- Generalize the causes and mode of transmission for leptospirosis
Leptospirosis (also known as Weil’s Syndrome, canicola fever, canefield fever, nanukayami fever, 7-day fever, Rat Catcher’s Yellows, Fort Bragg fever, black jaundice, and Pretibial fever) is caused by bacteria of the genus Leptospira, and affects humans as well as other animals. Symptoms can range from none to mild such as headaches, muscle pains, and fevers; to severe with bleeding from the lungs or meningitis. If the infection causes the person to turn yellow, have kidney failure and bleeding it is then known as Weil’s disease. If the infection causes lots of bleeding from the lungs it is known as severe pulmonary haemorrhage syndrome.
Leptospirosis is among the world’s most common diseases transmitted to people from animals. The infection is commonly transmitted to humans by allowing water that has been contaminated by animal urine to come in contact with unhealed breaks in the skin, eyes, or mucous membranes. Outside of tropical areas, leptospirosis cases have a relatively distinct seasonality, with most cases occurring in spring and autumn.
Leptospirosis is caused by a spirochaete bacterium called Leptospira spp. There are at least five serotypes of importance in the United States and Canada, all of which cause disease in dogs (Icterohaemorrhagiae, Canicola, Pomona, Grippotyphosa, and Bratislava).There are other (less common) infectious strains as well. Leptospirosis is transmitted by the urine of an infected animal and is contagious as long as it is still moist. Although rats, mice, and moles are important primary hosts, a wide range of other mammals (including dogs, deer, rabbits, hedgehogs, cows, sheep, raccoons, opossums, skunks, and certain marine mammals) are able to carry and transmit the disease as secondary hosts. Dogs may lick the urine of an infected animal off the grass or soil or drink from an infected puddle.
There have been reports of “house dogs” contracting leptospirosis from licking the urine of infected mice that enter the house. The type of habitats most likely to carry infectious bacteria are muddy riverbanks, ditches, gullies, and muddy livestock-rearing areas where there is regular passage of either wild or farm mammals. There is a direct correlation between the amount of rainfall and the incidence of leptospirosis, making it seasonal in temperate climates and year-round in tropical climates. Leptospirosis is also transmitted by the semen of infected animals. Humans become infected through contact with water, food, or soil containing urine from these infected animals. This may result from swallowing contaminated food and water or through skin contact. The disease is not known to be spread from person to person, and cases of bacterial dissemination in convalescence are extremely rare in humans. Leptospirosis is common among water-sport enthusiasts in specific areas, as prolonged immersion in water is known to promote the entry of the bacteria. Surfers and whitewater paddlers are at especially high risk in areas that have been shown to contain the bacteria, and can contract the disease by swallowing contaminated water, splashing contaminated water into their eyes or nose, or exposing open wounds to infected water.
- Leptospirosis symptoms can range from none to mild such as headaches, muscle pains, and fevers; to severe with bleeding from the lungs or meningitis.
- Outside of tropical areas, leptospirosis cases have a relatively distinct seasonality, with most cases occurring in spring and autumn.
- Leptospirosis is transmitted by the urine of an infected animal and is contagious as long as it remains moist.
- There is a direct correlation between the amount of rainfall and the incidence of leptospirosis, making it seasonal in temperate climates and year-round in tropical climates.
- leptospirosis: an acute, infectious, febrile disease of both humans and animals, caused by spirochetes of the genus Leptospira
Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics
The causative agents of leptospirosis are responsible for an emerging zoonotic disease worldwide. One of the major routes of transmission for leptospirosis is the natural environment contaminated with the urine of a wide range of reservoir animals. Soils and surface waters also host a high diversity of non-pathogenic Leptospira and species for which the virulence status is not clearly established. The genus Leptospira is currently divided into 35 species classified into three phylogenetic clusters, which supposedly correlate with the virulence of the bacteria. In this study, a total of 90 Leptospira strains isolated from different environments worldwide including Japan, Malaysia, New Caledonia, Algeria, mainland France, and the island of Mayotte in the Indian Ocean were sequenced. A comparison of average nucleotide identity (ANI) values of genomes of the 90 isolates and representative genomes of known species revealed 30 new Leptospira species. These data also supported the existence of two clades and 4 subclades. To avoid classification that strongly implies assumption on the virulence status of the lineages, we called them P1, P2, S1, S2. One of these subclades has not yet been described and is composed of Leptospira idonii and 4 novel species that are phylogenetically related to the saprophytes. We then investigated genome diversity and evolutionary relationships among members of the genus Leptospira by studying the pangenome and core gene sets. Our data enable the identification of genome features, genes and domains that are important for each subclade, thereby laying the foundation for refining the classification of this complex bacterial genus. We also shed light on atypical genomic features of a group of species that includes the species often associated with human infection, suggesting a specific and ongoing evolution of this group of species that will require more attention. In conclusion, we have uncovered a massive species diversity and revealed a novel subclade in environmental samples collected worldwide and we have redefined the classification of species in the genus. The implication of several new potentially infectious Leptospira species for human and animal health remains to be determined but our data also provide new insights into the emergence of virulence in the pathogenic species.
Conflict of interest statement
The authors have declared that no competing interests exist.
Fig 1. Phylogenetic tree based on the…
Fig 1. Phylogenetic tree based on the sequences of 1371 genes inferred as orthologous.
Fig 5. Distribution in functional categories of…
Fig 5. Distribution in functional categories of the predicted CDSs (%).
The points representing the…
Fig 6. Distribution of genes encoding lipoproteins.
Fig 6. Distribution of genes encoding lipoproteins.
