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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau „ (10) International Publication Number (43) International Publication Date Χ t it t t l December 20ll (0l.l2.20ll) 2U11/15U1U5 A2 (51) International Patent Classification: MAN, Karen B. [US/US]; 308 Ashton Lane, Mill Valley, CI2P 21/06 (2006.01) California 94941 (US). (21) International Application Number: (74) Agent: SCHERER, David C ; Bozicevic, Field & Fran PCT/US201 1/037969 cis LLP, 1900 University Avenue, Suite 200, East Palo Alto, California 94303 (US). (22) International Filing Date: 25 May 201 1 (25.05.201 1) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (25) Filing Language: English AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (26) Publication Language: English CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (30) Priority Data: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, 61/349,081 27 May 2010 (27.05.2010) US KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, 61/379,321 1 September 2010 (01 .09.2010) US ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 61/383,679 16 September 2010 (16.09.2010) us NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 61/415,321 18 November 2010 (18.1 1.2010) us SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 61/426,301 22 December 2010 (22.12.2010) us TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant (for all designated States except US): BIO- (84) Designated States (unless otherwise indicated, for every TIME INC. [US/US]; 1301 Harbor Bay Parkway, kind of regional protection available): ARIPO (BW, GH, Alameda, California 94502 (US). GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, (72) Inventors; and ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (75) Inventors/ Applicants (for US only): WEST, Michael D. TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, [US/US]; 308 Ashton Lane, Mill Valley, California EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, 9494 1 (US). STERNBERG, Hal [US/US]; 1735 Spruce LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, Street, #4, Berkeley, California 94709 (US). CHAP¬ SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). [Continued on next page] (54) Title: IMPROVED METHODS OF SCREENING EMBRYONIC PROGENITOR CELL LINES (57) Abstract: Aspects of the present invention include methods and compositions related to the production and use of numerous clonal lineages of embryonic progenitor cell lines derived from differentiating cultures of primor = dial stem cells with diverse molecular markers and having been cultured for > 2 1 doublings of clonal expansion. The robustness of these clonally-purified lines, their abil ity to expand for >40 passages while maintaining their ------ pattern of gene expression, lack of tumorigenicity, and their embryonic pattern of gene expression offers novel compositions and methods for modeling numerous differ = entiation pathways for the first time in vitro, and for the manufacture of purified product not existing in such a pu = rified state in nature or using other manufacturing modal ≡ ities. Representative progenitor cell lines described herein are capable of development into cutaneous adipocytes, blood-brain barrier cells, neuronal cells, or smooth mus cle cells each with therapeutic potential. < © o Fig. 1 o w o 2011/150105 A2 1II 11 II II 11 I III I I II I III I III I III II I II s e — with sequence listing part of description (Rule 5.2(a)) — without international search report and to be republished upon receipt of that report (Rule 48.2(g)) IMPROVED METHODS OF SCREENING EMBRYONIC PROGENITOR CELL LINES BACKGROUND Advances in stem cell technology, such as the isolation and propagation in vitro of primordial stem cells, including embryonic stem cells ("ES" cells including human ES cells ("hES" cells)) and related primordial stem cells including but not limited to, iPS, EG, EC, ICM, epiblast, or ED cells (including human iPS, EG, EC, ICM, epiblast, or ED cells), constitute an important new area of medical research. hES cells have a demonstrated potential to be propagated in the undifferentiated state and then to be induced subsequently to differentiate into likely any and all of the cell types in the human body, including complex tissues. In addition, many of these primordial stem cells are naturally telomerase positive in the undifferentiated state, thereby allowing the cells to be expanded indefinately. This expansion potential allows these primordial cells to be genetically modified followed by clonal expansion, thus permittting the production of numerous homogeneous populations of genetically modified primordial stem cells. Since the telomere length of many of these cells is comparable to that observed in sperm DNA (approximately 10-18 kb TRF length), differentiated cells derived from these immortal lines once they begin differentiation (generally associated with the repression of the expression of the catalytic component of telomerase (TERT)) display a long initial telomere length providing the cells with a long replicative capacity compared to fetal or adult-derived tissue. This has led to the suggestion that many diseases resulting from the dysfunction of cells may be amenable to treatment by the administration of hES-derived cells of various differentiated types (Thomson et al., Science 282:1145-1147 (1998)). Nuclear transfer studies have demonstrated that it is possible to transform a somatic differentiated cell back to a primordial stem cell state such as that of embryonic stem ("ES") cells (Cibelli et al., Nature Biotech 16:642-646 (1998)) or embryo-derived ("ED") cells. The development of technologies to reprogram somatic cells back to a totipotent ES cell state, such as by the transfer of the genome of the somatic cell to an enucleated oocyte and the subsequent culture of the reconstructed embryo to yield ES cells, often referred to as somatic cell nuclear transfer ("SCNT") or through analytical reprogramming technology, offers methods to transplant ES-derived somatic cells with a nuclear genotype of the patient (Lanza et al, Nature Medicine 5:975-977 (1999)). In addition to SCNT, other techniques exist to address the problem of transplant rejection, including the use of gynogenesis and androgenesis (see U.S. application nos. 60/161,987, filed October 28, 1999; 09/697,297, filed October 27, 2000; 09/995,659, filed November 29, 2001; 10/374,512, filed February 27, 2003; PCT application no. PCT/USOO/29551, filed October 27, 2000; the disclosures of which are incorporated by reference in their entirety). In the case of a type of gynogenesis designated parthenogenesis, pluripotent stem cells may be manufactured without antigens foreign to the gamete donor and therefore useful in manufacturin g cells that can be transplanted without rejection. In addition, parthenogenic stem cell lines can be assembled into a bank of cell lines homozygous in the HLA region (or corresponding MHC region of nonhuman animals) to reduce the complexity of a stem cell bank in regard to HLA haplotypes. In addition, cell lines or a bank of said cell lines can be produced that are hemizygous in the HLA region (or corresponding MHC region of nonhuman animals; see PCT application Ser. No. PCT/US2006/040985 filed October 20, 2006 entitled "Totipotent, Nearly Totipotent or Pluripotent Mammalian Cells Homozygous or Hemizygous for One or More Histocompatibility Antigen Genes", incorporated herein by reference). A bank of hemizygous cell lines provides the advantage of not only reducing the complexity inherent in the normal mammalian MHC gene pool, but it also reduces the gene dosage of the antigens to reduce the expression of said antigens without eliminating their expression entirely, thereby not stimulating a natural killer response. In addition to SCNT, parthenogenesis, and the construction of banks of cells with homozygous or hemizygous HLA alleles, other techniques exist to address the problem of transplant rejection, including the use of technologies to reprogram somatic cells using transcriptional regulators (see PCT application Ser. No. PCT/US2006/030632 filed on August 3, 2006 and titled "Improved Methods of Reprogramming Animal Somatic Cells", incorporated herein by reference). In regard to differentiating primordial stem cells into desired cell types, the potential to clonally isolate lines of human embryonic progenitor (hEP) cell lines provides a means to propagate novel highly purified cell lineages useful in the production of diverse secreted factors, for research, and for the manufacture of cell-based therapies (see PCT application Ser. No. PCT/US2006/013519 filed on April 11, 2006 and titled "Novel Uses of Cells With Prenatal Patterns of Gene Expression"; U.S. patent application Ser. No. 11/604,047 filed on November 21, 2006 and titled "Methods to Accelerate the Isolation of Novel Cell Strains from Pluripotent Stem Cells and Cells Obtained Thereby"; and U.S. patent application Ser. No. 12/504,630 filed on July 16, 2009 and titled "Methods to Accelerate the Isolation of Novel Cell Strains from Pluripotent Stem Cells and Cells Obtained Thereby", each incorporated herein by reference). Nevertheless, there remains a need for improved methods to discover the differentiation potential of said hEP cell lines when exposed to diverse differentiation-inducing factors or other differentiation conditions that induce such differentiation under conditions which are compatible in either a general laboratory setting or in a good manufacturing processes ("GMP") cell manufacturing facility where there is adequate documentation as to the purity and genetic normality of the cells at advanced passages (>1 8-21 doublings of clonal expansion).
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  • Synergistic and Antagonistic Drug Interactions in the Treatment of Systemic Fungal Infections

    Synergistic and Antagonistic Drug Interactions in the Treatment of Systemic Fungal Infections

    bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Synergistic and Antagonistic Drug Interactions in the Treatment of Systemic Fungal Infections Morgan A. Wambaugh, Steven T. Denham, Brianna Brammer, Miekan Stonhill, and Jessica C. S. Brown Division of Microbiology and Immunology, Pathology Department, University of Utah School of Medicine, Salt Lake City, UT 84132, USA [email protected]; [email protected] Keywords Cryptococcus neoformans, overlap2 method (O2M), synergy, antagonism, drug interactions, fluconazole, dicyclomine hydrochloride, berbamine hydrochloride, nafcillin sodium, Candida species Summary Invasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited number of efficacious drugs and poor drug availability. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits fungal cell growth instead of killing fungal cells, necessitating long treatments. To improve fluconazole treatments, we used our novel high-throughput method, the overlap2 method (O2M), to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. Although serendipitous identification of these interactions is rare, O2M allows us to screen molecules five times faster than testing combinations individually and greatly enriches for interactors. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole.