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Background: Heart transplantation is an emergency surgery requiring cardio-pulmonary bypass (CPB) and its timing is unpredictable. Patients on the transplant waiting list often have multiple reasons for being anticoagulated. Intraoperative removal of apixaban using CytoSorb seems to be an interesting solution for patients on DOACs requiring an emergency CPB intervention. The aim of this short communication is to describe the perioperative effects of the use of the CytoSorb hemoadsorption device during emergency CPB for a heart transplant patient. Methods: A 61-year-old male patient wait-listed for heart transplantation was admitted to our hospital to benefit from a heart transplantation. This patient, has an end-stage heart failure with multiple episodes of decompensation over the previous year. He was anticoagulated with a Vitamin K antagonist (VKA) due to atrial fibrillation and was switched to apixaban. Hemoadsorption by a CytoSorb cartridge was performed during the entire CPB duration. Anti-Factor Xa Activity (AFXaA) levels were taken before, during and after surgery in order to monitor anticoagulation. Results: Surgery consisted of an orthotopic heart transplantation with bi-caval anastomoses. At the time of anesthesia induction and after UFH administration, AFXaA levels were 330ng/mL and 317ng/mL, respectively. Thereafter, AFXaA decreased to 137ng/mL during CPB and to 57ng/mL after the end of CPB and protamine administration. After surgery, AFXaA levels stabilized over 50ng/mL over the next 14 hours. No primary graft dysfunction was observed, and during the post-operative period of 72 hours, the patient did not have any bleeding events requiring reintervention or transfusion. Conclusion: We observed that CytoSorb could be a potential solution to remove apixaban intraoperatively. If this efficacy is confirmed in larger trials, it would allow transplant candidates to be treated with DOACs without requiring a switch to VKAs.

Objectives: Patients on ticagrelor undergoing urgent cardiac surgery are at high risk for perioperative bleeding complications. We sought to determine whether intraoperative hemoadsorption could remove ticagrelor and lower circulating drug concentrations. Methods: The hemoadsorption device was incorporated in the cardiopulmonary bypass (CPB) circuit and remained active for the duration of the pump run. Blood samples were collected before and after CPB. The main objective of the current analysis was to compare mean total plasma ticagrelor levels (ng/mL) at baseline with ticagrelor levels obtained at the end of CPB. Plasma ticagrelor levels were measured at a certified outside laboratory (LabConnect). Data are presented as mean ± standard deviation. Results: A total of 11 patients undergoing urgent coronary artery bypass grafting at 3 institutions were included (mean age, 67.9 ± 9.9 years; 91% male; mean European System for Cardiac Operative Risk Evaluation II of 3.0 ± 3.3%; range, 0.7%-12.4%). Mean intraoperative hemoadsorption duration was 97.1 ± 43.4 minutes with a mean flow rate through the device of 422.9 ± 40.3 mL/min. Mean ticagrelor levels pre-CPB were 103.5 ± 63.8 ng/mL compared with mean post-CPB levels of 34.0 ± 17.5 ng/mL, representing a significant 67.1% reduction (P

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-X L inhibitor that selectively and potently induces apoptosis in BCL-X L -dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-X L inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp 3 -rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-X L . A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program., Competing Interests: The authors declare the following competing financial interest(s): AbbVie and Genentech participated in interpretation of data and review and approval of publication. L.W., G.A.D., A.S.J., Z-F.T., T. M. H., R.R.F., X.S., M.B., A.R.K., X.W., M.D.W., N.D.C., S.S., D.C.P., R.A.J., P.N., M.L.S., S.K.T., Y.X., J.X., H.Z., P.N.L., M.J.M., E.R.B., W.G., P.K., S.W.E., J.D.L., and A.J.S. are employees of AbbVie, Inc. C.H.P. was an employee of AbbVie, Inc at the time of the study. E.C. and W.J.F. are employees of Genentech, Inc. J.A.F., D.D., and D.S. were employees of Genentech, Inc. at the time of the study.