The "*" represent the level of significance between…
Fig 8. Phylogenetic tree based on the…
Fig 8. Phylogenetic tree based on the 16S rRNA and ppk sequences to evaluate the…
GLOBAL PATTERNS OF LEPTOSPIRA PREVALENCE IN VERTEBRATE RESERVOIR HOSTS
Leptospirosis is a widespread emerging bacterial zoonosis. As the transmission is believed to be predominantly waterborne, human incidence is expected to increase in conjunction with global climate change and associated extreme weather events. Providing more accurate predictions of human leptospirosis requires more detailed information on animal reservoirs that are the source of human infection. We evaluated the prevalence of Leptospira in vertebrates worldwide and its association with taxonomy, geographic region, host biology, ambient temperature, and precipitation patterns. A multivariate regression analysis with a meta-analysis-like approach was used to analyze compiled data extracted from 300 Leptospira-related peer reviewed papers. A fairly uniform Leptospira infection prevalence of about 15% was found in the majority of mammalian families. Higher prevalence was frequently associated with species occupying urban habitats, and this may explain why climatic factors were not significantly correlated with prevalence as consistently as expected. Across different approaches of the multiple regression analyses, the variables most frequently correlated with Leptospira infection prevalence were the host's ability to swim, minimum ambient temperature, and methodologic quality of the study. Prevalence in carnivores was not associated with any climatic variable, and the importance of environmental risk factors were indicated to be of lesser consequence in nonhuman mammals. The dataset is made available for further analysis.
Keywords: Climate change Leptospira leptospirosis meta-analysis multivariate regression analysis phylogeny reservoir hosts waterborne.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1B show workflow schematics for the identification of AAV variants. FIG. 1A depicts high-throughput detection of novel AAV variants in selected human tissues. Proviral capsid sequences are amplified using high-cycle PCR, followed by low-cycle PCR to barcode the amplicon libraries for multiplexed single-molecule, real-time (SMRT) sequencing. FIG. 1B shows a summary of the pipeline for bioinformatics analysis of sequencing data.
FIGS. 2A-2D show data relating to in vivo detection of FFLuc transgene activity with different administrations of selected AAV8 variants. FIG. 2A shows luciferase activities of different AAV8 variants were evaluated at week 6 after IV (intravenous), IM (intramuscular), or IN (intranasal) injection. FIGS. 2B-2D data relating to evaluation of FFLuc activity for each variant, B2 (FIG. 2B), B3 (FIG. 2C), and B61 (FIG. 2D), compared to AAV8 (mean±SD, n=3, t test).
FIGS. 3A-3B show data relating to evaluation of FFLuc transgene activity delivered by the AAV8 variant B61 compared to AAV9 at day 21 after neonatal injection. Luciferase activities and genome copies of brain (FIG. 3A) and spinal cord (FIG. 3B) were detected (mean±SD, n=5, t test).
FIGS. 4A-4B show data relating to in vivo detection of FFLuc transgene activity after right hindlimb intramuscular (IM) injection of the AAV8 variant B44 compared to AAV8. FIG. 4A shows whole animal Luciferase expression of variant B44 was evaluated at week 6 after IM injection. FIG. 4B shows evaluation of muscle (RTA, right tibialis anterior LTA, left tibialis anterior), liver, and heart. Luciferase activities (left bar graph) and relative ratios (right bar graph) for B44 compared to AAV8 (mean±SD, n=3).
FIG. 5 shows a phylogenic comparison of AAV8 variants (B2, B3, B61) to other AAV serotypes.
FIG. 6A shows a schematic depiction of a workflow for the in vivo characterization of novel AAV variants by high-throughput tropism screening.
FIG. 6B shows a schematic depiction of a workflow for the NHP characterization of novel AAV variants by high-throughput tropism screening.
FIG. 7 shows a scatter plot displaying the distribution of distinct AAV2 capsid variants (409 total) and AAV2/3 variants (194 total) harboring one or more single-amino-acid variants.
FIG. 8 shows diagrams of vector constructs used in the multiplexed screening of discovered capsid variants. Unique 6-bp barcodes were cloned into transgenes and packaged into candidate capsid variants.
FIG. 9 shows a schematic of an indexed transgene and high-throughput sequencing library design to assess capsid variant tropism profiling. The indexed and adapter cassette containing a 6-bp barcode (1° barcode) and a BstEII restriction site can be cloned into vector constructs using flanking BsrGI and SacI sites. Whole crude DNA from rAAV-treated tissues containing both host genome and vector genomes was cut with BstEII enzyme. The resulting 5′-overhang was used to specifically ligate to an adapter containing a second barcode, which allows for further multiplexed sequencing and streamlining and a 5′-biotin modification, which can be used to select for adapter-containing fragments using magnetic bead enrichment. Enriched material can then undergo PCR amplification using primers specific to adapter and transgene sequences to produce libraries for high-throughput sequencing. SEQ ID NOs.: 1719-1725 are shown from top to bottom.
FIGS. 10A-10D show transduction spread of rAAV2 and rAAVv66 following intrahippocampal injection. FIG. 10A shows native EGFP expression following rAAV2-CB6-Egfp or rAAVv66-CB6-Egfp injection via unilateral intrahippocampal administration. Scale bars=700 μm. FIG. 10B shows quantification of EGFP-positive surface normalized to DAPI-positive surface. Data is presented as the mean±SD n=3. ****P<0.0001. FIG. 10C shows coronal brain schematic depicting sub-anatomical regions of interest in both contralateral and ipsilateral hemispheres. Cornu ammonis (CA1, CA2, CA3, CA4), dentate gyrus (DG), corpus callosum (CC), and cortex (CTX). FIG. 10D shows high-magnification images of rAAVv66 transduced sub-anatomical regions. Scale bars=50 μm.
