Novel Acylguanidine-Based Inhibitor of HIV-1.

UNLABELLED: The emergence of transmissible HIV-1 strains with resistance to antiretroviral drugs highlights a continual need for new therapies. Here we describe a novel acylguanidine-containing compound, 1-(2-(azepan-1-yl)nicotinoyl)guanidine (or SM111), that inhibits in vitro replication of HIV-1, including strains resistant to licensed protease, reverse transcriptase, and integrase inhibitors, without major cellular toxicity. At inhibitory concentrations, intracellular p24(Gag) production was unaffected, but virion release (measured as extracellular p24(Gag)) was reduced and virion infectivity was substantially impaired, suggesting that SM111 acts at a late stage of viral replication. SM111-mediated inhibition of HIV-1 was partially overcome by a Vpu I17R mutation alone or a Vpu W22* truncation in combination with Env N136Y. These mutations enhanced virion infectivity and Env expression on the surface of infected cells in the absence and presence of SM111 but also impaired Vpu's ability to downregulate CD4 and BST2/tetherin. Taken together, our results support acylguanidines as a class of HIV-1 inhibitors with a distinct mechanism of action compared to that of licensed antiretrovirals. Further research on SM111 and similar compounds may help to elucidate knowledge gaps related to Vpu's role in promoting viral egress and infectivity. IMPORTANCE: New inhibitors of HIV-1 replication may be useful as therapeutics to counteract drug resistance and as reagents to perform more detailed studies of viral pathogenesis. SM111 is a small molecule that blocks the replication of wild-type and drug-resistant HIV-1 strains by impairing viral release and substantially reducing virion infectivity, most likely through its ability to prevent Env expression at the infected cell surface. Partial resistance to SM111 is mediated by mutations in Vpu and/or Env, suggesting that the compound affects host/viral protein interactions that are important during viral egress. Further characterization of SM111 and similar compounds may allow more detailed pharmacological studies of HIV-1 egress and provide opportunities to develop new treatments for HIV-1.

Mechanisms of Action of Novel Influenza A/M2 Viroporin Inhibitors Derived from Hexamethylene Amiloride.

The increasing prevalence of influenza viruses with resistance to approved antivirals highlights the need for new anti-influenza therapeutics. Here we describe the functional properties of hexamethylene amiloride (HMA)-derived compounds that inhibit the wild-type and adamantane-resistant forms of the influenza A M2 ion channel. For example, 6-(azepan-1-yl)-N-carbamimidoylnicotinamide ( 9: ) inhibits amantadine-sensitive M2 currents with 3- to 6-fold greater potency than amantadine or HMA (IC50 = 0.2 vs. 0.6 and 1.3 µM, respectively). Compound 9: competes with amantadine for M2 inhibition, and molecular docking simulations suggest that 9: binds at site(s) that overlap with amantadine binding. In addition, tert-butyl 4'-(carbamimidoylcarbamoyl)-2',3-dinitro-[1,1'-biphenyl]-4-carboxylate ( 27: ) acts both on adamantane-sensitive and a resistant M2 variant encoding a serine to asparagine 31 mutation (S31N) with improved efficacy over amantadine and HMA (IC50 = 0.6 µM and 4.4 µM, respectively). Whereas 9: inhibited in vitro replication of influenza virus encoding wild-type M2 (EC50 = 2.3 µM), both 27: and tert-butyl 4'-(carbamimidoylcarbamoyl)-2',3-dinitro-[1,1'-biphenyl]-4-carboxylate ( 26: ) preferentially inhibited viruses encoding M2(S31N) (respective EC50 = 18.0 and 1.5 µM). This finding indicates that HMA derivatives can be designed to inhibit viruses with resistance to amantadine. Our study highlights the potential of HMA derivatives as inhibitors of drug-resistant influenza M2 ion channels.

URINARY EXCRETION OF PLUTONIUM IN MAYAK WORKERS DURING AND AFTER CA-DTPA ADMINISTRATION.

