A calibration method for smartphone camera photophlethysmography.

Smartphone camera photoplethysmography (cPPG) enables non-invasive pulse oximetry and hemoglobin concentration measurements. However, the aesthetic-driven non-linearity in default image capture and preprocessing pipelines poses challenges for consistency and transferability of cPPG across devices. This work identifies two key parameters-tone mapping and sensor threshold-that significantly impact cPPG measurements. We propose a novel calibration method to linearize camera measurements, thus enhancing consistency and transferability of cPPG across devices. A benchtop calibration system is also presented, leveraging a microcontroller and LED setup to characterize these parameters for each phone model. Our validation studies demonstrate that, with appropriate calibration and camera settings, cPPG applications can achieve 74% higher accuracy than with default settings. Moreover, our calibration method proves effective across different smartphone models (N=4), and calibrations performed on one phone can be applied to other smartphones of the same model (N=6), enhancing consistency and scalability of cPPG applications.

Racially fair pupillometry measurements for RGB smartphone cameras using the far red spectrum.

Pupillometry is a measurement of pupil dilation commonly performed as part of neurological assessments. Prior work have demonstrated the potential for pupillometry in screening or diagnosing a number of neurological disorders including Alzheimer's Disease, Schizophrenia, and Traumatic Brain Injury. Unfortunately, the expense and inaccessibility of specialized pupilometers that image in the near infrared spectrum limit the measurement to high resource clinics or institutions. Ideally, this measurement could be available via ubiquitous devices like smartphones or tablets with integrated visible spectrum imaging systems. In the visible spectrum of RGB cameras, the melanin in the iris absorbs light such that it is difficult to distinguish the pupil aperature that appears black. In this paper, we propose a novel pupillometry technique to enable smartphone RGB cameras to effectively differentiate the pupil from the iris. The proposed system utilizes a 630 nm long-pass filter to image in the far red (630-700 nm) spectrum, where the melanin in the iris reflects light to appear brighter in constrast to the dark pupil. Using a convolutional neural network, the proposed system measures pupil diameter as it dynamically changes in a frame by frame video. Comparing across 4 different smartphone models, the pupil-iris contrast of N = 12 participants increases by an average of 451% with the proposed system. In a validation study of N = 11 participants comparing the relative pupil change in the proposed system to a Neuroptics PLR-3000 Pupillometer during a pupillary light response test, the prototype system acheived a mean absolute error of 2.4%.

Ultra-low-cost mechanical smartphone attachment for no-calibration blood pressure measurement.

We propose an ultra-low-cost at-home blood pressure monitor that leverages a plastic clip with a spring-loaded mechanism to enable a smartphone with a flash LED and camera to measure blood pressure. Our system, called BPClip, is based on the scientific premise of measuring oscillometry at the fingertip to measure blood pressure. To enable a smartphone to measure the pressure applied to the digital artery, a moveable pinhole projection moves closer to the camera as the user presses down on the clip with increased force. As a user presses on the device with increased force, the spring-loaded mechanism compresses. The size of the pinhole thus encodes the pressure applied to the finger. In conjunction, the brightness fluctuation of the pinhole projection correlates to the arterial pulse amplitude. By capturing the size and brightness of the pinhole projection with the built-in camera, the smartphone can measure a user's blood pressure with only a low-cost, plastic clip and an app. Unlike prior approaches, this system does not require a blood pressure cuff measurement for a user-specific calibration compared to pulse transit time and pulse wave analysis based blood pressure monitoring solutions. Our solution also does not require specialized smartphone models with custom sensors. Our early feasibility finding demonstrates that in a validation study with N = 29 participants with systolic blood pressures ranging from 88 to 157 mmHg, the BPClip system can achieve a mean absolute error of 8.72 and 5.49 for systolic and diastolic blood pressure, respectively. In an estimated cost projection study, we demonstrate that in small-batch manufacturing of 1000 units, the material cost is an estimated $0.80, suggesting that at full-scale production, our proposed BPClip concept can be produced at very low cost compared to existing cuff-based monitors for at-home blood pressure management.

Ultra-low-cost Mechanical Smartphone Attachment for No-Calibration Blood Pressure Measurement.

