839, respectively). Microscopic changes on the nasal sectioning noted in these animals were infrequent and considered incidental findings unrelated to administration of the test articles. In conclusion doses of up to 4% of REP8839 ointment as a single agent or 2% in the combination product, as well as 2% Bactroban Nasal, were not found to induce mucosal irritation when applied topically to the external nares twice a day for 21 consecutive days. Additionally, no delayed effects were observed in the recovery animals.

Influence of two different fluoride compounds and an in vitro pellicle on the amount of KOH-soluble fluoride and its retention after toothbrushing.

OBJECTIVE: To determine the influence of two different fluoride compounds and an in vitro pellicle on KOH-soluble fluoride formation - its retention and resistance to toothbrushing. MATERIAL AND METHODS: Forty bovine incisors were randomly assigned to four groups (A-D). Of five samples prepared per tooth, one remained untreated and served as a baseline control. Groups A and B were pretreated with artificial saliva and groups C and D with human saliva. Groups A and C were treated with amine fluoride and groups B and D with sodium fluoride. After treatment, samples were brushed with 25, 50, and 75 brushing strokes. The amount of KOH-soluble fluoride formed on the enamel samples was measured at baseline, after application, and after 25, 50, and 75 brushing strokes. Fluoride uptake was calculated by unpaired t-tests and fluoride retention by paired t-tests. RESULTS: No statistically significant differences in the KOH-soluble fluoride uptake of the groups that were pretreated (A vs B and C vs D) or treated equally (A vs C and B vs D) were observed. Retention of the KOH-soluble fluoride in the brushed samples was higher when the samples were pretreated with human saliva and treated with sodium fluoride than when the samples were pretreated with artificial saliva and treated with sodium fluoride. CONCLUSIONS: The fluoride compound and the acquired human in vitro pellicle have no influence on the uptake of KOH-soluble fluoride, but show a significant influence on abrasion resistance.

Tumor Contrast Enhancement and Whole-Body Elimination of the Manganese-Based Magnetic Resonance Imaging Contrast Agent Mn-PyC3A.

OBJECTIVES: The goals of this study were to compare the efficacy of the new manganese-based magnetic resonance imaging (MRI) contrast agent Mn-PyC3A to the commercial gadolinium-based agents Gd-DOTA and to Gd-EOB-DTPA to detect tumors in murine models of breast cancer and metastatic liver disease, respectively, and to quantify the fractional excretion and elimination of Mn-PyC3A in rats. METHODS: T1-weighted contrast-enhanced MRI with 0.1 mmol/kg Mn-PyC3A was compared with 0.1 mmol/kg Gd-DOTA in a breast cancer mouse model (n = 8) and to 0.025 mmol/kg Gd-EOB-DTPA in a liver metastasis mouse model (n = 6). The fractional excretion, 1-day biodistribution, and 7-day biodistribution in rats after injection of 2.0 mmol/kg [Mn]Mn-PyC3A or Gd-DOTA were quantified by Mn gamma counting or Gd elemental analysis. Imaging data were compared with a paired t test; biodistribution data were compared with an unpaired t test. RESULTS: The postinjection-preinjection increases in tumor-to-muscle contrast-to-noise ratio (ΔCNR) 3 minutes after injection of Mn-PyC3A and Gd-DOTA (mean ± standard deviation) were 17 ± 3.8 and 20 ± 4.4, respectively (P = 0.34). Liver-to-tumor ΔCNR values at 8 minutes postinjection of Mn-PyC3A and Gd-EOB-DTPA were 28 ± 9.0 and 48 ± 23, respectively (P = 0.11). Mn-PyC3A is eliminated with 85% into the urine and 15% into the feces after administration to rats. The percentage of the injected doses (%ID) of Mn and Gd recovered in tissues after 1 day were 0.32 ± 0.12 and 0.57 ± 0.12, respectively (P = 0.0030), and after 7 days were 0.058 ± 0.051 and 0.19 ± 0.052, respectively (P < 0.0001). CONCLUSIONS: Mn-PyC3A provides comparable tumor contrast enhancement to Gd-DOTA in a mouse breast cancer model and is more completely eliminated than Gd-DOTA; partial hepatobiliary elimination of Mn-PyC3A enables conspicuous delayed phase visualization of liver metastases.

High performance liquid chromatography with ultraviolet detection for the determination of SYUIQ-5, a novel telomerase inhibitor for cancer therapy: application to an enzyme kinetic study in rat liver microsomes.