FIGS. 11A-11P show transduction of major cell types of the brain by rAAVv66. (FIGS. 11A, 11E, 11I, 11M) Coronal sections of rAAVv66-CB6-Egfp transduced mouse brains. IF-stained sections with antibodies against NEUN (FIG. 11A neurons), GFAP (FIG. 11E astrocytes), IBA1 (FIG. 11I microglia), or OLIG2 (FIG. 11M oligodendrocytes) indicate the distribution of cell types across the brain. Native EGFP expression that colocalize with IF staining indicate positively transduced cell types. Scale bars=700 μm. (FIGS. 11B, 11F, 11J, 11N) 3D rendering of sub-anatomical regions of single representative frames from dashed line rectangle boxes within coronal section views (top panels) with single-cell representations from fields defined by dashed lined square boxes (bottom three panels). Left panels, total area EGFP and cell marker IF stains center panels, colocalized EGFP with total cell marker IF stains right panels, colocalized EGFP and cell marker IF stains. Scale bars=50 μm (top panels), 5 μm (bottom three panels). (FIGS. 11C, 11G, 11K, 11O) Quantification of cell type-specific IF staining across indicated hippocampal regions (x axes), normalized to DAPI signal. (FIGS. 11D, 11H, 11L, 11P) Quantification of cell type-specific transduction across indicated regions, normalized to total cell-type IF and DAPI signal. Data is presented as the mean±SD n=3. Cornu ammonis (CA1, CA2, CA3, CA4), dentate gyrus (DG), corpus callosum (CC), and cortex (CTX).
FIGS. 12A-12E show biophysical analyses of AAVv66. (FIGS. 12A-12B) Heatmap displays of differential scanning fluorimetry (DSF) analyses to query capsid protein unfolding (uncoating) and DNA accessibility (vector genome extrusion) at pHs 7, 6, 5, and 4. (FIGS. 12C-12E) Each defining amino acid residue of AAVv66 was converted to those of AAV2 by site-directed mutagenesis and examined for changes in (FIG. 12C) packaging yield, (FIG. 12D) capsid stability, and (FIG. 12E) genome release at pH 7. Values represent mean±SD. p values were determined by one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. n 3 3.
FIGS. 13A-13E show cryo-EM primary metrics, map reconstruction, and model generation of AAVv66. (FIG. 13A) Density map of AAVv66. Color scheme demarcates the topological distance from the center (A). (FIG. 13B) Ribbon structure of the refined AAVv66 capsid monomer. Amino acids differentiating from AAV2 are highlighted. The 2-fold (oval), 3-fold (triangle), and 5-fold (pentagon) symmetries are annotated. Part of AAVv66 electron density (dark grey mesh) and residues are shown for regions close to (FIG. 13C) L583, R487, Y533, and K532, (FIG. 13D) S446, D499, and S501, and (FIG. 13E) N407-T414.
FIG. 14 shows structural differences between AAVv66 and AAV2. At the center is the AAVv66 60-mer structure (grey). Amino acid residues unique to AAVv66 are highlighted in green, while amino acid residues for a single monomer that are in common with AAV2 are colored. Atomic models showing residue side chains of select regions with substantial difference between AAVv66 and AAV2. The alignments were made with using monomers of AAV2 (11p3) and AAVv66, with modeled side chains from neighboring residues displayed in grey. Annotations for amino acids shown are indicated as those belonging to AAVv66, the position number, and then AAV2.
FIGS. 15A-15C show differential capsid surface electrostatics between AAV2 and AAVv66. (FIG. 15A) Surface positive and negative charges are displayed for AAV2 and AAVv66 60-mer, trimer (3-fold symmetry), and pentamers (exterior and interior of the 5-fold symmetry) structures. Black arrows at the AAV2 60-mer and trimer structures indicate the approximate positions of R585 and R588 at a single 3-fold protrusion. (FIG. 15B) Zoom-in of amino acid residues at 585-588 of AAV2 and AAVv66. (FIG. 15C) Bar graphs of the zeta potentials of purified vectors as measured by a zetasizer. Values represent mean±SD, n=3.
FIG. 16 shows amino acid sequence of the AAVs/AAVv66 capsid. Amino acid differences between AAV2 and AAVv66 are highlighted. Variable region (VR) residues are denoted by short bars. The aA domain is demarcated by the dotted bar, and residues forming the b-sheets are marked with black arrows. Start positions for VP1, VP2, and VP3 are marked by greater-than symbol (>). The PLA domain within VP1 is denoted by a bar. The AAV2 strand corresponds to SEQ ID NO: 869, and the AAVv66 strand corresponds to SEQ ID NO: 66.
FIG. 17 shows AAVv66 produces higher vector yields than AAV2. Crude lysate PCR assays were performed on media and cellular lysates of HEK239 cells subjected to triple-transfection of pAAV and packaging plasmids for AAV2 or AAVv66. Values represent mean genome copies±SD, n=3.
FIG. 18 shows AAVv66 lacks strong heparin binding. Heparin competition assay showing transduction efficiency of AAV2-CB6-FLuc and AAVv66-CB6-FLuc in HEK293 cells in the presence of increasing amounts of heparin (x-axis). Luminescence values were scaled to values obtained for wells lacking heparin and set to 1 (y-axis). Values represent mean±SD, n=3. **, p<0.01 by 2-way ANOVA.
FIG. 19 shows in vitro infection efficiencies of AAV2, AAV3b, and AAVv66 in HEK293 cells. Vectors were packaged with CB6-FLuc. Cells were lysed 48-hr post-infection to assess the infectivity of vectors via detection of luciferase activity (RLU, relative light units). Data is displayed in log-scale. Values represent mean±SD, ***p<0.0001 by one-way ANOVA, n=3.
FIGS. 20A-20D show intravenous administration of AAVv66 vector shows transduction of the liver. Systemic injection of AAVv66-CB6-Fluc resulted in the transduction of the liver. rAAV2-CB6-Fluc or AAVv66-CB6-Fluc (1.0E11 GC/mouse) was injected into mice by tail vein administration. (FIG. 20A) After 14 days, mice were injected with luciferin substrate intraperitoneally and imaged. Although quantification of whole-body live bioluminescence of luciferase activity did not reveal significant differences in transduction of the liver between AAVv66-CB6-Fluc and AAV2-CB6-Fluc, isolation of liver tissues and quantification of luciferase activity and detection of vector genome copy by qPCR showed that AAVv66 is a significantly weaker transducer of liver than AAV2. (FIG. 20B) Total flux of the abdomen in acquired images was recorded. Tissues were harvested and assayed for luciferase activity (FIG. 20C) and vector genome abundance by qPCR (FIG. 20D). Values represent mean±SD, n=3. *, p<0.05 by Student's t test.