We propose BPClip, a less than $ 1 USD blood pressure monitor that leverages a plastic clip with a spring-loaded mechanism to enable any smartphone with a flash LED and a camera to measure blood pressure. Unlike prior approaches, our system measured systolic, mean, and diastolic blood pressure using oscillometric measurements that avoid cumbersome per-user calibrations and does not require specialized smartphone models with custom sensors.

At-Home Pupillometry using Smartphone Facial Identification Cameras.

With recent developments in medical and psychiatric research surrounding pupillary response, cheap and accessible pupillometers could enable medical benefits from early neurological disease detection to measurements of cognitive load. In this paper, we introduce a novel smartphone-based pupillometer to allow for future development in clinical research surrounding at-home pupil measurements. Our solution utilizes a NIR front-facing camera for facial recognition paired with the RGB selfie camera to perform tracking of absolute pupil dilation with sub-millimeter accuracy. In comparison to a gold standard pupillometer during a pupillary light reflex test, the smartphone-based system achieves a median MAE of 0.27mm for absolute pupil dilation tracking and a median error of 3.52% for pupil dilation change tracking. Additionally, we remotely deployed the system to older adults as part of a usability study that demonstrates promise for future smartphone deployments to remotely collect data in older, inexperienced adult users operating the system themselves.

Contemporary use of guideline-based higher potency P2Y12 receptor inhibitor therapy in patients with moderate-to-high risk non-ST-segment elevation myocardial infarction: Results from the Canadian ACS reflective II cross-sectional study.

BACKGROUND: After myocardial infarction, guidelines recommend higher-potency P2Y12 receptor inhibitors, namely ticagrelor and prasugrel, over clopidogrel. HYPOTHESIS: We aimed to determine the contemporary use of higher-potency antiplatelet therapy in Canadian patients with non-ST-elevation myocardial infarction (NSTEMI). METHODS: A total of 684 moderate-to-high risk NSTEMI patients were enrolled in the prospective Canadian ACS Reflective II registry at 12 Canadian hospitals and three clinics in five provinces between July 2016 and May 2018. Multivariable logistic regression modeling was performed to assess factors independently associated with higher-potency P2Y12 receptor inhibitor use at discharge. RESULTS: At hospital discharge, 78.3% of patients were treated with a P2Y12 receptor inhibitor. Among patients discharged on a P2Y12 receptor inhibitor, use of higher-potency P2Y12 receptor inhibitor was 61.4%. After adjustment, treatment in-hospital with PCI (OR 4.48, 95%CI 3.34-6.03, p < .0001) was most strongly associated with higher use of higher-potency P2Y12 receptor inhibitor, while oral anticoagulant use at discharge (OR 0.03, 95%CI 0.01-0.12, p < .0001), and atrial fibrillation (OR 0.40, 95%CI 0.17-0.98, p = .046) were most strongly associated with lower use of higher-potency P2Y12 receptor inhibitor. Use of higher-potency P2Y12 receptor inhibitor varied across provinces (range, 21.6%-78.9%). DISCUSSION: In contemporary Canadian practice, approximately 60% of moderate-to-high risk NSTEMI patients discharged on a P2Y12 receptor inhibitor are treated with a higher-potency P2Y12 receptor inhibitor. In addition to factors that increase risk of bleeding, interprovincial differences in practice patterns were associated with use of higher-potency P2Y12 receptor inhibitor at discharge. Opportunities remain for further optimization of evidence-based, guideline-recommended antiplatelet therapy use.

Recommendations for the content and management of Certificates of Analysis for reference standards from the GCC for bioanalysis.

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held in New Orleans, LA, USA on 5 April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysis for reference standards used in regulated bioanalysis. Similar challenges exist with critical reagents (e.g., capture and detection antibodies) used for assays supporting biologics. The recommendations provided in this publication are the minimum requirements for the content that GCC members believe should be included in Certificates of Analysis for reference standards obtained from commercial vendors, sponsors and compendial suppliers, for use in regulated bioanalytical studies. In addition, recommendations for internal standards, metabolites and critical reagents are discussed.

Provision of a DAPT Score to Cardiologists and Extension of Dual Antiplatelet Therapy Beyond 1 Year After ACS: Randomized Substudy of the Prospective Canadian ACS Reflective II Study.

