Granulocyte colony-stimulating factor utilization postautologous hematopoietic stem cell transplant in multiple myeloma patients: Does one size fit all?

PURPOSE: To evaluate a single institution's experience with granulocyte colony-stimulating factor after autologous hematopoietic stem cell transplant in myeloma patients to identify populations that benefit most from granulocyte colony-stimulating factor administration. METHODS: Retrospective chart reviews were conducted on patients 18+ years with multiple myeloma that underwent autologous hematopoietic stem cell transplant at UW Health from January 2012 to May 2016. Data collection included demographics, length of stay, time to engraftment, Eastern Cooperative Oncology Group performance status score, and hematopoietic cell transplantation-comorbidity index. The primary outcome was days from transplant to engraftment, defined as absolute neutrophil count > 500/mm3 for two consecutive days or absolute neutrophil count > 1000/mm3 once. A subset analysis was performed on patients whose date of engraftment was known. RESULTS: In total, 216 individual patients were included in the full cohort and 122 patients included in the subset analysis. Median time to engraftment between patients administered granulocyte colony-stimulating factor and the nongranulocyte colony-stimulating factor group was 12 versus 19 days (P < 0.001) in the full cohort and 12 versus 14 days (P < 0.001) in the subset analysis. The average length of stay posthematopoietic stem cell transplant in the granulocyte colony-stimulating factor group was 15 days versus 17 days in the nongranulocyte colony-stimulating factor group (P = 0.026) in the subset analysis. Additionally, no difference in time to engraftment was seen when stratified by age, Eastern Cooperative Oncology Group performance status score, or hematopoietic cell transplantation-comorbidity index. CONCLUSION: Our study supports use of granulocyte colony-stimulating factor posthematopoietic stem cell transplant in myeloma patients to decrease time to engraftment and length of stay. Consideration should be given to utilization in all patients in this population posthematopoietic stem cell transplant. Further research is needed to identify the populations that benefit most from granulocyte colony-stimulating factor administration.

Effects of losartan and allopurinol on cardiorespiratory regulation in obstructive sleep apnoea.

NEW FINDINGS: What is the central question of this study? In sleep apnoea, a putative link between intermittent hypoxia and hypertension is the generation of oxygen radicals by angiotensin II and xanthine oxidase within the chemoreflex arc and vasculature. We tested whether chemoreflex control of sympathetic outflow, hypoxic vasodilatation and blood pressure are altered by angiotensin blockade (losartan) and/or xanthine oxidase inhibition (allopurinol). What is the main finding and its importance? Both drugs lowered blood pressure without altering sympathetic outflow, reducing chemoreflex sensitivity or enhancing hypoxic vasodilatation. Losartan and allopurinol are effective therapies for achieving blood pressure control in sleep apnoea. ABSTRACT: Chemoreflex sensitization produced by chronic intermittent hypoxia in rats is attenuated by angiotensin II type 1 receptor (AT1 R) blockade. Both AT1 R blockade and xanthine oxidase inhibition ameliorate chronic intermittent hypoxia-induced endothelial dysfunction. We hypothesized that treatment with losartan and allopurinol would reduce chemoreflex sensitivity and improve hypoxic vasodilatation in patients with obstructive sleep apnoea. Eighty-six hypertensive patients with apnoea-hypopnoea index ≥25 events h-1 and no other cardiovascular, pulmonary, renal or metabolic disease were randomly assigned to receive allopurinol, losartan or placebo for 6 weeks. Treatment with other medications and/or continuous positive airway pressure remained unchanged. Tests of chemoreflex sensitivity and hypoxic vasodilatation were performed during wakefulness before and after treatment. Ventilation (pneumotachography), muscle sympathetic nerve activity (microneurography), heart rate (electrocardiography), arterial oxygen saturation (pulse oximetry), blood pressure (sphygmomanometry), forearm blood flow (venous occlusion plethysmography) and cerebral flow velocity (transcranial Doppler ultrasound) were measured during eupnoeic breathing and graded reductions in inspired O2 tension. Losartan and allopurinol lowered arterial pressure measured during eupnoeic breathing and exposure to acute hypoxia. Neither drug altered the slopes of ventilatory, sympathetic or cardiovascular responses to acute hypoxia. We conclude that losartan and allopurinol are viable pharmacotherapeutic adjuncts for achieving blood pressure control in hypertensive obstructive sleep apnoea patients, even those who are adequately treated with continuous positive airway pressure.

The effect of chain length and unsaturation on Mtb Dxr inhibition and antitubercular killing activity of FR900098 analogs.

Inhibition of the nonmevalonate pathway (NMP) of isoprene biosynthesis has been examined as a source of new antibiotics with novel mechanisms of action. Dxr is the best studied of the NMP enzymes and several reports have described potent Dxr inhibitors. Many of these compounds are structurally related to natural products fosmidomycin and FR900098, each bearing retrohydroxamate and phosphonate groups. We synthesized a series of compounds with two to five methylene units separating these groups to examine what linker length was optimal and tested for inhibition against Mtb Dxr. We synthesized ethyl and pivaloyl esters of these compounds to increase lipophilicity and improve inhibition of Mtb growth. Our results show that propyl or propenyl linker chains are optimal. Propenyl analog 22 has an IC50 of 1.07 µM against Mtb Dxr. The pivaloyl ester of 22, compound 26, has an MIC of 9.4 µg/mL, representing a significant improvement in antitubercular potency in this class of compounds.

Antibacterial and antitubercular activity of fosmidomycin, FR900098, and their lipophilic analogs.

The nonmevalonate pathway (NMP) of isoprene biosynthesis is an exciting new route toward novel antibiotic development. Inhibitors against several enzymes in this pathway are currently under examination. A significant liability of many of these agents is poor cell penetration. To overcome and improve our understanding of this problem, we have synthesized a series of lipophilic, prodrug analogs of fosmidomycin and FR900098, inhibitors of the NMP enzyme Dxr. Several of these compounds show improved antibacterial activity against a panel of organisms relative to the parent compound, including activity against Mycobacterium tuberculosis (Mtb). Our results show that this strategy can be an effective way for improving whole cell activity of NMP inhibitors.

Mechanism of Action of N-Acyl and N-Alkoxy Fosmidomycin Analogs: Mono- and Bisubstrate Inhibition of IspC from Plasmodium falciparum, a Causative Agent of Malaria.

Malaria is a global health threat that requires immediate attention. Malaria is caused by the protozoan parasite Plasmodium, the most severe form of which is Plasmodium falciparum. The methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential to the survival of many human pathogens, including P. falciparum, but is absent in humans, and thus shows promise as a new antimalarial drug target. The enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the MEP pathway. In addition to a divalent cation (Mg2+), the enzyme requires the substrates 1-deoxy-D-xylulose 5-phosphate (DXP) and NADPH to catalyze its reaction. We designed N-alkoxy and N-acyl fosmidomycin analogs to inhibit the activity of P. falciparum IspC in a bisubstrate manner. Enzyme assays reveal that the N-alkoxy fosmidomycin analogs have a competitive mode of inhibition relative to both the DXP- and NADPH-binding sites, confirming a bisubstrate mode of inhibition. In contrast, the N-acyl fosmidomycin analogs demonstrate competitive inhibition with respect to DXP but uncompetitive inhibition with respect to NADPH, indicating monosubstrate inhibitory activity. Our results will have a positive impact on the discovery of novel antimalarial drugs.

Clinical Outcomes and Economic Impact of Oritavancin for Gram-Positive Infections: A Single Academic Medical Center Health System Experience.

BACKGROUND: Vancomycin treatment of complicated Gram-positive infections is associated with laboratory monitoring, nephrotoxicity, and multiple daily dosing. Oritavancin, a lipoglycopeptide antibiotic with a once-weekly dosing strategy and similar but slightly broader spectrum of activity, presents several opportunities over vancomycin to improve compliance and convenience for the patient. Minimal real-world clinical and acquisition cost data in the inpatient setting and clinical data surrounding multiple dosing in the outpatient setting have limited oritavancin use despite its potential logistic advantages. OBJECTIVES: We describe inpatient and outpatient oritavancin administration, clinical outcomes, and economic impact. METHODS: This was a single-center, retrospective case series of patients treated with at least one dose of oritavancin between May 2015 and September 2017 at an academic medical center in the USA. A simplified cost-avoidance analysis was conducted assuming the patient had a national health insurance plan and focused on hospital days prevented. RESULTS: Seventy-five patients received oritavancin during the study period. The most common use of oritavancin was in patients with acute bacterial skin and skin structure infections (ABSSSI), defined as cellulitis, abscess or non-surgical wounds (n = 25, 33%), followed by surgical wound infections (n = 12, 16%) and osteomyelitis or septic arthritis (n = 10, 13%). Clinical cure or improvement was achieved in 68 patients (93.2%), while five patients (6.8%) failed treatment; adverse reactions were reported in nine patients (12%). Thirty-five patients received oritavancin as inpatients; 20 patients (57%) had at least one hospital day avoided due to inpatient oritavancin administration resulting in a total cost avoidance of US$343,654. CONCLUSION: In this series of 75 patients with Gram-positive infections, oritavancin treatment resulted in clinical cure or improvement in most patients, and was generally well tolerated. Inpatient administration may avoid costs and outpatient administration is a reasonable consideration for patients in which prolonged antibiotic therapy is necessary.

Structure-Activity Relationships of the MEPicides: N-Acyl and O-Linked Analogs of FR900098 as Inhibitors of Dxr from Mycobacterium tuberculosis and Yersinia pestis.

Despite continued research efforts, the threat of drug resistance from a variety of bacteria continues to plague clinical communities. Discovery and validation of novel biochemical targets will facilitate development of new drugs to combat these organisms. The methylerythritol phosphate (MEP) pathway to make isoprene units is a biosynthetic pathway essential to many bacteria. We and others have explored inhibitors of the MEP pathway as novel antibacterial agents. Mycobacterium tuberculosis, the causative agent of tuberculosis, and Yersinia pestis, resulting in the plague or "black death", both rely on the MEP pathway for isoprene production. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr) catalyzes the first committed step in the MEP pathway. We examined two series of Dxr inhibitors based on the parent structure of the retrohydroxamate natural product FR900098. The compounds contain either an extended N-acyl or O-linked alkyl/aryl group and are designed to act as bisubstrate inhibitors of the enzyme. While nearly all of the compounds inhibited both Mtb and Yp Dxr to some extent, compounds generally displayed more potent inhibition against the Yp homologue, with the best analogs displaying nanomolar IC50 values. In bacterial growth inhibition assays, the phosphonic acids generally resulted in poor antibacterial activity, likely a reflection of inadequate permeability. Accordingly, diethyl and dipivaloyloxymethyl (POM) prodrug esters of these compounds were made. While the added lipophilicity did not enhance Yersinia activity, the compounds showed significantly improved antitubercular activities. The most potent compounds have Mtb MIC values of 3-12 µg/mL. Taken together, we have uncovered two series of analogs that potently inhibit Dxr homologues from Mtb and Yp. These inhibitors of the MEP pathway, termed MEPicides, serve as leads for future analog development.

Kinetic characterization and allosteric inhibition of the Yersinia pestis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase).

The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development.

Design of Potential Bisubstrate Inhibitors against Mycobacterium tuberculosis (Mtb) 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr)-Evidence of a Novel Binding Mode.