The process development of a new synthetic route leading to an efficient and robust synthetic process for venetoclax (1: the active pharmaceutical ingredient (API) in Venclexta) is described. The redesigned synthesis features a Buchwald-Hartwig amination to construct the core ester 23c in a convergent fashion by connecting two key building blocks (4c and 26), which is then followed by a uniquely effective saponification reaction of 23c using anhydrous hydroxide generated in situ to obtain 2. Finally, the coupling of the penultimate core acid 2 with sulfonamide 3 furnishes drug substance 1 with consistently high quality. The challenges and solutions for the key Pd-catalyzed C-N cross-coupling will also be discussed in detail. The improved synthesis overcomes many of the initial scale-up challenges and was accomplished in 46% overall yield from 3,3-dimethyldicyclohexanone (6), more than doubling the overall yield of the first generation route. The new process was successfully implemented for producing large quantities of 1 with >99% area purity.

Recently, there has been an increasing focus on the pursuit of targets considered to be less druggable that offer potential for development of promising new therapeutic agents for the treatment of diseases with large unmet medical need, particularly in the areas of oncology and virology. However, conducting drug discovery campaigns in "beyond rule of 5" (bRo5) chemical space presents a significant drug design and development challenge to medicinal chemists to achieve acceptable oral pharmacokinetics. Retrospective analysis of past successes and failures in drug discovery bRo5 may shed light on the key principles that contribute to the oral bioavailability of successful bRo5 compounds and improve the efficiency of drug design for future projects. We present here highlights and case studies of lessons learned from discovery of bRo5 compounds. A simple multiparametric scoring function (AB-MPS) was devised that correlated preclinical PK results with cLogD, number of rotatable bonds, and number of aromatic rings.

Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, K i = 160 μM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.

Background: Hand and wrist injuries are one of the most common injuries seen in adults. The Patient-Rated Wrist Evaluation (PRWE) questionnaire has been developed as a patient-report outcome measure of pain and disability to evaluate the outcome after hand and wrist injuries., Objective: The aims of this study were (1) to evaluate the structural validity of the existing Dutch version of the PRWE (PRWE-NL) in patients with hand or wrist injuries and (2) to investigate the appropriateness of reporting subscale scores., Design: This was a retrospective analysis of cross-sectional data of 368 adult patients., Methods: Patients aged 18 to 65 years and treated either surgically or conservatively for an isolated hand or wrist injury were recruited. Patients were excluded if they were unable to speak or read Dutch. Confirmatory factor analyses were used to investigate structural validity, and Cronbach alpha (α) and omega (ω) coefficients were used to investigate internal consistency., Results: A series of confirmatory factor analyses revealed that all models (ie, a single-factor model, correlated 2- and 3-factor models, and 2 bifactor models) were associated with adequate model fit. However, inspection of the factor loadings, the explained common variance (ECV), and the different coefficient omega values revealed that the PRWE-NL should be considered a measure of a unidimensional trait. In addition, PRWE-NL subscales were associated with unacceptably low levels of reliability independently of the global PRWE-NL factor., Limitations: Although the sample size was adequate, the response rate was 37.1%. Participants were mainly patients with fractures of the wrist or hand, predominantly treated nonsurgically., Conclusion: This study suggests that the PRWE-NL measures a unidimensional trait. A single score should be used for the PRWE-NL, without subscale scores., (© 2016 American Physical Therapy Association.)

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics., (Copyright © 2015, American Association for the Advancement of Science.)

Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.