BACKGROUND: Extension of dual antiplatelet therapy (DAPT) beyond 1 year after acute coronary syndrome is associated with a reduction in ischemic events but also increased bleeding. The DAPT score identifies individuals likely to derive overall benefit or harm from DAPT extension. We sought to evaluate the impact of providing the DAPT score to treating physicians on the decision to extend DAPT beyond 1 year after non-ST-segment elevation myocardial infarction. METHODS: Moderate to high-risk non-ST-segment elevation myocardial infarction patients were enrolled from July 2016 to May 2018 in 13 Canadian hospitals by 52 cardiologists. Participating cardiologists were randomly assigned 1:1 to receive their individual patients' DAPT scores before the 1-year follow-up visit vs not receiving their patients' DAPT scores. Rates of DAPT extension were compared among the randomized groups. RESULTS: At 1 year, 370 of the 585 (63.2%) patients discharged on DAPT were receiving DAPT. Among patients on DAPT at 1 year, the median (25th, 75th percentile) DAPT score was 2 (1,3). DAPT was extended beyond 1 year in 36.2% randomly assigned to provision of DAPT score vs 35.7% in the control group (P = 0.93). In the subgroup of patients with DAPT score ≥ 2, DAPT extension was 49.5% in the DAPT score provision arm vs 40.4% in the control arm (P = 0.22); among patients with DAPT score < 2, DAPT termination was 78.6% in the DAPT score provision arm vs 70.6% in the control arm (P = 0.26) (P value for interaction = 0.1). CONCLUSIONS: In this exploratory randomized trial, provision of the DAPT score to treating physicians had no impact on the duration of DAPT treatment beyond 1 year.

INTRODUCTION: La prolongation de la bithérapie antiplaquettaire au-delà d'un an après un syndrome coronarien aigu est associée à la réduction des accidents ischémiques, mais aussi à l'augmentation des hémorragies. Le score de bithérapie antiplaquettaire permet de déterminer les individus susceptibles d'obtenir des avantages globaux ou des inconvénients de la prolongation de la bithérapie antiplaquettaire. Nous avons cherché à évaluer les répercussions de l'obtention du score de bithérapie antiplaquettaire par les médecins traitants sur la décision quant à la prolongation de la bithérapie antiplaquettaire au-delà d'un an après l'infarctus du myocarde sans élévation du segment ST. MÉTHODES: De juillet 2016 à mai 2018, 52 cardiologues de 13 hôpitaux du Canada ont inscrit des patients exposés à un risque modéré à élevé d'infarctus du myocarde sans élévation du segment ST. Nous avons réparti de façon aléatoire selon un rapport 1:1 les cardiologues participants qui recevaient les scores de bithérapie antiplaquettaire individuels de leurs patients avant la consultation de suivi après un an vs ceux qui ne recevaient pas les scores de bithérapie antiplaquettaire de leurs patients. Nous avons comparé les taux de prolongation de la bithérapie antiplaquettaire des groupes répartis de façon aléatoire. RÉSULTATS: Après un an, 370 (63,2 %) patients sur 585 qui avaient eu à la sortie de l'hôpital une bithérapie antiplaquettaire recevaient la bithérapie antiplaquettaire. Parmi les patients qui prenaient la bithérapie antiplaquettaire après un an, le score médian de bithérapie antiplaquettaire (25e, 75e percentiles) était de 2 (1, 3). La bithérapie antiplaquettaire était prolongée au-delà d'un an chez 36,2 % des patients répartis de façon aléatoire qui avaient un score de bithérapie antiplaquettaire vs 35,7 % dans le groupe témoin (P = 0,93). Dans le sous-groupe de patients qui avaient un score de bithérapie antiplaquettaire ≥ 2, la prolongation de la bithérapie antiplaquettaire était de 49,5 % dans le bras qui avait un score de bithérapie antiplaquettaire vs 40,4 % dans le bras témoin (P = 0,22); parmi les patients qui avaient un score de bithérapie antiplaquettaire < 2, la cessation de la bithérapie antiplaquettaire était de 78,6 % dans le bras qui avait un score de bithérapie antiplaquettaire vs 70,6 % dans le bras témoin (P = 0,26) (valeur P pour l'interaction = 0,1). CONCLUSIONS: Dans cet essai exploratoire à répartition aléatoire, l'obtention du score de la bithérapie antiplaquettaire par les médecins traitants n'a pas engendré de répercussions sur la durée de la bithérapie antiplaquettaire au-delà d'un an.

