A new chiral chromatography method has been developed to determine the chiral purity of MB-102. This method employs exchange of counter ions with the MB-102 molecule. Chromatography conditions including column temperature and concentration of counter ions were evaluated and optimized. This method may be used to monitor process development during formulation manufacture, is amenable to large scale production, and is suitable for the determination of agent chirality for reporting in regulatory submissions. Manufactured clinical supplies of both the drug substance and the drug product using this technique have been shown to routinely yield 99.9+ % D, D enantiomer. This new method is shown to be precise, accurate, and robust.

Critically ill patients undergoing continuous renal replacement therapy (CRRT) have medical conditions requiring extensive pharmacotherapy. Continuous renal replacement therapy impacts drug disposition. Few data exist regarding drug dosing requirements with contemporary CRRT modalities and effluent rates. The practical limitations of pharmacokinetic studies requiring numerous plasma and effluent samples, and lack of generalizability of observations from specific CRRT prescriptions, highlight gaps in bedside assessment of CRRT drug elimination and individualized dosing needs. We employed a porcine model using transdermal fluorescence detection of the glomerular filtration rate fluorescent tracer agent MB-102, with the aim to assess the relationship between systemic exposure of MB-102 and meropenem during CRRT. Animals underwent bilateral nephrectomies and received intravenous bolus doses of MB-102 and meropenem. Once MB-102 equilibrated in the animal, CRRT was initiated. Continuous renal replacement therapy prescriptions comprised four combinations of blood pump (low versus high) and effluent (low versus high) flow rates. Changes in transdermal detected MB-102 clearance occurred immediately with a change in CRRT rates. Blood side meropenem clearance mirrored transdermal MB-102 clearance (r 2: 0.95–0.97, p value all <0.001). We suggest transdermal MB-102 clearance provides real-time personalized assessment of drug elimination and could optimize prescription of drugs for critically ill patients requiring CRRT.

Dorshow, Richard B.; Shieh, Jeng-Jong; Riley, Ivan R.; Rogers, Thomas E. Author Information

OBJECTIVE To evaluate the effectiveness of a novel fluorescence tracer agent, MB-102, for conducting ocular angiography in dogs. ANIMALS 10 ophthalmologically normal dogs (2 to 4 years old) and 10 dogs with retinal degeneration or primary open-angle glaucoma (< 6 years old). PROCEDURES While anesthetized, all dogs received sodium fluorescein (20 mg/kg, IV) or MB-102 (20 or 40 mg/kg, IV) first and then the other dye in a second treatment session 2 days later in a randomized crossover design. Anterior fluorescence angiography was performed on one eye and posterior fluorescence angiography on the other. Imaging was performed with a full-spectrum camera and camera adaptor system. Filter sets that were tailored to match the excitation and emission characteristics of each angiographic fluorescent agent were used. RESULTS All phases and phase intervals during anterior and posterior segment angiography were identified, regardless of the dye used. However, agent fluorescence and visualization of the iridal blood vessels were hindered in some dogs, irrespective of agent, owing to the degree of iridal pigmentation present. No significant difference was noted between the 2 dyes in any phase or phase interval, and slight improvement in image contrast was observed with MB-102 during the venous phases owing to a reduction of vessel wall staining in both normal and diseased eyes. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that MB-102 would be useful for conducting ocular angiography in dogs.

MB-102 is a fluorescent tracer agent designed for measurement of point-of-care glomerular filtration rate (GFR) and is currently in clinical studies. MB-102 possesses a strong UV absorbance at 266 nm and 435 nm, and broad fluorescent emission at ~560 nm when excited at ~440 nm. The MB-102 formulation is stable at 2°C-8°C for >3 years. The pKa's of the 2 acid groups are 2.71 and 3.40. Both X-ray crystallography and HPLC confirmed the D, D chirality of MB-102 in solid, in solution, and in the drug formulation. Initial safety and toxicity was published previously [Bugaj and Dorshow, 2015], which enabled the commencement of clinical studies. In vitro studies showed that 4.1% of MB-102 is bound to human plasma proteins, compared to 6.0% for the accepted standard GFR agent iohexol. The blood-to-plasma ratio for MB-102 was 0.590, illustrating minimal distribution of MB-102 into red blood cells. The manufacture of MB-102 under good manufacturing practice yields the designed molecular structure at high purity (>95% wt/wt).

The intestinal mucosal barrier prevents macromolecules and pathogens from entering the circulatory stream. Tight junctions in this barrier are compromised in inflammatory bowel diseases, environmental enteropathy, and enteric dysfunction. Dual sugar absorption tests are a standard method for measuring gastrointestinal integrity, however, these are not clinically amenable. Herein, we report on a dual fluorophore system and fluorescence detection instrumentation for which gastrointestinal permeability is determined in a rat small bowel disease model from the longitudinal measured transdermal fluorescence of each fluorophore. This fluorophore technology enables a specimen-free, noninvasive, point-of-care gastrointestinal permeability measurement which should be translatable to human clinical studies.