Overexpression of prosurvival proteins such as Bcl-2 and Bcl-X L has been correlated with tumorigenesis and resistance to chemotherapy, and thus, the development of antagonists of these proteins may provide a novel means for the treatment of cancer. We recently described the discovery of 1 (ABT-737), which binds Bcl-2, Bcl-X L, and Bcl-w with high affinity, shows robust antitumor activity in murine tumor xenograft models, but is not orally bioavailable. Herein, we report that targeted modifications at three key positions of 1 resulted in a 20-fold improvement in the pharmacokinetic/pharmacodynamic relationship (PK/PD) between oral exposure (AUC) and in vitro efficacy in human tumor cell lines (EC 50). The resulting compound, 2 (ABT-263), is orally efficacious in an established xenograft model of human small cell lung cancer, inducing complete tumor regressions in all animals. Compound 2 is currently in multiple phase 1 clinical trials in patients with small cell lung cancer and hematological malignancies.

Background: The discovery of ABT-263, a rationally designed Bcl-2/Bcl-xL inhibitor at present in Phase I clinical trials for cancer, is described. Emphasis is placed on the specific hurdles overcome throughout the discovery process that relate to the nature of the targeted protein-protein interaction (PPI)., Objective/methods: This review draws on observations from the experience of discovering ABT-263 and discusses them within the framework of the larger issue of discovering drugs targeting PPIs. Issues discussed include the 'hot spot' paradigm, hit and lead generation, serum protein binding, structure-based design, and in particular, hydrophobicity and molecular size and their relation to pharmacokinetic/pharmacodynamic properties., Results/conclusion: Approaches to understanding obstacles thought of as being specifically attached to PPIs, and existing techniques to combat these obstacles, were very helpful in overcoming them. The example of ABT-263 provides evidence that the larger family of PPI targets is more tractable than may have been thought.

The molecular chaperone HSP90 has been shown to facilitate cancer cell survival by stabilizing key proteins responsible for a malignant phenotype. We report here the results of parallel fragment-based drug design approaches in the design of novel HSP90 inhibitors. Initial aminopyrimidine leads were elaborated using high-throughput organic synthesis to yield nanomolar inhibitors of the enzyme. Second site leads were also identified which bound to HSP90 in two distinct conformations, an 'open' and 'closed' form. Intriguingly, linked fragment approaches targeting both of these conformations were successful in producing novel, micromolar inhibitors. Overall, this study shows that, with only a few fragment hits, multiple lead series can be generated for HSP90 due to the inherent flexibility of the active site. Thus, ample opportunities exist to use these lead series in the development of clinically useful HSP90 inhibitors for the treatment of cancers.

Survivin is one of the most tumor-specific genes in the human genome and is an attractive target for cancer therapy. However, small-molecule ligands for survivin have not yet been described. Thus, an interrogation of survivin which could potentially both validate a small-molecule therapy approach, and determine the biochemical nature of any of survivin's functions has not been possible. Here we describe the discovery and characterization of a small molecule binding site on the survivin surface distinct from the Smac peptide-binding site. The new site is located at the dimer interface and exhibits many of the features of highly druggable, biologically relevant protein binding sites. A variety of small hydrophobic compounds were found that bind with moderate affinity to this binding site, from which one lead was developed into a group of compounds with nanomolar affinity. Additionally, a subset of these compounds are adequately water-soluble and cell-permeable. Thus, the structural studies and small molecules described here provide tools that can be used to probe the biochemical role(s) of survivin, and may ultimately serve as a basis for the development of small molecule therapeutics acting via direct or allosteric disruption of binding events related to this poorly understood target.

Overexpression of the antiapototic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional chemotherapy. Inhibition of these prosurvival proteins is an attractive strategy for cancer therapy. We recently described the discovery of a selective Bcl-xL antagonist that potentiates the antitumor activity of chemotherapy and radiation. Here we describe the use of structure-guided design to exploit a deep hydrophobic binding pocket on the surface of these proteins to develop the first dual, subnanomolar inhibitors of Bcl-xL and Bcl-2. This study culminated in the identification of 2, which exhibited EC50 values of 8 nM and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Compound 2 demonstrated single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2, and efficacy in a murine xenograft model of lymphoma when given both as a single agent and in combination with etoposide.