In most bacteria, the nonmevalonate pathway is used to synthesize isoprene units. Dxr, the second step in the pathway, catalyzes the NADPH-dependent reductive isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP). Dxr is inhibited by natural products fosmidomycin and FR900098, which bind in the DXP binding site. These compounds, while potent inhibitors of Dxr, lack whole cell activity against Mycobacterium tuberculosis (Mtb) due to their polarity. Our goal was to use the Mtb Dxr-fosmidomycin co-crystal structure to design bisubstrate ligands to bind to both the DXP and NADPH sites. Such compounds would be expected to demonstrate improved whole cell activity due to increased lipophilicity. Two series of compounds were designed and synthesized. Compounds from both series inhibited Mtb Dxr. The most potent compound (8) has an IC50 of 17.8 µM. Analysis shows 8 binds to Mtb Dxr via a novel, non-bisubstrate mechanism. Further, the diethyl ester of 8 inhibits Mtb growth making this class of compounds interesting lead molecules in the search for new antitubercular agents.

Inhibition of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr): a review of the synthesis and biological evaluation of recent inhibitors.

Isoprene biosynthesis is an essential component of metabolism. Two pathways are known for the production of five-carbon (isoprene) intermediates: the mevalonate and nonmevalonate pathways. As many pathogenic organisms rely exclusively on the nonmevalonate pathway (NMP) for isoprenoids and humans do not, the enzymes of this route have been recently explored as new therapeutic targets. The second and first-committed step in the NMP is catalyzed by 1-deoxy-Dxylulose- 5-phosphate reductoisomerase (Dxr) and has received significant attention as a novel drug target. This review describes the biochemistry and crystal structures of Dxr and the synthesis and biological activity of inhibitors to date, with a focus on compounds targeting E. coli, Plasmodium, and M. tuberculosis enzymes and intact cells. Most inhibitors for Dxr use natural products fosmidomycin and FR900098 as starting points. The review discusses several families of fosmidomycinrelated analogs including α-substituted, 'reverse' and modified hydroxamate, spacer-modified, and hydroxy-amide analogs. Also discussed are non-fosmidomycin-like inhibitors, the aryl phosphonates, and lipophilic prodrugs of fosmidomycin and FR900098 designed to increase cell penetration. A comprehensive SAR of inhibitors is presented.

Lipophilic prodrugs of FR900098 are antimicrobial against Francisella novicida in vivo and in vitro and show GlpT independent efficacy.

Bacteria, plants, and algae produce isoprenoids through the methylerythritol phosphate (MEP) pathway, an attractive pathway for antimicrobial drug development as it is present in prokaryotes and some lower eukaryotes but absent from human cells. The first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/MEP synthase). MEP pathway genes have been identified in many biothreat agents, including Francisella, Brucella, Bacillus, Burkholderia, and Yersinia. The importance of the MEP pathway to Francisella is demonstrated by the fact that MEP pathway mutations are lethal. We have previously established that fosmidomycin inhibits purified MEP synthase (DXR) from F. tularensis LVS. FR900098, the acetyl derivative of fosmidomycin, was found to inhibit the activity of purified DXR from F. tularensis LVS (IC(50)=230 nM). Fosmidomycin and FR900098 are effective against purified DXR from Mycobacterium tuberculosis as well, but have no effect on whole cells because the compounds are too polar to penetrate the thick cell wall. Fosmidomycin requires the GlpT transporter to enter cells, and this is absent in some pathogens, including M. tuberculosis. In this study, we have identified the GlpT homologs in F. novicida and tested transposon insertion mutants of glpT. We showed that FR900098 also requires GlpT for full activity against F. novicida. Thus, we synthesized several FR900098 prodrugs that have lipophilic groups to facilitate their passage through the bacterial cell wall and bypass the requirement for the GlpT transporter. One compound, that we termed "compound 1," was found to have GlpT-independent antimicrobial activity. We tested the ability of this best performing prodrug to inhibit F. novicida intracellular infection of eukaryotic cell lines and the caterpillar Galleria mellonella as an in vivo infection model. As a lipophilic GlpT-independent DXR inhibitor, compound 1 has the potential to be a broad-spectrum antibiotic, and should be effective against most MEP-dependent organisms.