The intestinal mucosal barrier prevents macromolecules and pathogens from entering the circulatory stream in a healthy gut. Tight junctions in this barrier are compromised in many intestinal disorders including inflammatory bowel diseases (such as Crohn's and ulcerative colitis), celiac disease, graft vs host disease, environmental enteropathy, and enteric dysfunction. Dual sugar absorption tests are a standard method for measuring gastrointestinal integrity in humans. The larger molecular weight sugar, lactulose, is minimally absorbed through a healthy gut. The smaller molecular weight sugar, mannitol or rhamnose, in contrast, is readily absorbed through both a healthy and inflamed gut. Thus, a higher ratio of lactulose to mannitol or rhamnose reflects increased intestinal permeability. However, several issues prevent the widespread use of the dual sugar assay including requirements for lengthy urine collection, transport of specimens under conditions free from contamination and bacterial growth, analysis by sophisticated laboratory equipment, and the associated lengthy turnaround time. In a recent publication, the feasibility of employing a dual fluorescent tracer agent assay to mimic the dual sugar absorption test without the need for specimen collection was reported. This dual fluorophore assay for GI permeability was demonstrated in an indomethacin-induced bowel disease model in rats. However, one of the fluorophores was not entirely biocompatible. Herein is reported a dual fluorophore system that is totally biocompatible, with emphasis on the transdermal detection of the fluorophores, thus enabling the use of the technology for noninvasive point-of-care gastrointestinal permeability determination.

The healthy gut restricts macromolecular and bacterial movement across tight junctions, while increased intestinal permeability accompanies many intestinal disorders. Dual sugar absorption tests, which measure intestinal permeability in humans, present challenges. Therefore, we asked if enterally administered fluorescent tracers could ascertain mucosal integrity, because transcutaneous measurement of differentially absorbed molecules could enable specimen-free evaluation of permeability. We induced small bowel injury in rats using high- (15 mg/kg), intermediate- (10 mg/kg), and low- (5 mg/kg) dose indomethacin. Then, we compared urinary ratios of enterally administered fluorescent tracers MB-402 and MB-301 to urinary ratios of sugar tracers lactulose and rhamnose. We also tested the ability of transcutaneous sensors to measure the ratios of absorbed fluorophores. Urinary fluorophore and sugar ratios reflect gut injury in an indomethacin dose dependent manner. The fluorophores generated smooth curvilinear ratio trajectories with wide dynamic ranges. The more chaotic sugar ratios had narrower dynamic ranges. Fluorophore ratios measured through the skin distinguished indomethacin-challenged from same day control rats. Enterally administered fluorophores can identify intestinal injury in a rat model. Fluorophore ratios are measureable through the skin, obviating drawbacks of dual sugar absorption tests. Pending validation, this technology should be considered for human use.

Gut dysfunction, often accompanied by increased mucosal permeability to gut contents, frequently accompanies a variety of human intestinal inflammatory conditions. These disorders include inflammatory bowel diseases (e.g., Crohn's Disease) and environmental enteropathy and enteric dysfunction, a condition strongly associated with childhood malnutrition and stunting in resource poor areas of the world. The most widely used diagnostic assay for gastrointestinal permeability is the lactulose to mannitol ratio (L:M) measurement. These sugars are administered orally, differentially absorbed by the gut, and then cleared from the body by glomerular filtration in the kidney. The amount of each sugar excreted in the urine is measured. The larger sugar, lactulose, is minimally absorbed through a healthy gut. The smaller sugar, mannitol, in contrast, is readily absorbed through both a healthy and injured gut. Thus a higher ratio of lactulose to mannitol reflects increased intestinal permeability. However, several issues prevent widespread use of the L: M ratio in clinical practice. Urine needs to be collected over time intervals of several hours, the specimen then needs to be transported to an analytical laboratory, and sophisticated equipment is required to measure the concentration of each sugar in the urine.In this presentation we show that fluorescent tracer agents with molecular weights similar to those of the sugars, selected from our portfolio of biocompatible renally cleared fluorophores, mimic the L:M ratio test for gut permeability. This fluorescent tracer agent detection technology can be used to overcome the limitations of the L: M assay, and is amenable to point-of-care clinical use.

We describe the synthesis, MMP-2 and 9 potency, and in vitro evaluation of a series of α-sulfone hydroxmate MMP inhibitors conjugated to a series of dyes with different absorption/emission lamina maxima’s that can be used to visualize tumors.

Studies directed toward the process research, development, and scale-up preparation of the potential αvβ3 integrin antagonist 1 are described. A convergent approach is detailed wherein tetrahydropyrimidine hydroxybenzoic acid 2 is linked to the β-amino acid ester 3 via a diisoproylcarbodiimide, catalytic HOBt coupling reaction. Saponification of the resulting ethyl ester, isolation of the corresponding zwitterion, H3PO4 salt formation, crystallization, and acetonitrile-to-acetone exchange give rise to 1 as a crystalline monohydrate in 67% overall yield from 4.