Development of a rationally designed potentiator of cancer chemotherapy, via inhibition of Bcl-X(L) function, is described. Lead compounds generated by NMR screening and directed parallel synthesis displayed sub-microM binding but were strongly deactivated in the presence of serum. The dominant component of serum deactivation was identified as domain III of human serum albumin (HSA); NMR solution structures of inhibitors bound to both Bcl-X(L) and HSA domain III indicated two potential optimization sites for separation of affinities. Modifications at both sites resulted in compounds with improved Bcl-X(L) binding and greatly increased activity in the presence of human serum, culminating in 73R, which bound to Bcl-X(L) with a K(i) of 0.8 nM. In a cellular assay 73R reversed the protection afforded by Bcl-X(L) overexpression against cytokine deprivation in FL5.12 cells with an EC(50) of 0.47 microM. 73R showed little effect on the viability of the human non small cell lung cancer cell line A549. However, consistent with the proposed mechanism, 73R potentiated the activity of paclitaxel and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in a mouse xenograft model.

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

A series of non-amide-linked 6-substituted-2-naphthamidine urokinase plasminogen activator (uPA) inhibitors are described. These compounds possess excellent binding activities and selectivities with significantly improved pharmacokinetic profiles versus previously described amide-linked inhibitors.

Several 8-substituted 2-naphthamidine-based inhibitors of the serine protease urokinase plasminogen activator (uPA) are described. Direct attachment of five-membered saturated or unsaturated rings improved inhibitor performance; substitution with sulfones further improved binding profiles. Combination of these substituents or of previously described NH-linked heteroaromatic rings with 6-phenyl amide substituents provided further enhancements to potency and selectivity.

The preparation and assessment of biological activity of 6-substituted 2-naphthamidine inhibitors of the serine protease urokinase plasminogen activator (uPA, or urokinase) is described. 2-Naphthamidine was chosen as a starting point based on synthetic considerations and on modeling of substituent vectors. Phenyl amides at the 6-position were found to improve binding; replacement of the amide with other two-atom linkers proved ineffective. The phenyl group itself is situated near the S1' subsite; substitutions off of the phenyl group accessed S1' and other distant binding regions. Three new points of interaction were defined and explored through ring substitution. A solvent-exposed salt bridge with the Asp60A carboxylate was formed using a 4-alkylamino group, improving affinity to K(i) = 40 nM. Inhibitors also accessed two hydrophobic regions. One interaction is characterized by a tight hydrophobic fit made with a small dimple largely defined by His57 and His99; a weaker, less specific interaction involves alkyl groups reaching into the broad prime-side protein binding region near Val41 and the Cys42-Cys58 disulfide, displacing water molecules and leading to small gains in activity. Many inhibitors accessed two of these three regions. Affinities range as low as K(i) = 6 nM, and many compounds had K(i) < 100 nM, while moderate to excellent selectivity was gained versus four of five members of a panel of relevant serine proteases. Also, some selectivity against trypsin was generated via the interaction with Asp60A. X-ray structures of many of these compounds were used to inform our inhibitor design and to increase our understanding of key interactions. In combination with our exploration of 8-substitution patterns, we have identified a number of novel binding interactions for uPA inhibitors.

A highly stereoselective total synthesis of (-)-bafilomycin A(1), the naturally occurring enantiomer of this potent vacuolar ATPase inhibitor, is described. The synthesis features the highly stereoselective aldol reaction of methyl ketone 8b and aldehyde 60c and a Suzuki cross-coupling reaction of the highly functionalized advanced intermediates 12 and 39. Vinyl iodide 12 was synthesized by a 14-step sequence starting from the readily available beta-alkoxy aldehyde 14, while the vinylboronic acid component 39 was synthesized by a nine-step sequence from beta-hydroxy-alpha-methyl butyrate 44 via a sequence involving the alpha-methoxypropargylation of chiral aldehyde 49 with the alpha-methoxypropargylstannane reagent 54. Syntheses of fragments 12 and 39 also feature diastereoselective double asymmetric crotylboration reactions to set several of the critical stereocenters. The Suzuki cross-coupling of 12 and 39 provided seco ester 40, which following conversion to the seco acid underwent smooth macrolactonization to give 41. The success of the macrocyclization required that C(7)-OH be unprotected. The Mukaiyama aldol reaction between aldehyde 60c and the TMS enol ether generated from 8b provided aldol 65 with high diastereoselectivity. Finally, all silicon protecting groups were removed by treatment of the penultimate intermediate 65 with TAS-F (tris(dimethylamino)sulfonium difluorotrimethylsilicate), thereby completing the total synthesis of (-)-bafilomycin A(1).