A sensitive assay for the determination of SYUIQ-5, a novel telomerase inhibitor and anti-tumor drug, in rat liver microsomes was developed by using high-performance liquid chromatography with ultraviolet detection. SYUIQ-5 was incubated in vitro with liver microsomes from rats pre-treated with control vehicle, beta-naphthofIavone, phenobarbital, 20% ethanol or dexamethasone. The analytes were extracted with diethyl ether and separated a C(18) 5-microm analytical column. Elution was conducted with 30 mM dipotassium hydrogen phosphate (pH 8.0)-methanol-triethylamine (30:70:0.05, v/v/v) at a flow-rate of 1.0 ml/min and the detection of UV absorbance was conducted at 278 nm. Intra-day and inter-day precision and accuracy of the method were within 10%. The mean analytical recoveries of SYUIQ-5 ranged from 78.8 to 95.3%. The linearity of the calibration curve was in the range of 1.0-80.0 microM. The lower limit of quantification (LOQ) was 1.0 microM. Kinetic analysis showed that beta-naphthofIavone and dexamethasone significantly induced SYUIQ-5 metabolism, suggesting that cytochrome P450 1A and 3A are the major contributor to SYUIQ-5 metabolism in rat liver microsomes.

1-Acyl-1H-[1,2,4]triazole-3,5-diamine analogues as novel and potent anticancer cyclin-dependent kinase inhibitors: synthesis and evaluation of biological activities.

A series of 1-acyl-1H-[1,2,4]triazole-3,5-diamine analogues were synthesized as cyclin-dependent kinase (CDK) inhibitors. These compounds showed potent and selective CDK1 and CDK2 inhibitory activities and inhibited in vitro cellular proliferation in various human tumor cells. Representative compound 3b demonstrated in vivo efficacy in a human melanoma A375 xenograft model in nude mice.

Pharmacodynamics and pharmacokinetics of SQ109, a new diamine-based antitubercular drug.

SQ109 is a novel [1,2]-diamine-based ethambutol (EMB) analog developed from high-throughput combinatorial screening. The present study aimed at characterizing its pharmacodynamics and pharmacokinetics. The antimicrobial activity of SQ109 was confirmed in vitro (Mycobacterium tuberculosis-infected murine macrophages) and in vivo (M. tuberculosis-infected C57BL/6 mice) and compared to isoniazid (INH) and EMB. SQ109 showed potency and efficacy in inhibiting intracellular M. tuberculosis that was similar to INH, but superior to EMB. In vivo oral administration of SQ109 (0.1-25 mg kg(-1) day(-1)) to the mice for 28 days resulted in dose-dependent reductions of mycobacterial load in both spleen and lung comparable to that of EMB administered at 100 mg kg(-1) day(-1), but was less potent than INH at 25 mg kg(-1) day(-1). Monitoring of SQ109 levels in mouse tissues on days 1, 14 and 28 following 28-day oral administration (10 mg kg(-1) day(-1)) revealed that lungs and spleen contained the highest concentration of SQ109, at least 10 times above its MIC. Pharmacokinetic profiles of SQ109 in mice following a single administration showed its C(max) as 1038 (intravenous (i.v.)) and 135 ng ml(-1) (p.o.), with an oral T(max) of 0.31 h. The elimination t(1/2) of SQ109 was 3.5 (i.v.) and 5.2 h (p.o.). The oral bioavailability was 4%. However, SQ109 displayed a large volume of distribution into various tissues. The highest concentration of SQ109 was present in lung (>MIC), which was at least 120-fold (p.o.) and 180-fold (i.v.) higher than that in plasma. The next ranked tissues were spleen and kidney. SQ109 levels in most tissues after a single administration were significantly higher than that in blood. High tissue concentrations of SQ109 persisted for the observation period (10 h). This study demonstrated that SQ109 displays promising in vitro and in vivo antitubercular activity with favorable targeted tissue distribution properties.

2,2-Difluoro-5-hexyne-1,4-diamine: a potent enzyme-activated inhibitor of ornithine decarboxylase.

2,2-Difluoro-5-hexyne-1,4-diamine was prepared in an eight-step sequence from ethyl 2,2-difluoro-4-pentenoate and tested as an inhibitor of mammalian ornithine decarboxylase. It produces a time-dependent inhibition of the enzyme in vitro which shows saturation kinetics, with KI = 10 microM and tau 1/2 = 2.4 min. In rats, it produces a rapid, long-lasting, and dose-dependent decrease of ornithine decarboxylase activity in the ventral prostate, testis, and thymus. In contrast with the nonfluorinated analogue 5-hexyne-1,4-diamine (Danzin et al. Biochem. Pharmacol. 1983, 32, 941), 2,2-difluoro-5-hexyne-1,4-diamine is not a substrate of mitochondrial monoamine oxidase.

