High-Speed Optical Characterization of Protein-and-Nanoparticle-Stabilized Microbubbles for Ultrasound-Triggered Drug Release.

OBJECTIVE: Ultrasound-triggered bubble-mediated local drug delivery has shown potential to increase therapeutic efficacy and reduce systemic side effects, by loading drugs into the microbubble shell and triggering delivery of the payload on demand using ultrasound. Understanding the behavior of the microbubbles in response to ultrasound is crucial for efficient and controlled release. METHODS: In this work, the response of microbubbles with a coating consisting of poly(2-ethyl-butyl cyanoacrylate) (PEBCA) nanoparticles and denatured casein was characterized. High-speed recordings were taken of single microbubbles, in both bright field and fluorescence. RESULTS: The nanoparticle-loaded microbubbles show resonance behavior, but with a large variation in response, revealing a substantial interbubble variation in mechanical shell properties. The probability of shell rupture and the probability of nanoparticle release were found to strongly depend on microbubble size, and the most effective size was inversely proportional to the driving frequency. The probabilities of both rupture and release increased with increasing driving pressure amplitude. Rupture of the microbubble shell occurred after fewer cycles of ultrasound as the driving pressure amplitude or driving frequency was increased. CONCLUSION: The results highlight the importance of careful selection of the driving frequency, driving pressure amplitude and duration of ultrasound to achieve the most efficient ultrasound-triggered shell rupture and nanoparticle release of protein-and-nanoparticle-stabilized microbubbles.

Progression of monoclonal gammopathy of undetermined significance to multiple myeloma is associated with enhanced translational quality control and overall loss of surface antigens.

BACKGROUND: Despite significant advancements in treatment strategies, multiple myeloma remains incurable. Additionally, there is a distinct lack of reliable biomarkers that can guide initial treatment decisions and help determine suitable replacement or adjuvant therapies when relapse ensues due to acquired drug resistance. METHODS: To define specific proteins and pathways involved in the progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM), we have applied super-SILAC quantitative proteomic analysis to CD138 + plasma cells from 9 individuals with MGUS and 37 with MM. RESULTS: Unsupervised hierarchical clustering defined three groups: MGUS, MM, and MM with an MGUS-like proteome profile (ML) that may represent a group that has recently transformed to MM. Statistical analysis identified 866 differentially expressed proteins between MM and MGUS, and 189 between MM and ML, 177 of which were common between MGUS and ML. Progression from MGUS to MM is accompanied by upregulated EIF2 signaling, DNA repair, and proteins involved in translational quality control, whereas integrin- and actin cytoskeletal signaling and cell surface markers are downregulated. CONCLUSION: Compared to the premalignant plasma cells in MGUS, malignant MM cells apparently have mobilized several pathways that collectively contribute to ensure translational fidelity and to avoid proteotoxic stress, especially in the ER. The overall reduced expression of immunoglobulins and surface antigens contribute to this and may additionally mediate evasion from recognition by the immune apparatus. Our analyses identified a range of novel biomarkers with potential prognostic and therapeutic value, which will undergo further evaluation to determine their clinical significance.

Ultrasound and Microbubbles Increase the Uptake of Platinum in Murine Orthotopic Pancreatic Tumors.

OBJECTIVE: Currently available cytotoxic treatments have limited effect on pancreatic ductal adenocarcinoma (PDAC) because desmoplastic stroma limits drug delivery. Efforts have been made to overcome these barriers by drug targeting the tumor microenvironment. Results so far are promising, but without clinical impact. Our aim was to investigate whether ultrasound and microbubbles could improve the uptake and therapeutic response of conventional chemotherapy. METHODS: Orthotopic pancreatic tumors growing in mice were treated with commercially available FOLFIRINOX (fluorouracil, irinotecan, oxaliplatin and calcium folinate) and SonoVue microbubbles combined with focused ultrasound. Tumor uptake of platinum (Pt) was measured by inductively coupled plasma mass spectroscopy (ICP-MS), and tumor volumes were measured by ultrasound imaging. DISCUSSION: Uptake of Pt, the active ingredient of oxaliplatin, was significantly increased after ultrasound treatment of orthotopic PDAC tumors. Multiple injections with FOLFIRONOX increased the amount of Pt in tumors. However, the enhanced accumulation did not improve therapeutic response. Increased uptake of Pt confirms that ultrasound and microbubbles have potential in clinical practice with existing drugs. CONCLUSION: The lack of therapeutic response, despite increased uptake in tumor tissue, emphasizes the importance of studying how to overcome stromal barriers.