FIGS. 21A-21D show intramuscular administration of AAVv66 vector shows transduction of muscle. Intramuscular injection of AAVv66 into the tibialis anterior resulted in very little difference in transduction capacity when compared with the transduction of AAV2. AAV2-CB6-FLuc or AAVv66-CB6-FLuc (4.0E10 GC/mouse) was injected into mice by intramuscular administration into one hindlimb (tibialis anterior). (FIG. 21A) After 14 days, mice were injected with luciferin substrate intraperitoneally and imaged. (FIG. 21B) Total flux of the injected hindlimb in acquired images was recorded. Tissues were harvested and assayed for luciferase activity (FIG. 21C) and vector genome abundance by qPCR (FIG. 21D). Values represent mean±SD, n=3. *, p<0.05 by Student's t test.
FIGS. 22A-22D show immunological characterization of AAVv66. Mice were intramuscularly administrated by AAV2-CB6-Egfp vector (1E11 GC/mouse). Four weeks after administration, sera were collected for testing neutralizing antibody (NAb) titers against AAV2 or AAVv66 infection. NAb50 values for AAV2 (FIG. 22A) and AAVv66 (FIG. 22B) are defined as the titer dilution that can block 50% of the total transduction achievable by the vector packaged with the LacZ reporter gene. Left, NAb table summaries of individual animals tested. Right, transduction efficiencies were plotted against various serum dilutions. Values represent mean±SD. Dashed lines indicate mean NAb50 serum titers. (FIG. 22C) After the four-week period, mice were intramuscularly administrated with AAV2-hA1AT or AAVv66-hA1AT (1E11 GC/mouse) on the contralateral hindlimb. Serum A1AT levels were measured by ELISA at weeks 5, 6, 7, and 8. Values represent mean±SD, n=3. n.s., not significant *, p<0.05 **, p<0.01 and ***, p<0.001 by 2-way ANOVA on cross-sectional data points. (FIG. 22D) Rabbit anti-AAV serum cross-reactivity. Rabbit antisera raised against AAV serotypes was tested for NAb to AAVv66 versus the homologous AAV serotype to assess relative cross reactivity. Log 2 values represent highest antibody dilution to achieve 50% inhibition of transduction.
FIGS. 23A-23B show cryo-EM primary metrics, map reconstruction, and model generation of AAVv66. (FIG. 23A) Cryo-electron micrograph of AAVv66. The scale bar represents 100 Å. (FIG. 23B) Fourier shell correlation for even and odd particles (FSC_part) for AAVv66.
FIG. 24 shows RMSD (Å) statistics comparing AAVv66 to AAV2 or AAV3b. Summary of the total and regional RMSD (Å) between AAVv66 and AAV2 (1LP3) or AAV3b (3KIC) measured across all alpha-carbon pairs indicated (AAV2 numbering) calculated by the rms_cur function within PyMOL. Full capsid structures of AAV2, 3b, and AAVv66 were aligned through optimized fit within the cryo-EM density map of AAVv66. Using a custom script within PyMOL, the distance values (Å) between individual alpha-carbon pairs for either AAV2 (upper) or AAV3b (lower) were quantitatively transformed for representation as both color and radial thickness for the corresponding residues of AAVv66.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Organ weights from intact rats treated with a compound of formula III presented as a percentage of intact control. *P-value <0.05 versus intact controls.
FIG. 2: Organ weights from castrated, compound of formula III-treated rats presented as a percentage of intact control. *P-value <0.05 versus intact controls.
FIG. 3: Organ weight maintenance dose-response curves for compound of formula III in castrated rats compared to oxandrolone.
FIG. 4: Organ weight maintenance dose-response curves for compound of formula III in castrated rats. Emax and ED50 values for the levator ani (closed triangles), prostate (open circles), and seminal vesicles (closed squares) were obtained by nonlinear regression analysis using the sigmoid Emax model in WinNonlin®.
FIG. 5: Organ weights from castrated rats after delayed dosing of compound of formula III presented as a percentage of intact control. *P-value <0.05 versus intact controls.
FIG. 6: Organ weight regrowth dose-response curves following delayed dosing of compound of formula III in castrated rats. Emax and ED50 values for the levator ani (closed triangles), prostate (open circles), and seminal vesicles (closed squares) were obtained by nonlinear regression analysis using the sigmoid Emax model in WinNonlin®.
FIG. 7: Cholesterol reduction by compound of formula III in rats.
FIG. 8: Total lean mass increase of all subjects with 0.1 mg, 0.3 mg, 1 mg, and 3 mg dose of Compound III.
FIG. 9: Total fat mass change of all subjects with 0.1 mg, 0.3 mg, 1 mg, and 3 mg dose of Compound III.
FIG. 10: Insulin resistance results (including insulin, glucose and HOMA-IR levels) of Avandia®, glipizide and compound of formula III.
FIG. 11: Improvement of soleus strength in ovariectomized (OVX) rats treated with compound of formula III.
FIG. 12: Trabecular bone mineral density determined by pQCT analysis of the distal femur 12A. Rat distal femur representative reconstructions 12B. BV/TV analysis of the distal femur 12C. Trabecular number of the distal femur 12D.
FIG. 13: plots circulating levels of compound of formula III in plasma in male and female dogs.
FIG. 14: depicts recruitment of AR in response to DHT or SARM. FIG. 14A is a Ven diagram showing the number of promoters significantly recruiting AR over vehicle in response to DHT, SARM or DHT and SARM. FIG. 14B illustrates classification of genes assayed with known function (1023) whose promoters were occupied by AR in response to DHT (open bars), SARM (filled bars) or promoters common to DHT or SARM (hatched bars). FIG. 14C depicts computational identification of androgen responsive AR direct target gene promoters in response to DHT, SARM or DHT and SARM. Human and orthologous mouse sequences determined from the AR promoter array experiment were searched for the presence of ARE.