A new, general synthetic route to multidentate N,S ligands for use in technetium-99m radiopharmaceuticals. Preparation of diamido disulfur, diamino dithiol, and tripodal N3S3 prototypes. Comparative biodistributions of [99mTcvO-DADS]- analogues which contain 5,5,5- and 5,7,5-membered chelate ring systems.

A new, versatile synthetic route to a variety of tetradentate N2S2 and hexadentate N3S3 ligands for use in technetium-99m radiopharmaceuticals has been developed. The key reaction employs 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) for coupling an appropriate di- or triamine with S-protected thioglycolic acid. Selected DADS (diamido disulfur) and DADT (diamino dithiol) and analogues derived from ethanediamine-1,2 and butanediamine-1,4, as well as a tripodal N3S3 analogue, were synthesized in high yield. The labeling of DADS analogue ligands derived from butanediamine-1,4 (DADS-bn) with 99mTc results in a single radiochemical product, or the expected number of stereoisomeric products. FAB MS analysis, using 99Tc, indicates that DADS-bn ligands form a tetradentate 5,7,5-membered ring chelate system with the monooxo Tc(V) core: [TcO-DAD-bn]-. In rat biodistribution studies the DADS-en and DADS-bn complexes show very similar biological activity. Phenyl substituents on the 7-membered ring give rise to a 99mTc complex of increased lipophilicity, increased liver uptake, and minimal hepatobillary clearance. Thus, new classes of DADS ligand analogues which contain a 5,7,5-membered chelate ring system are readily synthesized and labeled with 99mTc to form stable complexes. The presence of the 7-membered chelate ring allows for facile introduction of pendant groups into the ligand system, and thus for a ready route to control the biodistribution of the resulting 99mTc radiopharmaceutical. The 99mTc labeling of the DADT analogues derived from butanediamine-1,4 provided to be erratic, despite the use of a variety of labeling techniques; the larger ring system of DADT-bn apparently does not favor the monooxo Tc(V) core and appears to generate a mixture of mono- and dioxo Tc(V) cores.

Technetium-99m-MRP20, a potential brain perfusion agent: in vivo biodistribution and SPECT studies in normal male volunteers.

The lipophilic neutral complex [99mTc][TcO(MRP20)] [MRP20 is (N-(2(1H pyrolylmethyl]N'-(4-pentene-3-one-2)ethane-1,2-diamine)] is known to cross the blood-brain barrier in non-primate animals. We report here its in vivo biodistribution, radiation dosimetry, and single-photon emission computed tomography (SPECT) characteristics in man. Following i.v. administration of 15-25 mCi of the tracer, the maximum uptake of activity in the brain peaked at 1 min p.i. Fifteen minutes p.i., the percentage retained in the brain was 5.2 +/- 1.6, which remained fairly constant over 24 hr. Blood clearance was relatively slow with an apparent affinity of the compound for the cellular fraction of the blood, however, soft-tissue and facial activity cleared at a rate four times faster than that of the brain. SPECT images obtained at 15 min, 1 hr, 3 hr, 7 hr, and 24 hr p.i. showed no redistribution of the tracer within the brain. The dosimetry is favorable for administration of 25-30 mCi of MRP20. Our results indicate that this compound is rapidly extracted and retained by the brain and may be used for SPECT imaging of regional blood flow.

Technetium-99m-MRP20, a potential brain perfusion agent: in vivo biodistribution and SPECT studies in non-primate animals.

MRP20 (N-(2(1H pyrolylmethyl]N'-(4-pentene-3-one-2] ethane-1,2-diamine) complexes with technetium-99m, yielding a neutral, lipophilic species. This compound has been characterized as [TcO(MRP20)]. Biologic investigation of [99mTc][TcO(MRP20)] in female rats showed 2.35% ID in the brain 30 min p.i. with no significant wash-out over 3 hr. A single-photon emission computed tomography (SPECT) study in a dog demonstrated rapid tracer uptake in the brain, reaching a maximum within 1 min, with 2.24% i.d. 15 min p.i., decreasing to 1.7% after 4 hr. The complex undergoes hydrolysis in vitro forming a cationic species. This is possibly the trapping mechanism in the brain in vivo. The main excretory route of [99mTc][TcO(MRP20)] is via the hepatobiliary tract. There is evidence of some "in vivo" cell labeling and soft-tissue uptake.