A multi-pulse ultrasound technique for imaging of thick-shelled microbubbles demonstrated in vitro and in vivo.

Contrast enhanced ultrasound is a powerful diagnostic tool and ultrasound contrast media are based on microbubbles (MBs). The use of MBs in drug delivery applications and molecular imaging is a relatively new field of research which has gained significant interest during the last decade. MBs available for clinical use are fragile with short circulation half-lives due to the use of a thin encapsulating shell for stabilization of the gas core. Thick-shelled MBs can have improved circulation half-lives, incorporate larger amounts of drugs for enhanced drug delivery or facilitate targeting for use in molecular ultrasound imaging. However, methods for robust imaging of thick-shelled MBs are currently not available. We propose a simple multi-pulse imaging technique which is able to visualize thick-shelled polymeric MBs with a superior contrast-to-tissue ratio (CTR) compared to commercially available harmonic techniques. The method is implemented on a high-end ultrasound scanner and in-vitro imaging in a tissue mimicking flow phantom results in a CTR of up to 23 dB. A proof-of-concept study of molecular ultrasound imaging in a soft tissue inflammation model in rabbit is then presented where the new imaging technique showed an enhanced accumulation of targeted MBs in the inflamed tissue region compared to non-targeted MBs and a mean CTR of 13.3 dB for stationary MBs. The presence of fluorescently labelled MBs was verified by confocal microscopy imaging of tissue sections post-mortem.

Formulation and characterisation of drug-loaded antibubbles for image-guided and ultrasound-triggered drug delivery.

The aim of this study was to develop high load-capacity antibubbles that can be visualized using diagnostic ultrasound and the encapsulated drug can be released and delivered using clinically translatable ultrasound. The antibubbles were developed by optimising a silica nanoparticle stabilised double emulsion template. We produced an emulsion with a mean size diameter of 4.23 ± 1.63 µm where 38.9 ± 3.1% of the droplets contained a one or more cores. Following conversion to antibubbles, the mean size decreased to 2.96 ± 1.94 µm where 99% of antibubbles were <10 µm. The antibubbles had a peak attenuation of 4.8 dB/cm at 3.0 MHz at a concentration of 200 × 103 particles/mL and showed distinct attenuation spikes at frequencies between 5.5 and 13.5 MHz. No increase in subharmonic response was observed for the antibubbles in contrast to SonoVue®. High-speed imaging revealed that antibubbles can release their cores at MIs of 0.6. In vivo imaging indicated that the antibubbles have a long half-life of 68.49 s vs. 40.02 s for SonoVue®. The antibubbles could be visualised using diagnostic ultrasound and could be disrupted at MIs of ≥0.6. The in vitro drug delivery results showed that antibubbles can significantly improve drug delivery (p < 0.0001) and deliver the drug within the antibubbles. In conclusion antibubbles are a viable concept for ultrasound guided drug delivery.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Sonopermeation Enhances Uptake and Therapeutic Effect of Free and Encapsulated Cabazitaxel.

Delivery of drugs and nanomedicines to tumors is often heterogeneous and insufficient and, thus, of limited efficacy. Microbubbles in combination with ultrasound have been found to improve delivery to tumors, enhancing accumulation and penetration. We used a subcutaneous prostate cancer xenograft model in mice to investigate the effect of free and nanoparticle-encapsulated cabazitaxel in combination with ultrasound and microbubbles with a lipid shell or a shell of nanoparticles. Sonopermeation reduced tumor growth and prolonged survival (26%-100%), whether the free drug was co-injected with lipid-shelled microbubbles or the nanoformulation was co-injected with lipid-shelled or nanoparticle-shelled microbubbles. Coherently with the improved therapeutic response, we found enhanced uptake of nanoparticles directly after ultrasound treatment that lasted several weeks (2.3 × -15.8 × increase). Neither cavitation dose nor total accumulation of nanoparticles could explain the variation within treatment groups, emphasizing the need for a better understanding of the tumor biology and mechanisms involved in ultrasound-mediated treatment.