FIG. 15: depicts recruitment of SRC-1 in response to DHT or SARM. FIG. 15A illustrates recruitment to PSA enhancer as measured by realtime quantitative PCR. Values are reported as the ratio of target detected in the immunoprecipitated (IP) DNA pool to target detected in the total input DNA pool. Open bars are vehicle treated, filled bars are DHT treated and hatched bars are SARM treated. FIG. 15B depicts is a Ven diagram showing the number of promoters significantly recruiting SRC-1 over vehicle in response to DHT or SARM or DHT and SARM. FIG. 15C depicts classification of genes assayed with known function (1015) whose promoters were occupied by SRC-1 in response to DHT (open bars), SARM (filled bars) or promoters common to DHT and SARM (hatched bars). FIG. 15D illustrates computational identification of androgen responsive elements in SRC-1 target gene promoters in response to DHT, SARM or DHT and SARM. Human and orthologous mouse sequences determined from the SRC-1 promoter array experiment were searched for the presence of ARE.
FIG. 16: Validation of promoter array. FIG. 16A. Validation of AR recruitment to various promoters. LNCaP cells were maintained in 1% csFBS for 6 days to reduce the basal transcription factor recruitment and were treated with vehicle (open bars), 100 nM DHT (filled bars) or SARM (hatched bars) for 60 min. ChIP assay was performed with AR antibody and recruitment to various promoters showing significance from the array were measured using realtime rtPCR primers and probes (Table 16). Values are reported as the ratio of target DNA detected in the IP DNA pool to target DNA detected in the total input DNA pool. The experiments were performed in triplicate. FIG. 16B. Measurement of gene transcription of promoters to which AR was recruited. Gene transcription was measured by treating LNCaP cells maintained in 1% csFBS (STAT5B, SHC-1, GAS7, APIG1, AXIN1, ATM and MSX-1) or full serum (NFkB1E). The cells were treated with vehicle (open bars), DHT (filled bars) or SARM (hatched bars). RNA was extracted and realtime rtPCR was performed using TaqMan primers and probe and normalized to 18S. The experiments were performed in triplicate. Cells were treated for 24 hrs. * indicate significance at P<0.05 from vehicle treated samples. IP-Immunoprecipitation ChIP-Chromatin Immunoprecipitation.
FIG. 17: Change from baseline to Day 113/EOS in stair climb power: MITT population.
EOS=end of study MITT=modified intent-to-treat.
FIG. 18: Change from baseline to Day 113/EOS in stair climb time: MITT population. EOS=end of study MITT=modified intent-to-treat.
FIG. 19: Correlation between stair climb power and QoL per FAACT questionnaire in NSCLC patients.
FIG. 20 depicts Study A—Platinum+Taxane plus add on Lean body mass efficacy endpoint. FIG. 20A: MMRM analysis through Day 84 visit FIG. 20B: MMRM analysis through Day 147 visit.
FIG. 21 depicts Study A—Platinum+Taxane plus add on body weight efficacy endpoints. FIG. 21A: MMRM analysis through Day 84 visit FIG. 21B: MMRM analysis through Day 147 visit.
FIG. 22 depicts Study A—Platinum+Taxane plus add on Stair climb test (% power change) efficacy endpoints. FIG. 22A: MMRM analysis through Day 84 visit FIG. 22B: MMRM analysis through Day 147 visit.
FIG. 23 depicts Study B—Platinum+nontaxane plus add on lean body mass efficacy endpoint. FIG. 23A: MMRM analysis through Day 84 visit FIG. 23B: MMRM analysis through Day 147 visit.
FIG. 24 depicts Study B—Platinum+nonaxane plus add on body weight endpoint. FIG. 24A: MMRM analysis through Day 84 visit FIG. 24B: MMRM analysis through Day 147 visit.
FIG. 25 depicts Study B—Platinum+nonaxane plus add on stair climb test (% power change) efficacy endpoint. FIG. 25A: MMRM analysis through Day 84 visit FIG. 25B: MMRM analysis through Day 147 visit.
FIG. 26 shows plasma concentrations of compound III were lower in Study B LBM nonresponders.
FIG. 27 shows LBM nonresponders who reported nausea and vomiting had lower Compound III levels in Study B. Compound III levels were similar in LBM responders and non-responders who did NOT report nausea and vomiting
FIG. 28 shows Study B Platinum+Nontaxane subjects had lower hemoglobin levels. WHO definition of anemia Men ≦13 g/dL Women ≦12 g/dL.
FIG. 29 depicts LBM benefit not affected by hemoglobin concentrations.
FIG. 30 shows physical function benefit of new muscle is associated with hemoglobin concentrations.
FIG. 31 depicts pooled survival analysis.
FIG. 32 shows Day 84 LBM response is associated with longer survival landmark analyses.
FIG. 33 shows Day 42 LBM response is associated with longer survival landmark analyses.
FIG. 34 depicts survival by arm and LBM response.
FIG. 35 depicts 10% SCP response by ≧1 kg LBM response in post-hoc analyses.
FIG. 36 depicts 10% SCP response by ARM by ≧1 kg LBM response in post-hoc analyses.
15.22D: Leptospirosis - Biology
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Citation: Faggion Vinholo T, Ribeiro GS, Silva NF, Cruz J, Reis MG, Ko AI, et al. (2020) Severe leptospirosis after rat bite: A case report. PLoS Negl Trop Dis 14(7): e0008257. https://doi.org/10.1371/journal.pntd.0008257
Editor: Melissa J. Caimano, University of Connecticut Health Center, UNITED STATES
Published: July 9, 2020
Copyright: © 2020 Faggion Vinholo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by grants from the Fogarty International Center (R25 TW009338, R01 TW009504) and National Institute of Allergy and Infectious Diseases (F31 AI114245, R01 AI121207) from the National Institutes of Health the UK Medical Research Council (MR/P0240841), the Wellcome Trust (102330/Z/13/Z), and the Fulbright Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
What are the symptoms of Leptospirosis?