Two populations of muscarinic binding sites in the chick heart distinguished by affinities for ligands and selective inactivation.

1. By measuring the binding of N-[3H-methyl]-scopolamine ([3H]-NMS) and of unlabelled subtype-specific muscarinic antagonists, two populations of muscarinic binding sites can be distinguished in the membranes of cardiac ventricles taken from 1-day-old chicks. One of them, corresponding to approximately 80% of [3H]-NMS binding sites, has higher affinities for AF-DX116 (pKi = 6.42) and methoctramine (pKi = 7.33); the rate of [3H]NMS dissociation from these sites is fast. The other population, corresponding to approximately 20% of [3H]-NMS binding sites, has lower affinities for AF-DX116 (pKi = 5.00) and methoctramine (pKi = 6.19); the rate of [3H]-NMS dissociation from these sites is slow. Both populations have high affinities for pirenzepine, but the affinity of the former (major) population is lower (pKi = 7.99) than that of the latter (minor) population (pKi = 10.14). 2. Since it has been shown earlier that two mRNAs for muscarinic receptors are expressed in the chick heart, one of them close to the genetically defined m2 and the other to the m4 subtype, we propose that the major population of binding sites with high affinities for AF-DX116 and methoctramine and the lower affinity for pirenzepine represents the M2-like receptors, while the minor population represents the M4-like receptors. 3. It proved possible to obtain isolated samples of either population by selectively protecting the M2-like sites with AF-DX116 and the M4-like sites with pirenzepine, and by inactivating the unprotected sites with benzilylcholine mustard. The properties of the isolated populations corresponded to those derived from the analysis of [3H]-NMS binding to the original mixed population.4 Alcuronium exerted positive allosteric action on the binding of [3H]-NMS both to the M2-like and the M4-like population and severely slowed down [3H]-NMS dissociation from them; its affinity for the M2-like sites was 3-10 times higher.

Targeting superoxide dismutase to renal proximal tubule cells inhibits nephrotoxicity of cisplatin and increases the survival of cancer-bearing mice.

Because cis-diamminedichloroplatinum(II) (cisplatin) which generates reactive oxygen species induces renal dysfunction, administration of a large dose for killing cancer cells is highly limited. We recently synthesized a cationic superoxide dismutase (SOD) (hexamethylenediamine-conjugated SOD, AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. Treatment of Ehrlich ascites tumor cells (EATC)-bearing mice with cisplatin sufficient for killing tumor cells increased their motality. The motality of cisplatin-treated EATC-bearing mice was markedly decreased by AH-SOD. These results suggest that targeting SOD to renal proximal tubule cells might permit the administration of high doses of cisplatin and related anticancer agents without causing renal injury.

M2 muscarinic receptor subtype in the feline medial pontine reticular formation modulates the amount of rapid eye movement sleep.

Rapid eye movement (REM) sleep is generated, in part, by activating muscarinic cholinergic receptors (mAChRs) in the medial pontine reticular formation (mPRF). Molecular cloning has identified five mAChR subtypes, and this study tested the hypothesis that the M2 subtype in the mPRF modulates the amount of REM sleep. This hypothesis cannot be tested directly, due to lack of subtype selective muscarinic agonists. However, the amount of REM sleep can be enhanced by mPRF microinjection of a muscarinic agonist, and the relative potencies of muscarinic antagonists to block the REM sleep enhancement can be determined. Two muscarinic antagonists, methoctramine and 4-DAMP, were studied. Six concentrations of each antagonist were microinjected into the mPRF of conscious cat 15 min prior to the agonist bethanechol. Nonlinear regression analysis was used to calculate the dose of antagonist that caused a 50% inhibition (ID50) of bethanechol-induced REM sleep. Bethanechol significantly increased (442%) the amount of time spent in REM sleep. Both methoctramine and 4-DAMP significantly blocked the bethanechol-induced REM sleep increase, with an ID50 of 1.8 microM and 0.6 microM, respectively. The ID50 ratio for methoctramine-to-4-DAMP (3.0) was similar to the affinity ratio of methoctramine-to-4-DAMP only at the M2 subtype (3.5), suggesting that the M2 subtype in the mPRF modulates the amount of REM sleep. This study also tested the null hypothesis that sleep-dependent respiratory depression evoked by mPRF cholinomimetics would not be antagonized by pretreatment of the mPRF with muscarinic antagonists. Neither methoctramine nor 4-DAMP antagonized the bethanechol-induced decrease in respiratory rate.