12th GCC Closed Forum: critical reagents; oligonucleotides; CoA; method transfer; HRMS; flow cytometry; regulatory findings; stability and immunogenicity.

The 12th GCC Closed Forum was held in Philadelphia, PA, USA, on 9 April 2018. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: critical reagents; oligonucleotides; certificates of analysis; method transfer; high resolution mass spectrometry; flow cytometry; recent regulatory findings and case studies involving stability and nonclinical immunogenicity. Conclusions and consensus from discussions of these topics are included in this article.

Recommendations for classification of commercial LBA kits for biomarkers in drug development from the GCC for bioanalysis.

Over the last decade, the use of biomarker data has become integral to drug development. Biomarkers are not only utilized for internal decision-making by sponsors; they are increasingly utilized to make critical decisions for drug safety and efficacy. As the regulatory agencies are routinely making decisions based on biomarker data, there has been significant scrutiny on the validation of biomarker methods. Contract research organizations regularly use commercially available immunoassay kits to validate biomarker methods. However, adaptation of such kits in a regulated environment presents significant challenges and was one of the key topics discussed during the 12th Global Contract Research Organization Council for Bioanalysis (GCC) meeting. This White Paper reports the GCC members' opinion on the challenges facing the industry and the GCC recommendations on the classification of commercial kits that can be a win-win for commercial kit vendors and end users.

11th GCC Closed Forum: cumulative stability; matrix stability; immunogenicity assays; laboratory manuals; biosimilars; chiral methods; hybrid LBA/LCMS assays; fit-for-purpose validation; China Food and Drug Administration bioanalytical method validation.

The 11th Global CRO Council Closed Forum was held in Universal City, CA, USA on 3 April 2017. Representatives from international CRO members offering bioanalytical services were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The second CRO-Pharma Scientific Interchange Meeting was held on 7 April 2017, which included Pharma representatives' sharing perspectives on the topics discussed earlier in the week with the CRO members. The issues discussed at the meetings included cumulative stability evaluations, matrix stability evaluations, the 2016 US FDA Immunogenicity Guidance and recent and unexpected FDA Form 483s on immunogenicity assays, the bioanalytical laboratory's role in writing PK sample collection instructions, biosimilars, CRO perspectives on the use of chiral versus achiral methods, hybrid LBA/LCMS assays, applications of fit-for-purpose validation and, at the Global CRO Council Closed Forum only, the status and trend of current regulated bioanalytical practice in China under CFDA's new BMV policy. Conclusions from discussions of these topics at both meetings are included in this report.

Significant proteins affecting cerebral vasospasm using complementary ICPMS and MALDI-MS.

Cerebral vasospasm (CV) following subarachnoid hemorrhagic stroke affects more than one million people each year. The etiology and prevention of CV is currently of great interest to researchers in various fields of medical science. More recently, the idea that selenium could be playing a major role in the onset of cerebral vasospasm has come into the spotlight. This study focused on using newly established metallomics techniques in order to explore the proteome associated with CV and if selenium might affect the discovered proteins. Size exclusion chromatography coupled to inductively coupled plasma mass spectrometry, along with LC-MALDI-TOF/TOF were both essential in determining protein identifications in three different sample types; a control (normal, healthy patient, CSF control), SAH stroke patients (no vasospasm, CSF C) and SAH CV patients (CSF V). The results of this study, although preliminary, indicate the current methods are applicable and warrant further application to these clinically important targets.

Deciphering the human platelet sheddome.