Referred to by some sources as a “mimic disease,” cases of Leptospirosis can be difficult to identify. It is often confused with dengue fever, influenza, meningitis, hepatitis and other viral hemorrhagic fevers. 16 According to the CDC, Leptospirosis can cause a wide range of symptoms:
- High fever
- Muscle aches
- Jaundice (yellow skin and eyes)
- Red eyes
- Abdominal pain
- Rash 17
It is common that the illness develops after the individual can clearly connect their symptoms with exposure to a potentially contaminated source. The CDC explains, “The time between a person’s exposure to a contaminated source and becoming sick is 2 days to 4 weeks.” 18
Course of the Disease
The course of the disease typically involves two phases: a septic state and immune response. The first phase generally involves the abrupt onset of fever, chills, headache, muscle aches, vomiting, or diarrhea, and lasts 3-7 days. After a one to three day period with minimal symptoms, the immune response begins and antibodies appear in the blood. Many symptoms return, and the individual may continue to be sick for days or weeks longer.
A more severe form of Leptospirosis, comprising 5-10% of cases, is known as Weill’s Disease. According to the NCBI, it… “has a fatality rate of 5-10% and, the rate increases to 20-40% with hepatorenal involvement and jaundice.” 19 (Heptorenal- involves a rapid decline in kidney function. 20 )
The disease involves the symptoms mentioned above, along with severe damage to the liver, kidneys and/or other organs. Recovery can take years, and long-term effects have been observed.
High Risk Groups
As with other zoonotic diseases (spread from animals to humans), the CDC explains:
- Children younger than 5
- Adults older than 65
- People with weakened immune systems 21
In another aspect, the present disclosure provides compositions comprising a MON described herein and optionally an excipient. A composition described herein may further comprise a solvent (e.g., a suitable solvent described herein, such as water or DMSO). The solvent may be encapsulated inside a MON and/or be present outside of any MON in the composition.
In still another aspect, the present disclosure provides compositions comprising a BCPMON described herein and optionally an excipient. In another aspect, the present disclosure provides compositions comprising a thermoplastic elastomer described herein and optionally an excipient. In another aspect, the present disclosure provides compositions comprising a macromonomer described herein and optionally an excipient.
The excipient included in a composition described herein may be a pharmaceutically acceptable excipient, cosmetically acceptable excipient, dietarily acceptable excipient, or nutraceutically acceptable excipient.
A composition described herein may further comprise an agent (e.g., a pharmaceutical agent or diagnostic agent). In a composition described herein, an agent may form an adduct (e.g., through covalent attachment and/or non-covalent interactions) with a MON described herein (including a MON moiety of a BCPMON described herein). In certain embodiments, a composition described herein is useful in the delivery of the agent (e.g., an effective amount of the agent) to a subject, tissue, or cell.
A composition described herein may further comprise a fluid (e.g., a solvent, e.g., water, DMSO, acetonitrile, or a mixture thereof)
Compositions of the disclosure may improve or increase the delivery of an agent described herein to a subject, tissue, or cell. In certain embodiments, the compositions increase the delivery of the agent to a target tissue or target cell. In certain embodiments, the target tissue is liver, spleen, or lung. In certain embodiments, the target tissue is pancreas, kidney, uterus, ovary, heart, thymus, fat, or muscle. In certain embodiments, the target cell is a liver cell, spleen cell, lung cell, pancreas cell, kidney cell, uterus cell, ovary cell, heart cell, thymus cell, or muscle cell. In certain embodiments, the compositions selectively deliver the agent to the target tissue or target cell (e.g., the compositions deliver the agent to the target tissue in a greater quantity in unit time than to a non-target tissue or deliver the agent to the target cell in a greater quantity in unit time than to a non-target cell).
The delivery of an agent described herein may be characterized in various ways, such as the exposure, concentration, and bioavailability of the agent. The exposure of an agent in a subject, tissue, or cell may be defined as the area under the curve (AUC) of the concentration of the agent in the subject, tissue, or cell after administering or dosing the agent. In general, an increase in exposure may be calculated by first taking the difference in: (1) a first AUC, which is the AUC measured in a subject, tissue, or cell administered or dosed with a composition described herein and (2) a second AUC, which is the AUC measured in a subject, tissue, or cell administered or dosed with a control composition and then by dividing the difference by the second AUC. Exposure of an agent may be measured in an appropriate animal model. The concentration of an agent and, when appropriate, its metabolite(s), in a subject, tissue, or cell is measured as a function of time after administering or dosing the agent.
Concentration of an agent, and, when appropriate, of its metabolite(s), in a subject, tissue, or cell, may be measured as a function of time in vivo using an appropriate animal model. In certain embodiments, the concentration of the agent is the concentration of the agent in a target tissue or target cell. One exemplary method of determining the concentration of an agent involves dissecting of a tissue. The concentration of the agent may be determined by HPLC or LC/MS analysis.
In some embodiments, a composition of the disclosure increases the delivery of an agent described herein to a subject, tissue, or cell due to the presence of a MON described herein. In some embodiments, a composition of the disclosure increases the delivery of an agent described herein to a subject, tissue, or cell due to the presence of a BCPMON described herein. In some embodiments, the composition increases the delivery of the agent due to the presence of an adduct formed between the MON (including a MON moiety of a BCPMON) and the agent. In some embodiments, the presence of a MON or BCPMON described herein increase the delivery of the agent by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 2-fold, at least 3-fold, at least 10-fold, at least 30-fold, at least 100-fold, at least 300-fold, or at least 1000-fold. In certain embodiments, a MON or BCPMON described herein is present in the composition in an amount sufficient to increase the delivery of the agent by an amount described herein when administered in the composition compared to the delivery of the agent when administered in the absence of the MON or BCPMON.
Compositions described herein may deliver an agent selectively to a tissue or cell. In certain embodiments, the tissue or cell to which the agent is selectively delivered is a target tissue or target cell, respectively. In certain embodiments, the compositions deliver at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 100%, at least 3-fold, at least 10-fold, at least 30-fold, at least 100-fold, at least 300-fold, or at least 1000-fold more amount of the agent in unit time to a target tissue than to a non-target tissue or to a target cell than to a non-target cell. The amount of agent may be measured by the exposure, concentration, and/or bioavailability of the agent in a tissue or cell as described herein.
The compositions described herein (e.g., pharmaceutical compositions) including one or more agents (e.g., pharmaceutical agents) may be useful in treating and/or preventing a disease. In certain embodiments, the compositions are useful in gene therapy. In certain embodiments, the compositions are useful for treating and/or preventing a genetic disease. In certain embodiments, the compositions are useful for treating and/or preventing a proliferative disease. In certain embodiments, the compositions are useful for treating and/or preventing cancer. In certain embodiments, the compositions are useful for treating and/or preventing a benign neoplasm. In certain embodiments, the compositions are useful for treating and/or preventing pathological angiogenesis. In certain embodiments, the compositions are useful for treating and/or preventing an inflammatory disease. In certain embodiments, the compositions are useful for treating and/or preventing an autoimmune disease. In certain embodiments, the compositions are useful for treating and/or preventing a hematological disease. In certain embodiments, the compositions are useful for treating and/or preventing a neurological disease. In certain embodiments, the compositions are useful for treating and/or preventing a gastrointestinal disease. In certain embodiments, the compositions are useful for treating and/or preventing a liver disease. In certain embodiments, the compositions are useful for treating and/or preventing a spleen disease. In certain embodiments, the compositions are useful for treating and/or preventing a respiratory disease. In certain embodiments, the compositions are useful for treating and/or preventing a lung disease. In certain embodiments, the compositions are useful for treating and/or preventing hepatic carcinoma, hypercholesterolemia, refractory anemia, or familial amyloid neuropathy. In certain embodiments, the compositions are useful for treating and/or preventing a painful condition. In certain embodiments, the compositions are useful for treating and/or preventing a genitourinary disease. In certain embodiments, the compositions are useful for treating and/or preventing a musculoskeletal condition. In certain embodiments, the compositions are useful for treating and/or preventing an infectious disease. In certain embodiments, the compositions are useful for treating and/or preventing a psychiatric disorder. In certain embodiments, the compositions are useful for treating and/or preventing a metabolic disorder.
The agents may be provided in an effective amount in a composition described herein. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is an amount effective for treating a disease described herein. In certain embodiments, the effective amount is an amount effective for preventing a disease described herein.
An effective amount of an agent may vary from about 0.001 mg/kg to about 1000 mg/kg in one or more dose administrations for one or several days (depending on the mode of administration). In certain embodiments, the effective amount per dose varies from about 0.001 to about 1000 mg/kg, from about 0.01 to about 750 mg/kg, from about 0.1 to about 500 mg/kg, from about 1.0 to about 250 mg/kg, and from about 10.0 to about 150 mg/kg.
In certain embodiments, a composition described herein is in the form of gels. In certain embodiments, the gels result from self-assembly of the components of the composition. The agent to be delivered by the gel may be in the form of a gas, liquid, or solid. The MONs and/or BCPMONs described herein may be combined with polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, lipidoids, etc. to form gels. The gels may be further combined with an excipient to form the composition. The gels are described in more detail herein.
The compositions described herein (e.g., pharmaceutical compositions) can be prepared by any method known in the art (e.g., pharmacology). In certain embodiments, such preparatory methods include the steps of bringing a MON or BCPMON described herein into association with an agent described herein (i.e., the “active ingredient”), optionally with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.
Compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A unit dose is a discrete amount of the composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
Relative amounts of the active ingredient, the excipient (e.g., the pharmaceutically or cosmetically acceptable excipient), and/or any additional ingredients in a composition described herein will vary, depending upon the identity, size, and/or condition of the subject to whom the composition is administered and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.
Excipients used in the manufacture of provided compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.
Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.
Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.
Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij® 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F-68, Poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and mixtures thereof.
Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and mixtures thereof.
Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.
Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, sodium sulfite, and mixtures thereof.
Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, and dipotassium edetateke), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, tartaric acid and salts and hydrates thereof, and mixtures thereof.
Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, thimerosal, and mixtures thereof.
Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, sorbic acid, and mixtures thereof.
Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, phenylethyl alcohol, and mixtures thereof.
Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, phytic acid, and mixtures thereof.
Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, Neolone®, Kathon®, Euxyl®, and mixtures thereof.
Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.
Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.
Additionally, the composition may further comprise an apolipoprotein. Previous studies have reported that Apolipoprotein E (ApoE) was able to enhance cell uptake and gene silencing for a certain type of materials. See, e.g., Akinc, A., et al., Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther. 18(7): p. 1357-64. In certain embodiments, the apolipoprotein is ApoA, ApoB, ApoC, ApoE, or ApoH, or an isoform thereof.
Liquid dosage forms for oral and parenteral administration include emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In certain embodiments, the emulsions, microemulsions, solutions, suspensions, syrups and elixirs are or cosmetically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.
Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, excipient or carrier (e.g., pharmaceutically or cosmetically acceptable excipient or carrier) such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.
Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the formulation art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.
Dosage forms for topical and/or transdermal administration of a composition of this disclosure may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches. Generally, the active ingredient is admixed under sterile conditions with a carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.
Suitable devices for use in delivering intradermal compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Ballistic powder/particle delivery devices which use compressed gas to accelerate the agent in powder form through the outer layers of the skin to the dermis are suitable.
Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
A composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
Compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.
Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a composition described herein. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
A composition described herein can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are also contemplated as being within the scope of this disclosure.
Although the descriptions of compositions provided herein are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
Metal organic nanostructures and BCPMONs described herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder, the activity of the specific active ingredient employed, the specific composition employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, route of administration, and rate of excretion of the specific active ingredient employed, the duration of the treatment, drugs used in combination or coincidental with the specific active ingredient employed, and like factors well known in the medical arts.