Chelation therapy is sometimes used after potential exposures to plutonium to increase urinary excretion of the radionuclide to improve the accuracy of bioassay measurements. The purpose of this report is to describe the enhancement of urinary excretion of plutonium during and after the administration of the trisodium salt of calcium diethylenetriaminepentaacetate (Ca-DTPA) daily for 3 d to a group of male and female plutonium workers from the Mayak Production Association in Ozyorsk, Russia. One-hundred and two cases (18 females and 84 males) were selected where urinary contents of plutonium, prior to chelation, exceeded the detection threshold. Daily urine collections were obtained during the 3 d of Ca-DTPA treatments. In addition, 58 of these cases had urine bioassays at 1-45 d after chelation. The daily enhancement over baseline values excretion of plutonium was found to be 50.4×/1.4 (geometric mean and geometric standard deviation); 58.9×/1.2; 72.9×/1.4 in the first, second and third days of Ca-DTPA administration. The mean enhancement for the 3-d period was 60.1×/1.7. The rate of plutonium excretion from 1 to 45 d after chelation decreased with a half-period of 3.9 d and the chelation enhancement factor (Кenh-i) is described by the function Кenh-i = (0.79 ± 0.24) + (42.9 ± 1.2) × e-(0.18 ± 0.01) × day.

The effects of chronic diseases on plutonium urinary excretion in former workers of the Mayak Production Association.

The radiochemical analysis of plutonium activity in urine is the main method for indirect estimation of doses of internal exposure from plutonium incorporation in professional workers. It was previously shown that late-in-life acute diseases, particularly those that affect the liver, can promote accelerated rates of release of plutonium from the liver with enhanced excretion rates. This initial study examines the relationships of some chronic diseases on plutonium excretion as well as the terminal relative distribution of plutonium between the liver and skeleton. Fourteen cases from former workers at the Mayak Production Association (Mayak PA) who provided from 4-9 urine plutonium bioassays for plutonium, had an autopsy conducted after death, and had sufficient clinical records to document their health status were used in this study. Enhanced plutonium excretion was associated with more serious chronic diseases, including cardiovascular diseases and other diseases that involved the liver. These chronic diseases were also associated with relatively less plutonium found in the liver relative to the skeleton determined by analyses conducted after autopsy. These data further document health conditions that affect plutonium biokinetics and organ deposition and retention patterns and suggest that health status should be considered when conducting plutonium bioassays as these may alter subsequent dosimetry and risk models.

THE MAYAK WORKER DOSIMETRY SYSTEM (MWDS-2016): INTERNAL DOSIMETRY RESULTS AND COMPARISON WITH MWDS-2013.

Differences in results from the new Mayak Worker Dosimetry System (MWDS-2016) vs the previous MWDS-2013 are described. Statistical characteristics are shown for the distribution of accumulated absorbed doses to organs for 8340 workers with bioassay data. Differences in mean values of accumulated doses and their relative standard uncertainties calculated by MWDS-2016 and MWDS-2013 were analysed separately for various types of industrial compounds of plutonium, specifically nitrates, mixtures and oxides. Within the range of accumulated doses >1 mGy, lung doses for nitrates and mixtures decreased by 41 and 15%, respectively, and remained at the same level for oxides. Accumulated liver doses within the range >1 mGy increased for nitrates and mixtures by 13 and 8%, respectively, and decreased for oxides by 7%.

Uncertainties analysis for the plutonium dosimetry model, doses-2005, using Mayak bioassay data.

The Doses-2005 model is a combination of the International Commission on Radiological Protection (ICRP) models modified using data from the Mayak Production Association cohort. Surrogate doses from inhaled plutonium can be assigned to approximately 29% of the Mayak workers using their urine bioassay measurements and other history records. The purpose of this study was to quantify and qualify the uncertainties in the estimates for radiation doses calculated with the Doses-2005 model by using Monte Carlo methods and perturbation theory. The average uncertainty in the yearly dose estimates for most organs was approximately 100% regardless of the transportability classification. The relative source of the uncertainties comes from three main sources: 45% from the urine bioassay measurements, 29% from the Doses-2005 model parameters, and 26% from the reference masses for the organs. The most significant reduction in the overall dose uncertainties would result from improved methods in bioassay measurement with additional improvements generated through further model refinement. Additional uncertainties were determined for dose estimates resulting from changes in the transportability classification and the smoking toggle. A comparison was performed to determine the effect of using the model with data from either urine bioassay or autopsy data; no direct correlation could be established. Analysis of the model using autopsy data and incorporation of results from other research efforts that have utilized plutonium ICRP models could improve the Doses-2005 model and reduce the overall uncertainty in the dose estimates.