Activated platelets shed surface proteins, potentially modifying platelet function as well as providing a source of bioactive fragments. Previous studies have identified several constituents of the platelet sheddome, but the full extent of shedding is unknown. Here we have taken a global approach, analyzing protein fragments in the supernate of activated platelets using mass spectroscopy and looking for proteins originating from platelet membranes. After removing plasma proteins and microparticles, 1048 proteins were identified, including 69 membrane proteins. Nearly all of the membrane proteins had been detected previously, but only 10 had been shown to be shed in platelets. The remaining 59 are candidates subject to confirmation. Based on spectral counts, protein representation in the sheddome varies considerably. As proof of principle, we validated one of the less frequently detected proteins, semaphorin 7A, which had not previously been identified in platelets. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with α-granules. Finally, cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function.

Stable isotope dilution multidimensional liquid chromatography-tandem mass spectrometry for pancreatic cancer serum biomarker discovery.

A novel approach to pancreatic cancer biomarker discovery has been developed, which employs a stable isotope labeled proteome (SILAP) standard coupled with extensive multidimensional separation coupled with tandem mass spectrometry (MS/MS). Secreted proteins from CAPAN-2 human pancreatic cancer derived cells were collected after conducting stable isotope labeling by amino acids in cell culture (SILAC). The resulting SILAP standard contained <0.5% of individual unlabeled proteins. Pooled sera from patients with early stage pancreatic cancer or controls were prepared, and an equal amount of the SILAP standard was added to each sample. Proteins were separated by isoelectric focusing (IEF) prior to two-dimensional liquid chromatography (2D-LC)-MS/MS analysis. A total of 1065 proteins were identified of which 121 proteins were present at 1.5-fold or greater concentrations in the sera of patients with pancreatic cancer. ELISA validation of these findings was successfully performed for two proteins, ICAM-1 and BCAM. Results of these studies have provided proof of principle that a SILAP standard derived from the CAPAN-2 secreted proteome can be used in combination with extensive multidimensional LC-MS/MS for the identification and relative quantitation of potential biomarkers of pancreatic cancer. This technique allows for the detection of low-abundance proteins, and focuses only on biologically relevant proteins derived from pancreatic cancer cells.

Duplex-promoted platination of adenine-N3 in the minor groove of DNA: challenging a longstanding bioinorganic paradigm.

The interactions of [Pt(en)Cl(ACRAMTU-S)](NO3)2 (PT-ACRAMTU, en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) with adenine in DNA have been studied using a combination of analytical and high-resolution structural methods. For the first time, a cytotoxic platinum(II) complex has been demonstrated to form adducts in the minor groove of DNA through platination of the adenine-N3 endocyclic nitrogen. An acidic depurination assay was developed that allowed the controlled and selective (pH 2, 60 degrees C, 12 h) release of platinum-modified adenine from drug-treated nucleic acid samples. From the digested mixtures, three adducts were isolated by semipreparative reverse phase high-performance liquid chromatography and studied by electrospray ionization mass spectrometry (in-line LC-MS), variable-pH 1H NMR spectroscopy, and, where applicable, X-ray crystallography. The three species were identified as the N7 (A-I), N3 (A-II), and N1 (A-III) linkage isomers of [Pt(en)(ACRAMTU-S)(adenine)]3+ (A). Incubations carried out with the single- and double-stranded model sequences, d(TA)5 and d(TA)15, as well as native DNA indicate that the adduct profiles (A-I:A-II:A-IIIratios) are sensitive to the nature of the nucleic acid template. A-II was found to be a double-strand specific adduct. The crystal structure of this adduct has been determined, providing ultimate evidence for the N3 connectivity of platinum. A-II crystallizes in the triclinic space group P in the form of centrosymmetric dimers, {[Pt(en)(ACRAMTU-S)(adenine-N3)]2}6+. The cations are stabilized by a combination of adenine-adenine base pairing (N6...N1 2.945(5) A) and mutual acridine-adenine base stacking. Tandem mass spectra and 1H chemical shift anomalies indicate that this type of self-association is not merely a crystal packing effect but persists in solution. The monofunctional platination of adenine at its N7, N3, and N1 positions in a significant fraction of adducts breaks a longstanding paradigm in platinum-DNA chemistry, the requirement for nucleophilic attack of guanine-N7 as the principal step in cross-link formation. The biological consequences and potential therapeutic applications of the unique base and groove recognition of PT-ACRAMTU are discussed.