The compositions described herein can be administered by any suitable route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual by intratracheal instillation, bronchial instillation, and/or inhalation and/or as an oral spray, nasal spray, and/or aerosol. In certain embodiments, the compositions are administered by oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).
The exact amount of an agent required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular agent, mode of administration, and the like. The desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
In certain embodiments, an effective amount of an agent for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of an agent per unit dosage form.
In certain embodiments, the agents described herein may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic and/or prophylactic effect.
It will be appreciated that dose ranges as described herein provide guidance for the administration of provided compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
Compositions described herein may further include a hydrophilic polymer (e.g., polyethylene glycol (PEG)). The compositions described herein may further include a lipid (e.g., a steroid, a substituted or unsubstituted cholesterol, or a polyethylene glycol (PEG)-containing material). In certain embodiments, the lipid included in the compositions is a triglyceride, a driglyceride, a PEGylated lipid, dimyristoyl-PEG2000 (DMG-PEG2000), a phospholipid (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), dioleoylphosphatidylethanolamine (DOPE), a substituted or unsubstituted cholesterol, a steroid an apolipoprotein, or a combination thereof. In certain embodiments, the compositions include two components selected from the group consisting of the following components: a hydrophilic polymer, a triglyceride, a driglyceride, a PEGylated lipid, a phospholipid, a steroid, a substituted or unsubstituted cholesterol, and an apolipoprotein. In certain embodiments, the compositions include three components selected from the group consisting of the following components: a hydrophilic polymer, a triglyceride, a driglyceride, a PEGylated lipid, a phospholipid, a steroid, a substituted or unsubstituted cholesterol, and an apolipoprotein. In certain embodiments, the compositions include at least four components selected from the group consisting of the following components: a hydrophilic polymer, a triglyceride, a driglyceride, a PEGylated lipid, a phospholipid, a steroid, a substituted or unsubstituted cholesterol, and an apolipoprotein. In certain embodiments, the compositions include a hydrophilic polymer, a phospholipid, a steroid, and a substituted or unsubstituted cholesterol. In certain embodiments, the compositions include PEG, DSPC, and substituted or unsubstituted cholesterol. In certain embodiments, the additional materials are approved by a regulatory agency, such as the U.S. FDA, for human and/or veterinary use.
Compositions described herein may be useful in other applications, e.g., non-medical applications. Nutraceutical compositions described herein may be useful in the delivery of an effective amount of a nutraceutical, e.g., a dietary supplement, to a subject in need thereof. Cosmetic compositions described herein may be formulated as a cream, ointment, balm, paste, film, or liquid, etc., and may be useful in the application of make-up, hair products, and materials useful for personal hygiene, etc. Compositions described herein may be useful for other non-medical applications, e.g., such as an emulsion, emulsifier, or coating, useful, for example, as a food component, for extinguishing fires, for disinfecting surfaces, for oil cleanup, and/or as a bulk material.
Effect of VSTM5 in a model of allogeneic islet transplantation in diabetic mice. To test the effect of immunoinhibitory VSTM5 targeting antibodies on transplant rejection, a model of allogeneic islet transplantation is used. Diabetes is induced in C57BL/6 mice by treatment with streptozotocin. Seven days later, the mice are transplanted under the kidney capsule with pancreatic islets which are isolated from BALB/c donor mice. Recipient mice are treated with immunoinhibitory VSTM5 targeting antibodies or with mIgG2a as a negative control. Tolerance with ECDI-fixed donor splenocytes is used as the positive control for successful modulation islet graft rejection. Recipient mice are monitored for blood glucose levels as a measure of graft acceptance/rejection (Luo et al., PNAS, Sep. 23, 2008 105(38) 14527-14532).
Effect of VSTM5 in the Hya-Model of Skin Graft Rejection.
In humans and certain strains of laboratory mice, male tissue is recognized as non-self and destroyed by the female immune system via recognition of histocompatibility-Y chromosome encoded antigens (Hya). Male tissue destruction is thought to be accomplished by cytotoxic T lymphocytes in a helper-dependent manner.
To test the effect of immunoinhibitory VSTM5 targeting antibodies on transplantation, the Hya model system is used, in which female C57BL/6 mice receive tail skin grafts from male C57BL/6 donors.
In this study, female C57BL/6 mice are engrafted with orthotopic split-thickness tail skin from age matched male C57BL/6 mice. The mice are treated with immunoinhibitory VSTM5 targeting antibodies s, isotype control mIgG2a. Immunodominant Hya-encoded CD4 epitope (Dby) attached to female splenic leukocytes (Dby-SP) serve as positive control for successful modulation of graft rejection (Martin et al., J Immunol. 2010 Sep. 15 185(6): 3326-3336). Skin grafts are scored daily for edema, pigment loss and hair loss. Rejection is defined as complete hair loss and more than 80% pigment loss.
In addition, T cell recall responses of cells isolated from spleens and draining lymph nodes at different time points are studied in response to CD4 specific epitope (Dby), CD8 epitopes (Uty and Smcy) or irrelevant peptide (OVA 323-339) while anti CD3 stimulation is used as positive control for proliferation and cytokine secretion.
Effect of Immunoinhibitory VSTM5 Targeting Antibodies on Graft Rejection
The effect of immunoinhibitory VSTM5 targeting antibodies on graft rejection is further studied in a murine model of syngeneic bone marrow cells transplantation using the Hya model system described above. Male hematopoietic cells expressing the CD45.1 marker are transplanted to female host mice which express the CD45.2 congenic marker. Female hosts are treated with immunoinhibitory VSTM5 targeting antibodies or with isotype control mIgG2a. The female hosts are followed over time and the presence of CD45.1 + cells is monitored.
The invention has been described and various embodiments provided relating to manufacture and selection of desired anti-VSTM5 antibodies for use as therapeutics and diagnostic methods for various diseases. Different embodiments and sub-embodiments may optionally be combined herein in any suitable manner, beyond those explicit combinations and sub combinations shown herein. The invention is now further described by the claims which follow.