We describe a simple 1H NMR analysis that permits the stereochemistry of beta-hydroxy ketones to be assigned by visual inspection of the ABX patterns for the alpha-methylene unit of the beta-hydroxy ketone in the 1H NMR spectra. This method has been verified by application to a wide range of beta-hydroxy ketones deriving from aldol reactions of chiral aldehydes with a variety of chiral and achiral methyl ketone enolates (see Tables 1 and 2). The stereochemistry of 54 of these compounds have been assigned by rigorous chemical methods.

Highly stereoselective syntheses of aldols 8a-c corresponding to the C(13)-C(25) segment of bafilomycin A(1) were developed by routes involving fragment assembly aldol reactions of chiral aldehyde 6a and the chiral methyl ketones 7. A remote chelation effect plays a critical role in determining the stereoselectivity of the key aldol coupling of 6a and the lithium enolate of 7b. The protecting group for C(23)-OH of the chiral aldehyde fragment also influences the selectivity of the lithium enolate aldol reaction. In contrast, the aldol reaction of 6a and the chlorotitanium enolates of 7a,c were much less sensitive to the nature of the C(15)-hydroxyl protecting group. Studies of the reactions of chiral aldehydes with Takai's (gamma-methoxyallyl)chromium reagent 40 are also described. The stereoselectivity of these reactions is also highly dependent on the protecting groups and stereochemistry of the chiral aldehyde substrates.

In search of a uroselective alpha1A subtype selective antagonist, a novel series of 6-OMe hexahydrobenz[e]isoindoles attached to a bicyclic heterocyclic moiety via a two-carbon linker was synthesized. It was found that in contrast to the previously described series of tricyclic heterocycles,(1) this bicyclic series has very specific requirements for the heterocyclic attachments. The most important structural features contributing to the alpha1A/alpha1B selectivity of these compounds were identified. In vitro functional assays for the alpha1 adrenoceptor subtypes were used to further characterize the most selective compounds, and in vivo models of vascular vs prostatic tone were used to assess uroselectivity. Compound 48 showed the highest degree of selectivity in the radioligand binding assays (56-fold), in the in vitro functional tests (80-fold), and for in vivo prostate selectivity (960-fold).

In search of a uroselective agent that exhibits a high level of selectivity for the alpha(1A) receptor, a novel series of tricyclic hexahydrobenz[e]isoindoles was synthesized. A generic pharmacophoric model was developed requiring the presence of a basic amine core and a fused heterocyclic side chain separated by an alkyl chain. It was shown that the 6-OMe substitution with R, R stereochemistry of the ring junction of the benz[e]isoindole and a two-carbon spacer chain were optimal. In contrast to the highly specific requirements for the benz[e]isoindole portion and linker chain, a wide variety of tricyclic fused heterocyclic attachments were tolerated with retention of potency and selectivity. In vitro functional assays for the alpha(1) adrenoceptor subtypes were used to further characterize these compounds, and in vivo models of vascular vs prostatic tone were used to assess uroselectivity.