Platinum-intercalator conjugates: from DNA-targeted cisplatin derivatives to adenine binding complexes as potential modulators of gene regulation.

Nuclear DNA is the cellular target for many cancer treatments, and DNA-directed chemotherapies continue to play an important role in drug discovery in the postgenomic era. The majority of DNA-targeted anticancer agents bind through covalent interactions, non-covalent intercalation or groove binding, or hybrid binding modes. The sequence and regiospecificity of these interactions and the resulting structural alterations within the biopolymer play an important role in the mechanism of action of these drugs. DNA-binding proteins and/or DNA-processing enzymes, which also interact with DNA in a sequence- and groove-specific manner, are mediators of the cytotoxic effect produced by these agents. Thus one major goal in the design of new clinical agents of this type is to produce new types of adducts on DNA, which may lead to unprecedented cell kill mechanisms. Platinum-intercalator conjugates are such a class of hybrid agents acting through a dual DNA binding mode. The platinum center (usually a cis-diaminedichloroPt(II) unit) dominates the DNA adduct profiles in the majority of these species-the result of the metal's tendency to form cross-links in runs of consecutive guanine bases in the major groove of DNA. This paradigm has been broken recently for the first time with the design of cytotoxic platinum-acridinylthiourea conjugates, a class of adenine-affinic minor-groove directed agents. This review summarizes major advancements in the chemistry and biology of platinum-intercalators from 1984 to 2004, with emphasis being placed on the interplay between chemical structure, mechanism of DNA binding, and biological properties.

Structure-activity relationships in platinum-acridinylthiourea conjugates: effect of the thiourea nonleaving group on drug stability, nucleobase affinity, and in vitro cytotoxicity.

The synthesis, cytotoxicity, and nucleoside binding of some platinum-acridinylthiourea conjugates derived from the prototypical compound [PtCl(en)(ACRAMTU)](NO3)2 ("PT-ACRAMTU"; en=ethane-1,2-diamine, ACRAMTU=1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, protonated form) are reported. To establish structure-activity relationships within this class of compounds, systematic changes were made to the thiourea nonleaving group, which links the intercalator to platinum. Three new derivatives of ACRAMTU, one di-, one tri-, and one tetraalkylated, were generated, where the degree of alkylation indicates the number of alkyl groups attached to the SCN2 framework. Subsequent reaction of the tri- and tetraalkylated derivatives with activated [PtCl2(en)] yielded the corresponding platinum conjugates. The dialkylated thiourea gave an unstable complex, which was not included in the studies. The crystal structure of PT-ACRAMTU x MeOH has been determined. In the solid state, one axial position of the square-planar platinum coordination sphere is partially shielded by the bulky thiourea group, providing a strong rationale for the kinetic inertness of the compound. The cytotoxicity of the prototype, the two new conjugates, and cisplatin was assessed in ovarian (A2780, A2780/CP), lung (NCI-H460), and colon (RKO) cancer cell lines using clonogenic survival assays. The derivatives containing trialkylated thiourea groups showed activity similar or superior to cisplatin, with IC50 values in the low micromolar concentration range. The complex modified with the tetraalkylated (bulkiest) thiourea was significantly less active, possibly due to the greatly decreased rate of binding to nucleobase nitrogen (1H NMR spectroscopy), but was most efficient at overcoming cross resistance to cisplatin in A2780/CP. Possible consequences of the reported structural modifications for the mechanism of action of these agents are discussed.

Unprecedented monofunctional metalation of adenine nucleobase in guanine- and thymine-containing dinucleotide sequences by a cytotoxic platinum-acridine hybrid agent.

We have investigated the reactions of [PtCl(en)(ACRAMTU-S)](NO(3))(2) (2) (en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation, 1), the prototype of a new class of cytotoxic DNA-targeted agents, with 2'-deoxyguanosine (dGuo) and random-sequence native DNA by in-line liquid chromatography/mass spectrometry (LC/MS) and NMR spectroscopy ((1)H, (195)Pt) to identify the covalent adducts formed by platinum. In the mononucleoside model system, two adducts are observed, [Pt(en)(ACRAMTU)(dGuo)](3+) (P1, major) and [Pt(en)(dGuo)(2)](2+) (P2, minor). The reaction, which proceeds significantly slower (half-life 11-12 h at 37 degrees C, pH 6.5) than analogous reactions with cisplatin and reactions of 2 with double-stranded DNA, results in the unexpected displacement of the sulfur-bound acridine ligand in approximately 15% of the adducts. This reactivity is not observed in double-stranded DNA, rendering 1 a typical nonleaving group in reactions with this potential biological target. In enzymatic digests of calf thymus DNA treated with 2, three adducts were identified: [Pt(en)(ACRAMTU)(dGuo)](3+) (A1, approximately 80%), [Pt(en)(ACRAMTU)[d(GpA)]](2+) (A2, approximately 12%), and [Pt(en)(ACRAMTU)[d(TpA)]](2+) (A3, approximately 8%). A1 and P1 proved to be identical species. In the dinucleotide adducts A2 and A3, complex 2 covalently modifies adenine at GA and TA base steps, which are high-affinity intercalation sites of the acridine derivative 1. A2 and A3, which may be formed in the minor groove of DNA, are the first examples of monofunctional adenine adducts of divalent platinum formed in double-stranded DNA. The analysis of the adduct profile indicates that the sequence specificity of 1 plays an important role in the molecular recognition between DNA and the corresponding conjugate, 2. Possible biological consequences of the unusual adduct profile are discussed.

Thermally inert metal ammines as light-inducible DNA-targeted agents. Synthesis, photochemistry, and photobiology of a prototypical rhodium(III)-intercalator conjugate.

The recent discovery of the promising tumor cell kill by a novel platinum-acridine conjugate [Martins, E. T.; et al. J. Med. Chem. 2001, 44, 4492] has prompted us to explore the utility of analogous light-activatable rhodium(III) compounds as photocytotoxic agents. Here, the design and synthesis of [Rh(NH(3))(5)L](n)(+) complexes are described with L = 1,1,3,3-tetramethylthiourea (tmtu) or 1-[2-(acridin-9-ylamino)ethyl]-1,3,3-trimethylthiourea (2). The intercalator-based DNA-affinic carrier ligand 2 was synthesized from N-acridin-9-yl-N'-methylethane-1,2-diamine and dimethylthiocarbamoyl chloride and isolated as the hydrotriflate salt 2(CF(3)SO(3)). [Rh(NH(3))(5)(tmtu)](3+) (1) and [Rh(NH(3))(5)(2)](4+) (3) were obtained from the reactions of the trifluoromethanesulfonato complex [Rh(NH(3))(5)(OSO(2)CF(3))](CF(3)SO(3))(2) with the appropriate thiourea in noncoordinating solvents. All compounds were characterized by (1)H NMR and UV-vis spectroscopies and by elemental analyses. The single-crystal X-ray structures of 1(CF(3)SO(3))(3) x 2MeOH, 2(CF(3)SO(3)), and 3(CF(3)SO(3))(4) x H(2)O have been determined. Ligand-field photolysis of thermally inert 1 (lambda(max) = 378 nm) resulted in the aquation of 2 equiv of ammine ligand without noticeable release of sulfur-bound tmtu ((1)H NMR spectroscopy, NH(3)-sensitive electrode measurements). This was confirmed by (15)N[(1)H] NMR spectroscopy using (15)N-labeled [Rh((15)NH(3))(5)(tmtu)](3+) (1), which also indicated photoisomerization of the [RhN(5)S] moiety. Despite greatly accelerated ligand exchange, rhodium in 1 and 3 did not show light-enhanced formation of covalent adducts in calf thymus DNA. "Dark binding" levels of 3 in native DNA were slightly higher than for nontargeted 1, but significantly lower than those observed for analogous platinum-acridine. Agarose gel electrophoresis revealed photocleavage of supercoiled pUC19 plasmid DNA in the presence of hybrid 3 and its individual constituents 1 and 2. Simple 1 induced single-strand breaks while 3 produced complete degradation of the DNA after 24 h of continuous irradiation. Acridine 2 alone produced double-strand breaks. The extent of DNA damage observed for 1-3 correlates with the photocytotoxicity of the compounds in human leukemia cells, suggesting that DNA might be the cellular target of these agents.