RON kinase: A target for treatment of cancer-induced bone destruction and osteoporosis.

Bone destruction occurs in aging and numerous diseases, including osteoporosis and cancer. Many cancer patients have bone osteolysis that is refractory to state-of-the-art treatments, which block osteoclast activity with bisphosphonates or by inhibiting the receptor activator of nuclear factor κB ligand (RANKL) pathway. We previously showed that macrophage-stimulating protein (MSP) signaling, which is elevated in about 40% of breast cancers, promotes osteolytic bone metastasis by activation of the MSP signaling pathway in tumor cells or in the bone microenvironment. We show that MSP signals through its receptor, RON tyrosine kinase, expressed on host cells, to activate osteoclasts directly by a previously undescribed pathway that is complementary to RANKL signaling and converges on proto-oncogene, non-receptor tyrosine kinase SRC (SRC). Genetic or pharmacologic inhibition of RON kinase blocked cancer-mediated bone destruction and osteoporosis in several mouse models. Furthermore, the RON kinase inhibitor BMS-777607/ASLAN002 altered markers of bone turnover in a first-in-human clinical cancer study, indicating the inhibitor's potential for normalizing bone loss in patients. These findings uncover a new therapeutic target for pathogenic bone loss and provide a rationale for treatment of bone destruction in various diseases with RON inhibitors.

Efficacy of orally administered amphipathic polyaminocarboxylic acid chelators for the removal of plutonium and americium: comparison with injected Zn-DTPA in the rat.

Chelators are used to promote excretion of actinides and some other metals, but few are orally effective. The relative efficacies of orally administered triethylenetetraminepentaacetic acids (TT) with varying lipophilic properties on the removal of 241Am and 239Pu and comparison with parenteral Zn-DTPA was determined. The actinides were administered to adult rats 2 weeks prior to initiation of 30 d of chelation treatment. The TT compounds were given orally while Zn-DTPA was given twice weekly by injection. Total body content of 241Am was measured before and during the treatment period and organ contents of 241Am and 239Pu were measured at the end of the study. Significant reductions in 241Am occurred within the first week, with Zn-DTPA being the most effective. By 3 weeks, the most lipophilic chelator, C22TT was as effective as Zn-DTPA. After 30 d, reductions in organ content of 239Pu and 241Am directly correlated with increasing lipophilicity of the TT chelators. Oral C22TT was as effective as injected Zn-DTPA in liver and bone, the major organs of actinide deposition. The removal of 239Pu from the liver and reduction of redeposition of 239Pu in newly formed bone by C22TT was confirmed by neutron-induced autoradiographs. The amphipathic TT chelators may be useful as orally administered alternatives to current parenteral DTPA for the removal of actinide elements from the body, particularly for longer-term therapeutic applications.

Characterization of spinal cord white matter by suppressing signal from hindered space. A Monte Carlo simulation and an ex vivo ultrahigh-b diffusion-weighted imaging study.

Signal measured from white matter in diffusion-weighted imaging is difficult to interpret because of the heterogeneous structure of white matter. Characterization of the white matter will be straightforward if the signal contributed from the hindered space is suppressed and purely restricted signal is analyzed. In this study, a Monte Carlo simulation (MCS) of water diffusion in white matter was performed to understand the behavior of the diffusion-weighted signal in white matter. The signal originating from the hindered space of an excised pig cervical spinal cord white matter was suppressed using the ultrahigh-b radial diffusion-weighted imaging. A light microscopy image of a section of white matter was obtained from the excised pig cervical spinal cord for the MCS. The radial diffusion-weighted signals originating from each of the intra-axonal, extra-axonal, and total spaces were studied using the MCS. The MCS predicted that the radial diffusion-weighted signal remains almost constant in the intra-axonal space and decreases gradually to about 2% of its initial value in the extra-axonal space when the b-value is increased to 30,000s/mm2. The MCS also revealed that the diffusion-weighted signal for a b-value greater than 20,000s/mm2 is mostly from the intra-axonal space. The decaying behavior of the signal-b curve obtained from ultrahigh-b diffusion-weighted imaging (bmax∼30,000s/mm2) of the excised pig cord was very similar to the decaying behavior of the total signal-b curve synthesized in the MCS. A mono-exponential plus constant fitting of the signal-b curve obtained from a white matter pixel estimated the values of constant fraction and apparent diffusion coefficient of decaying fraction as 0.32±0.05 and (0.16±0.01)×10-3mm2/s, respectively, which agreed well with the results of the MCS. The signal measured in the ultrahigh-b region (b>20,000s/mm2) is mostly from the restricted (intra-axonal) space. Integrity and intactness of the axons can be evaluated by assessing the remaining signal in the ultrahigh-b region.

Skeletal function and structure: implications for tissue-targeted therapeutics.

Osteoporosis, arthritis, and periodontal disease are common diseases of the skeleton, all of which could benefit from new therapeutic strategies, including targeted drug delivery. While bone is a rigid structure, it is not inert, with the cells of the skeleton being able to repair damage and respond to alterations in mechanical stimuli and various endocrine agents. Several important factors related to bone physiology that could influence the success of a pharmacological treatment include heterogeneity in bone remodeling activities throughout the skeleton, differences in blood supply and local vascularization, and the "blood-bone" barrier. The structural qualities of bone, especially the presence of hydroxyapatite crystals in the bone mineral and the established binding of certain molecules to this mineral phase, including tetracyclines, bisphosphonates, and other chelators, provide unique opportunities to treat skeletal diseases using targeted drug delivery. Additional opportunities exist in targeting sites with contrasting bone surface activities, including surfaces that are inactive, forming new bone or being resorbed. The ultimate key to developing new bone-targeted therapies is to understand and exploit the physiological characteristics at the desired target sites.

Bone-targeting macromolecular therapeutics.

Musculoskeletal diseases such as osteoporosis are recognized as major public health problems worldwide. Many novel therapeutic agents have been identified for the treatment of these diseases. However, the majority of them are not specific to hard tissue, resulting significant toxicity. Bone-targeting drug delivery systems based on water-soluble polymers can specifically direct candidate drugs to bone thereby reducing side effects due to non-specific tissue interactions. Incorporation of a targeting moiety, a drug release mechanism, drug selection and optimization of the polymer carrier are all essential elements in the development of bone-targeting macromolecular therapeutics. Successful clinical application of this approach can significantly contribute to the development of treatments for many musculoskeletal diseases.

Does the severity of radionuclide-induced skeletal malignancy depend upon radiation dose?

The severity of effect as a function of skeletal radiation dose for induced skeletal malignancy was investigated among a relatively large group of beagle dogs singly injected as young adults with soluble radionuclide. Bone-seeking radionuclides considered were 239Pu, 226Ra, 228Ra, 224Ra, 228Th, and 90Sr. Variables included skeletal radiation dose, tumor growth rate, maximum tumor volume, sex of the animals, growth period of the tumor, degree of calcification of the tumor, skeletal location of the tumor, year of death, and occurrence of metastases. Except for a significant relationship between tumor volume and metastatic process and for growth rate and tumor volume, no significant dependence of any two of these factors could be established. It is concluded from available data on skeletal malignancies among beagles exposed to bone-seeking radionuclides that the severity of the disease is not dependent upon skeletal radiation dose.

Prostaglandin E--a powerful anabolic agent for generalized or site-specific bone formation.

Prostaglandins are locally secreted, rapidly metabolized, biologically active fatty acids first identified in the prostate. The role of prostaglandins in the inflammatory response has been widely studied. However, some prostaglandins, particularly those of the E series (PGEs), can suppress inflammation, making it difficult to understand the local events and their sequence. This bimodal potential of the PGEs has been poorly understood in skeletal biology, causing the initial report of PGEs as mediators of bone resorption to persist for more than two decades, despite ample evidence to the contrary. This resulted in part from the power of any initial report to overrule subsequent conflicting views and in part on the exclusive reliance on in vitro data to explain in vivo phenomena. Over a decade ago, the potential of PGEs as authentic anabolic skeletal agents was demonstrated convincingly in vivo by both systemic and local delivery. The potential clinical applications of the PGEs in skeletal biology have not yet been developed. Our purpose is to review the reasons for the delayed discovery of the true skeletal effects of the PGEs and to describe applications for this technology. With the development of appropriate delivery systems, one can anticipate widespread clinical applications of the PGEs to accelerate skeletal repair, and to treat skeletal pathologies and trauma.

Comparisons of the skeletal locations of putative plutonium-induced osteosarcomas in humans with those in beagle dogs and with naturally occurring tumors in both species.

Osteosarcomas occur from exposures to bone-seeking, alpha-particle-emitting isotopes, particularly plutonium. The skeletal distribution of putative 239Pu-induced osteosarcomas reported in Mayak Metallurgical and Radiochemical Plutonium Plant workers is compared with those observed in canine studies, and these are compared with distributions of naturally occurring osteosarcomas in both species. In the Mayak workers, 29% and 71% of the osteosarcomas were in the peripheral and central skeleton, respectively, with the spine having the most tumors (36%). An almost identical distribution of plutonium-induced osteosarcomas was reported for dogs injected with 239Pu as young adults. This distribution of osteosarcomas is quite different from the distributions of naturally occurring osteosarcomas for both species. In the Cooperative Osteosarcoma Study Group in humans (1,736 osteosarcomas from all ages), over 91% of the tumors occurred in the peripheral skeleton. In the Mayo Clinic group of older individuals (>40 years old), over 60% of the osteosarcomas appeared in the peripheral skeleton. The distribution of naturally occurring osteosarcomas in the canine is similar to that in the adult human. The similarities of the distributions of plutonium-associated osteosarcomas in the Mayak workers with those found in experimental studies suggest that many of the reported osteosarcomas may have been associated with plutonium exposures. These results also support the experimental paradigm that plutonium osteosarcomas have a preference for well vascularized cancellous bone sites. These sites have a greater initial deposition of plutonium, but also greater turnover due to elevated bone remodeling rates.

Cortical bone dynamics, strength, and densitometry after induction of emphysema in hamsters.

Recent evidence suggests that patients suffering from chronic obstructive pulmonary disease are also at an increased risk of developing osteoporosis. The pathophysiological mechanism(s) linking these progressive diseases is unknown. The goal of this investigation was to determine whether there were alterations in bone mineral density and content, cortical bone structure and strength, and indexes of bone formation and resorption in the elastase-induced emphysematous hamster. At 3 wk after induction of emphysema, the femoral bone mineral content was 8% less (P = 0.026) and the femoral fracture strength was 6% less (P = 0.032) in the emphysematous hamster than in controls. The cortical area was 8.4% less (P = 0.013) and the periosteal mineral appositional rate was 27% less (P = 0.05) than in controls. Additionally, the endocortical eroded surface in the emphysematous group was about twice that in the control group (P = 0.003). Differences in some indexes of bone formation and resorption, paralleled by differences in bone structure and strength, were observed 3 wk after induction of emphysema. These differences in skeletal metabolism and strength may help explain some of the skeletal changes associated with chronic obstructive pulmonary disease in humans.

Rapid improvements in cortical bone dynamics and structure after lactation in established breeder rats.

The mineral requirements for milk production during lactation usually result in a decrease in maternal skeletal mass during this period. The purpose of this study was to characterize changes in cortical bone formation, resorption, and structure after lactation in established breeder rats. Rats were taken at the end of the second pregnancy, second lactation, and at two, four, and six weeks after the pups were weaned. Age-related, nulliparous groups were included for comparison. Cortical bone structure and bone formation and resorption were measured at the tibiofibular junction using histomorphometric methods. As expected, there were decreases in cortical bone area, width, and minimum cortical thickness with an increase in marrow cavity area during lactation. Bone formation rates were essentially zero on the periosteal and endocortical surfaces at the end of lactation, while eroded (resorption) endocortical surface was greatly increased compared with the end of pregnancy. At or immediately after weaning, there was a rapid reversal of resorption to formation surface on the endocortical envelope similar to the events of true bone remodeling. Likewise, there was a commencement of bone formation on the periosteal surface. The volume- and surface-referent bone rates measured on the endocortical surface were substantially elevated over those measured at the end of the second pregnancy or in the nulliparous animals. Peak bone formation rates were observed on both the endocortical and periosteal surfaces by four weeks after weaning. These results show that the postlactation period is profoundly anabolic for cortical bone in the established breeder rat. The rapid and dramatic increases in bone formation likely serve to restore bone lost during lactation and to prepare the maternal skeleton for the next reproductive cycle.