A careful orchestration of protecting groups is an essential requirement for the total synthesis of the macrolide antibiotic bafilomycin A 1 (1). Key steps were the Suzuki cross-coupling reaction of two advanced, suitably protected intermediates prior to closure of the macrocycle, as well as a highly stereoselective methyl ketone aldol reaction., (© 1999 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)

Purpose: To better understand wound healing after glaucoma filtration surgery by measuring the production of type I and type III collagen in cultured Tenon's fibroblasts and determine the effect of ascorbic acid on collagen subtype production., Methods: An ELISA-type dot blot assay was used to directly measure the production of types I and III collagen by subconfluent cultures of fibroblasts from human Tenon's capsule. Because ascorbic acid is both high in aqueous humor and necessary for the production of collagen, we measured the dose response of type I and type III collagen production to ascorbic acid., Results: Ascorbic acid stimulated an increase in collagen production that reached a maximum level at 100 micrograms/ml. This is approximately half of the ascorbic acid concentration found in human aqueous humor. Unlike previous reports, we found no toxic effects from ascorbic acid at concentrations as high as 250 micrograms/ml over a 24-hour period. The lack of toxicity may result from the use of serum-free media in the assay., Conclusions: This culture system will be useful for exploring factors that may alter collagen production and could potentially affect wound healing.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) regulates the expression of c-myc protooncogene in HL-60 promyelocytic leukemia cells (Reitzma, P. H., Rothberg, P. G., Astrin, S. M., Trial, J., Barshavit, Z., Hall, A., Teitelbaum, S. U., and Kahn, A. J. (1983) Nature 306, 492-494). The regulation of c-myc expression occurs at least in part at the transcriptional level (Simpson, R. U., Hsu, T., Begley, D. A., Mitchell, B. S., and Alizadeh, B. N. (1987) J. Biol. Chem. 262, 4104-4108). Also, 1,25-(OH)2D3 stimulates an increase in protein kinase C (PKC) levels and inhibitors of PKC block 1,25-(OH)2D3-induced differentiation of HL-60 cells (Martell, R. E., Simpson, R. U., and Taylor, J. M. (1987) J. Biol. Chem. 262, 5570-5575). In this report we demonstrated that sphinganine, an inhibitor of PKC that is mechanistically and structurally distinct from 1-(5-isoquinoline sulfonyl)-2-methylpiperazine-HCl (H-7), also blocks 1,25-(OH)2D3 induction of HL-60 cell differentiation. The effect of inhibitors of PKC on 1,25-(OH)2D3 regulation of c-myc transcription was examined. H-7 (18 microM) and sphinganine (3 and 6 microM) blunted 1,25-(OH)2D3-induced reduction of c-myc transcription as assessed by nuclear run-off assays. We showed that c-myc/beta-actin ratios (cpm/cpm, % of control mean +/- S.E.) were as follows: ethanol control, 100 +/- 14%; 50 nM 1,25-(OH)2D3, 17 +/- 5%; 50 nM 1,25-(OH)2D3 and 6 microM H-7, 13 +/- 6%; 50 nM 1,25-(OH)2D3 and 18 microM H-7, 53 +/- 6% 50 nM 1,25-(OH)2D3 and 18 microM N-[2-guanidinoethyl]-5-isoquinoline sulfonamide (HA-1004), 10 +/- 8%; 50 nM 1,25-(OH)2D3 and 6 microM sphinganine, 49 +/- 8%. No significant differences in c-myc transcription between control, 18 microM H-7, 18 microM HA-1004, and 3 or 6 microM sphinganine-treated cells were observed. The block in c-myc transcription was beyond exon 1, and regulation of exon 1 transcription by 1,25-(OH)2D3 was not detected. Furthermore, we demonstrated that expression of markers for HL-60 cell differentiation was more rapidly induced by 25 nM 12-O-tetradecanoylphorbol-13-acetate than 50 nM 1,25-(OH)2D3, suggesting that direct activation of PKC by phorbol esters may make processes required for 1,25-(OH)2D3 induction of differentiation unnecessary. In summary, these data suggest that a primary effect of 1,25-(OH)2D3 on HL-60 cells is to regulate PKC levels, and regulation of c-myc transcription by 1,25-(OH)2D3 is a result of this action.