Ultrasound-Mediated Delivery of Chemotherapy into the Transgenic Adenocarcinoma of the Mouse Prostate Model.

Ultrasound (US) in combination with microbubbles (MB) has had promising results in improving delivery of chemotherapeutic agents. However, most studies are done in immunodeficient mice with xenografted tumors. We used two phenotypes of the spontaneous transgenic adenocarcinoma of the mouse prostate (TRAMP) model to evaluate if US + MB could enhance the therapeutic efficacy of cabazitaxel (Cab). Cab was either injected intravenously as free drug or encapsulated into nanoparticles. In both cases, Cab transiently reduced tumor and prostate volume in the TRAMP model. No additional therapeutic efficacy was observed combining Cab with US + MB, except for one tumor. Additionally, histology grading and immunostaining of Ki67 did not reveal differences between treatment groups. Mass spectrometry revealed that nanoparticle encapsulation of Cab increased the circulation time and enhanced the accumulation in liver and spleen compared with free Cab. The therapeutic results in this spontaneous, clinically relevant tumor model differ from the improved therapeutic response observed in xenografts combining US + MB and chemotherapy.

Ultrasound-mediated delivery enhances therapeutic efficacy of MMP sensitive liposomes.

To improve therapeutic efficacy of nanocarrier drug delivery systems, it is essential to improve their uptake and penetration in tumour tissue, enhance cellular uptake and ensure efficient drug release at the tumour site. Here we introduce a tumour targeting drug delivery system based on the ultrasound-mediated delivery of enzyme sensitive liposomes. These enzyme sensitive liposomes are coated with cleavable poly(ethylene glycol) (PEG) which will be cleaved by two members of the enzyme matrix metalloproteinase family (MMP-2 and MMP-9). Cleavage of the PEG coat can increase cellular uptake and will destabilize the liposomal membrane which can result in accelerated drug release. The main aim of the work was to study the effect of focused ultrasound and microbubbles on the delivery and therapeutic efficacy of the MMP sensitive liposome. The performance of the MMP sensitive liposome was compared to a non-MMP sensitive version and Doxil-like liposomes. In vitro, the cellular uptake and cytotoxicity of the liposomes were studied, while in vivo the effect of ultrasound and microbubbles on the tumour accumulation, biodistribution, microdistribution, and therapeutic efficacy were investigated. For all tested liposomes, ultrasound and microbubble treatment resulted in an improved tumour accumulation, increased extravasation, and increased penetration of the liposomes from blood vessels into the extracellular matrix. Surprisingly, penetration depth was independent of the ultrasound intensity used. Ultrasound-mediated delivery of free doxorubicin and the Doxil-like and MMP sensitive liposome resulted in a significant reduction in tumour volume 28 days post the first treatment and increased median survival. The MMP sensitive liposome showed better therapeutic efficacy than the non-MMP sensitive version indicating that cleaving the PEG-layer is important. However, the Doxil-like liposome outcompeted the MMP and non-MMP sensitive liposome, both with and without the use of ultrasound and microbubbles.

Effect of Ultrasound on the Vasculature and Extravasation of Nanoscale Particles Imaged in Real Time.

Ultrasound and microbubbles have been found to improve the delivery of drugs and nanoparticles to tumor tissue. To obtain new knowledge on the influence of vascular parameters on extravasation and to elucidate the effect of acoustic pressure on extravasation and penetration of nanoscale particles into the extracellular matrix, real-time intravital multiphoton microscopy was performed during sonication of tumors growing in dorsal window chambers. The impact of vessel diameter, vessel structure and blood flow was characterized. Fluorescein isothiocyanate-dextran (2 MDa) was injected to visualize blood vessels. Mechanical indexes (MI) of 0.2-0.8 and in-house-made, nanoparticle-stabilized microbubbles or Sonovue were applied. The rate and extent of penetration into the extracellular matrix increased with increasing MI. However, to achieve extravasation, smaller vessels required MIs (0.8) higher than those of blood vessels with larger diameters. Ultrasound changed the blood flow rate and direction. Interestingly, the majority of extravasations occurred at vessel branching points.

Ultrasound-mediated delivery and distribution of polymeric nanoparticles in the normal brain parenchyma of a metastatic brain tumour model.

The treatment of brain diseases is hindered by the blood-brain barrier (BBB) preventing most drugs from entering the brain. Focused ultrasound (FUS) with microbubbles can open the BBB safely and reversibly. Systemic drug injection might induce toxicity, but encapsulation into nanoparticles reduces accumulation in normal tissue. Here we used a novel platform based on poly(2-ethyl-butyl cyanoacrylate) nanoparticle-stabilized microbubbles to permeabilize the BBB in a melanoma brain metastasis model. With a dual-frequency ultrasound transducer generating FUS at 1.1 MHz and 7.8 MHz, we opened the BBB using nanoparticle-microbubbles and low-frequency FUS, and applied high-frequency FUS to generate acoustic radiation force and push nanoparticles through the extracellular matrix. Using confocal microscopy and image analysis, we quantified nanoparticle extravasation and distribution in the brain parenchyma. We also evaluated haemorrhage, as well as the expression of P-glycoprotein, a key BBB component. FUS and microbubbles distributed nanoparticles in the brain parenchyma, and the distribution depended on the extent of BBB opening. The results from acoustic radiation force were not conclusive, but in a few animals some effect could be detected. P-glycoprotein was not significantly altered immediately after sonication. In summary, FUS with our nanoparticle-stabilized microbubbles can achieve accumulation and displacement of nanoparticles in the brain parenchyma.

Ultrasound Improves the Delivery and Therapeutic Effect of Nanoparticle-Stabilized Microbubbles in Breast Cancer Xenografts.

Compared with conventional chemotherapy, encapsulation of drugs in nanoparticles can improve efficacy and reduce toxicity. However, delivery of nanoparticles is often insufficient and heterogeneous because of various biological barriers and uneven tumor perfusion. We investigated a unique multifunctional drug delivery system consisting of microbubbles stabilized by polymeric nanoparticles (NPMBs), enabling ultrasound-mediated drug delivery. The aim was to examine mechanisms of ultrasound-mediated delivery and to determine if increased tumor uptake had a therapeutic benefit. Cellular uptake and toxicity, circulation and biodistribution were characterized. After intravenous injection of NPMBs into mice, tumors were treated with ultrasound of various pressures and pulse lengths, and distribution of nanoparticles was imaged on tumor sections. No effects of low pressures were observed, whereas complete bubble destruction at higher pressures improved tumor uptake 2.3 times, without tissue damage. An enhanced therapeutic effect was illustrated in a promising proof-of-concept study, in which all tumors exhibited regression into complete remission.

Nanoparticle delivery to the brain--By focused ultrasound and self-assembled nanoparticle-stabilized microbubbles.

The blood-brain barrier (BBB) constitutes a significant obstacle for the delivery of drugs into the central nervous system (CNS). Nanoparticles have been able to partly overcome this obstacle and can thus improve drug delivery across the BBB. Furthermore, focused ultrasound in combination with gas filled microbubbles has opened the BBB in a temporospatial manner in animal models, thus facilitating drug delivery across the BBB. In the current study we combine these two approaches in our quest to develop a novel, generic method for drug delivery across the BBB and into the CNS. Nanoparticles were synthesized using the polymer poly(butyl cyanoacrylate) (PBCA), and such nanoparticles have been reported to cross the BBB to some extent. Together with proteins, these nanoparticles self-assemble into microbubbles. Using these novel microbubbles in combination with focused ultrasound, we successfully and safely opened the BBB transiently in healthy rats. Furthermore, we also demonstrated that the nanoparticles could cross the BBB and deliver a model drug into the CNS.

Nanoparticle-stabilized microbubbles for multimodal imaging and drug delivery.

Microbubbles (MBs) are routinely used as contrast agents for ultrasound imaging. The use of ultrasound in combination with MBs has also attracted attention as a method to enhance drug delivery. We have developed a technology platform incorporating multiple functionalities, including imaging and therapy in a single system consisting of MBs stabilized by polyethylene glycol (PEG)-coated polymeric nanoparticles (NPs). The NPs, containing lipophilic drugs and/or contrast agents, are composed of the widely used poly(butyl cyanoacrylate) (PBCA) polymer and prepared in a single step. MBs stabilized by these NPs are subsequently prepared by self-assembly of NPs at the MB air-liquid interface. Here we show that these MBs can act as contrast agents for conventional ultrasound imaging. Successful encapsulation of iron oxide NPs inside the PBCA NPs is demonstrated, potentially enabling the NP-MBs to be used as magnetic resonance imaging (MRI) and/or molecular ultrasound imaging contrast agents. By precise tuning of the applied ultrasound pulse, the MBs burst and the NPs constituting the shell are released. This could result in increased local deposit of NPs into target tissue, providing improved therapy and imaging contrast compared with freely distributed NPs.

Ultrasound-enhanced drug delivery in prostate cancer xenografts by nanoparticles stabilizing microbubbles.

The delivery of nanoparticles to solid tumors is often ineffective due to the lack of specificity towards tumor tissue, limited transportation of the nanoparticles across the vascular wall and poor penetration through the extracellular matrix of the tumor. Ultrasound is a promising tool that can potentially improve several of the transportation steps, and the interaction between sound waves and microbubbles generates biological effects that can be beneficial for the successful delivery of nanocarriers and their contents. In this study, a novel platform consisting of nanoparticle-stabilized microbubbles has been investigated for its potential for ultrasound-enhanced delivery to tumor xenografts. Confocal laser scanning microscopy was used to study the supply of nanoparticles from the vasculature and to evaluate the effect of different ultrasound parameters at a microscopic level. The results demonstrated that although the delivery is heterogeneous within tumors, there is a significant improvement in the delivery and the microscopic distribution of both nanoparticles and a released model drug when the nanoparticles are combined with microbubbles and ultrasound. The mechanisms that underlie the improved delivery are discussed.

Acoustic backing in 3-D integration of CMUT with front-end electronics.

Capacitive micromachined ultrasonic transducers (CMUTs) have shown promising qualities for medical imaging. However, there are still some problems to be investigated, and some challenges to overcome. Acoustic backing is necessary to prevent SAWs excited in the surface of the silicon substrate from affecting the transmit pattern from the array. In addition, echoes resulting from bulk waves in the substrate must be removed. There is growing interest in integrating electronic circuits to do some of the beamforming directly below the transducer array. This may be easier to achieve for CMUTs than for traditional piezoelectric transducers. We will present simulations showing that the thickness of the silicon substrate and thicknesses and acoustic properties of the bonding material must be considered, especially when designing highfrequency transducers. Through simulations, we compare the acoustic properties of 3-D stacks bonded with three different bonding techniques; solid-liquid interdiffusion (SLID) bonding, direct fusion bonding, and anisotropic conductive adhesives (ACA). We look at a CMUT array with a center frequency of 30 MHz and three silicon wafers underneath, having a total silicon thickness of 100 µm. We find that fusion bonding is most beneficial if we want to prevent surface waves from damaging the array response, but SLID and ACA are also promising if bonding layer thicknesses can be reduced.

Successful treatment of relapsed lymphomatoid granulomatosis with bexarotene.

Lymphomatoid granulomatosis is a rare lymphoproliferative disorder involving the lungs, skin and other organs. Advanced-stage disease does not tend to respond well to cytotoxic chemotherapy and is associated with a poor prognosis. We present a case of successful treatment of relapsed lymphomatoid granulomatosis with bexarotene, a novel retinoid agent.