Familial resemblance of bone mineralization, calcium intake, and physical activity in early-adolescent daughters, their mothers, and maternal grandmothers.

OBJECTIVE: To describe familial relationships among bone mineral density (BMD), calcium intake, and physical activity in early-adolescent daughters, their premenopausal mothers, and postmenopausal maternal grandmothers. SUBJECTS: Healthy, early-adolescent daughter and premenopausal mother pairs (n=72) were enrolled in the study. In addition, a cohort of 22 postmenopausal maternal grandmothers were measured for comparison of related triads (n=22). DESIGN: Cross-sectional measurements of hip (three sites) and lumbar spine BMD by dual energy x-ray absorptiometry (DXA), body height and weight, menstrual function, current calcium intake, and current and past physical activity patterns were assessed using recalls and questionnaires. STATISTICAL ANALYSIS: Correlational analysis was used to establish relationships between bone characteristics and body size, menstrual function, calcium intake, and physical activity. Multiple regression analyses with backward elimination were used to examine heritability of bone characteristics in daughter-mother and mother-grandmother pairs and daughter-mother-grandmother triads. Quick cluster analysis and cross-tabulation with Pearson's chi(2) were used to evaluate familial patterns for bone characteristics and lifestyle practices. RESULTS: Height, weight, and lumbar spine BMD were significantly correlated within mother-daughter pairs. Current and past calcium intakes were not related within pairs or triads or to BMD in the daughters or the grandmothers. A weak inverse relationship between calcium intake and the hip trochanter and lumbar spine BMD was observed in the mothers (R(2)=-0.25; P=.05). Physical activity, independent of calcium intake, was strong predictor of BMD for daughters and mothers. Among the daughters, the hertiability estimates for trochanter and lumbar spine BMD were 0.56 and 0.70, respectively (P<.01). The heritability estimate for premenopausal mothers were significant for lumbar spine BMD (h(2)=0.66; P<.01). Daughter-mother-grandmother triads with low physical activity had low femoral neck BMD whereas those with high physical activity had high femoral neck BMD (P<.001). APPLICATIONS: Making physical activity a part of the daily routine, in addition to an adequate intake of calcium and bone-related nutrients, is an important goal for maintaining or improving bone health for women of all ages.

Acyclonucleoside iron chelators of 1-(2-hydroxyethoxy)methyl-2-alkyl-3-hydroxy-4-pyridinones: potential oral iron chelation therapeutics.

The method of synthesizing acyclonucleoside iron chelators is both convenient and cost effective compared to that of synthesizing ribonucleoside iron chelators. The X-ray crystal structural analysis shows that the 2-hydroxyethoxymethyl group does not affect the geometry of the iron chelating sites. Therefore, the iron binding and removal properties of the acyclonucleoside iron chelators should remain similar to the ribonucleoside iron chelators, which is confirmed by the titration and competition reaction of the acyclonucleoside chelators with iron and ferritin, respectively. The acyclonucleoside iron chelators are more lipophilic with measured n-octanol and Tris buffer distribution coefficients than ribonucleoside iron chelators.

Does malignant hematopoietic disease result from internal exposure to 239Pu?

Neither Fisher's Exact Test nor linear regression analysis (dose-response relationship) using average skeletal dose from 239Pu as a surrogate for marrow dose could establish a statistically significant association between malignant hematopoietic disease (MHD including leukemia and aleukemia) and exposure to 239Pu among dogs in the Utah beagle colony. In addition, when dog-years at risk was the parameter used to estimate the rate of expected malignant hematopoietic tumors in dogs exposed to 239Pu, the estimate was within the range of the 95% confidence limits of the control dogs for six cases (and also within the 95% confidence limits for five cases when the single case of lymphoma was omitted). It was concluded that if there was an effect of 239Pu exposure on the occurrence of MHD in this beagle experiment, it was not very meaningful. These data could not establish a significant susceptibility to neoplasia of either myeloid or lymphoid tissue of 239Pu exposure.