A 28-year-old Hispanic male presented with a 3-month history of nosebleeds, fatigue, jaundice, easy bruising, weight loss, ascites and lower extremity oedema. Physical examination revealed hepatosplenomegaly, an area of bruising over the left thigh and right buttock and 3–4 telangiectases on the hands but none on the lips or oropharynx. Laboratory studies showed: aspartate transaminase 43 U/l [normal range (NR) 13–40 U/l], alanine transaminase 20 U/l (NR 10–40), gamma glutamyl transferase 203 U/l (NR 8–78), bilirubin 106 lmol/l (NR 3AE4–22AE1), activated partial thromboplastin time 34AE9 s (NR 23AE5–33AE5), prothrombin time 19AE4 s (NR 9AE2–12AE8), and International Normalized Ratio 2AE1 (NR 0AE9–1AE3). All viral and autoimmune hepatitis serologies were negative. The full blood cell count showed haemoglobin concentration 103 g/l, platelets 82 · 10/l, and white blood cells 4AE3 · 10/l with an unremarkable differential. Abdominal computed tomography showed an enlarged, nodular liver with numerous hypervascular masses, a few hypervascular splenic lesions and multiple lytic bone lesions. Bone marrow aspirate and core biopsy showed unremarkable, trilineage haematopoiesis with approximately 10–20% of the marrow space replaced by an anastomosing, benign, vascular lesion infiltrating the cellular marrow (top left, 100·; top right, 400·). Cells lining the vascular network were CD34(+) (bottom left, 200·). Transjugular liver biopsy showed a similar vascular process (bottom right, 200·). It extensively infiltrated throughout a cirrhotic liver, in the parenchymal nodules and in the bands of fibrous tissue encircling them. With the patient’s clinical, radiological, and pathological findings, a diagnosis of hereditary haemorrhagic telangiectasia (HHT) was made. HHT, or Osler-Weber-Rendu syndrome, is an autosomal dominant syndrome that may involve three different genes in the transforming growth factor-b signalling pathway. Defects in this pathway may result in abnormal vessel repair. The syndrome is defined by recurrent, spontaneous epistaxis, telangiectases, visceral arteriovenous malformations (AVMs), and a positive family history. Lung, gastrointestinal tract and liver are the three most common sites for AVMs. In the present case, AVMs involved the liver and bone marrow and probably the spleen. Bone marrow involvement is rare and has not been well reported in the literature.

Fleas of prairie dogs have been implicated in the transmission of Bartonella spp. We used PCR to test DNA extracts from 47 fleas of prairie dogs from 6 states. We amplified DNA from 5 unique genotypes of Bartonella spp. and 1 Rickettsia sp. from 12 fleas collected in North Dakota, Oklahoma, Texas, and Wyoming. Sequences from the Bartonella spp. were similar, but not identical, to those from prairie dogs and their fleas in Colorado.

Bartonella spp. are putatively vector-borne bacterial agents of humans and animals. Fleas have been incriminated as vectors of Bartonella spp. and are suspected of transmitting Bartonella of rodents and bats, but some of these Bartonella spp. have not yet been directly detected in wild caught fleas. We report the molecular detection of Bartonella tribocorum, Bartonella vinsonii subsp. vinsonii, and two novel genotypes of Bartonella from the fleas Xenopsylla cheopis, Ctenophthalmus pseudagyrtes, Sternopsylla texanus, or Orchopeas howardi.

The integrin alpha(v)beta(3), vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. We recently disclosed the discovery of a tripeptide Arg-Gly-Asp (RGD) mimic, which has been shown to be a potent inhibitor of the integrin alpha(v)beta(3) and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. In other investigations involving RGD mimics, only compounds containing the S-isomers of the beta-amino acids have been shown to be potent. We were surprised to find the potencies of analogs containing enantiomerically pure S-isomers of beta-amino acids which were only marginally better than the corresponding racemic mixtures. We therefore synthesized RGD mimics containing R-isomers of beta-amino acids and found them to be relatively potent inhibitors of alpha(v)beta(3). One of the compounds was examined in tumor models in mice and has been shown to significantly reduce the rate of growth and the size of tumors.

The integrin alpha(v)beta(3) is expressed in a number of cell types and is thought to play a major role in several pathological conditions. Various small molecules that inhibit the integrin have been shown to suppress tumor growth and retinal angiogenesis. The tripeptide Arg-Gly-Asp (RGD), a common binding motif in several ligands that bind to alpha(v)beta(3), has been depeptidized and optimized in our efforts toward discovering a small molecule inhibitor. We recently disclosed the synthesis and biological activity of several small molecules that did not contain any peptide bond and mimic the tripeptide RGD. The phenethyl group in one of the lead compounds was successfully replaced with a cyclopropyl moiety. The new lead compound was optimized for potency, selectivity, and for its ADME properties. We describe herein the discovery, synthesis, and optimization of cyclopropyl containing analogs that are potent and selective inhibitors of alpha(v)beta(3).