Automated analysis of strain rate and strain: feasibility and clinical implications.

BACKGROUND: This study evaluated 3 new automated methods, based on a combination of speckle tracking and tissue Doppler, for the analysis of strain rate (SR) and strain. Feasibility and values for peak systolic strain rate (SR s ) and end-systolic strain (S es ) were assessed. METHODS: Thirty patients with myocardial infarction and 30 normal subjects were examined. Customized software with automatic definition of segments was used for automated measurements. SR s and SR es were measured over each segment simultaneously and identified automatically. The study compared tissue Doppler-based SR and strain measurements without (method 1) and with segment tracking (method 2) to speckle tracking-based measurements (method 3). For tracking, speckle tracking and tissue Doppler were used in combination. Standard manual analysis was used as a reference. RESULTS: The automated analysis (16 segments, 3 apical views) required 2 minutes; manual analysis took 11 minutes. Accuracy was compared in 56 segments (28 mid-infarcted and 28 normal) from 28 patients and was 93.9% for method 1, 93.8% for method 2, 95.8% for method 3, and 96.2% for the manual method. In the normal group, mean SR s (0.27 s -1 ) was less with method 3 than with the other methods ( P < .001). CONCLUSIONS: Our findings indicate that automated analysis of SR and strain, with some manual adjustment, is feasible and quicker than manual analysis. Diagnostic accuracy was similar with all methods. SR s was lower in the speckle tracking-based method than in the Doppler-based methods.

Effects of losartan or atenolol in hypertensive patients without clinically evident vascular disease: a substudy of the LIFE randomized trial.

BACKGROUND: Cardiovascular morbidity and mortality are reduced by treatment with the angiotensin II AT(1)-receptor antagonist losartan compared with conventional treatment with the beta-blocker atenolol in patients with hypertension and electrocardiogram-defined left ventricular hypertrophy, many of whom had known vascular disease. OBJECTIVE: To determine whether losartan reduces cardiovascular event rates in lower-risk hypertensive patients without clinically evident vascular disease. DESIGN: Subgroup analysis of a randomized trial. SETTING: The Losartan Intervention for Endpoint reduction in hypertension (LIFE) study. PATIENTS: 6886 men and women (57% women) 55 to 80 years of age (average, 66 years) with essential hypertension (sitting blood pressure, 160 to 200/95 to 115 mm Hg [average, 174/98 mm Hg]) and electrocardiogram-defined left ventricular hypertrophy who did not have clinically evident vascular disease. INTERVENTION: Patients were randomly assigned to once-daily double-blind treatment with losartan or atenolol. MEASUREMENTS: An end point committee ascertained end points (cardiovascular death, stroke, or myocardial infarction). RESULTS: Blood pressure was reduced similarly by losartan and atenolol. The primary composite end point occurred in 282 losartan-treated patients (17.5 per 1000 patient-years) and 355 atenolol-treated patients (21.8 per 1000 patient-years; relative risk, 0.81 [95% CI, 0.69 to 0.95]; P = 0.008). Cardiovascular death occurred in 103 losartan-treated patients and 132 atenolol-treated patients (relative risk, 0.80 [CI, 0.62 to 1.04]; P = 0.092), stroke (nonfatal and fatal) occurred in 125 losartan-treated patients and 193 atenolol-treated patients (relative risk, 0.66 [CI, 0.53 to 0.82]; P < 0.001), and myocardial infarction (nonfatal and fatal) occurred in 110 losartan-treated patients and 100 atenolol-treated patients (relative risk, 1.14 [CI, 0.87 to 1.49]; P > 0.2). New-onset diabetes occurred less often in patients treated with losartan (n = 173) than in patients treated with atenolol (n = 254) (relative risk, 0.69 [CI, 0.57 to 0.84]; P < 0.001). Benefits of losartan treatment were numerically smaller, but not significantly so, in patients with preexisting vascular disease. CONCLUSION: In hypertensive patients without clinically evident vascular disease, losartan was more effective than atenolol in preventing cardiovascular morbidity and death, predominantly stroke, independent of blood pressure reduction.