Drug Cocktail Research Articles

Facile construction of bioreducible crosslinked polypeptide micelles for enhanced cancer combination therapy.

We have synthesized a biodegradable polypeptide block copolymer to construct a facile pH and redox-responsive polymeric micelle asan advanced therapeutic system for cancer therapy. We have designed a corona-crosslinked triblock copolymer (poly (ethylene glycol)-b-poly(aspartic acid)-b-poly(tyrosine) (PEG-b-pAsp-b-pTyr)) micelles co-loaded with docetaxel and lonidamine (cl-M/DL). The corona of triblock polymer was crosslinked to maintain its structural integrity in the physiological environment. The mitochondrial targeting LND is expected to generate ROS, oxidative stress and thereby synergize the chemotherapeutic efficacy of DTX in killing cancer cells. Consistently, cl-M/DL exhibited excellent antitumor efficacy in xenograft tumor model with remarkable tumor regression. Overall, we demonstrated the construction of bioreducible nanosystem for the effective synergistic delivery of DTX/LND in tumor tissues towards cancer treatment.

Prognosis and treatment of FOLFOX therapy related interstitial pneumonia: a plea for multimodal immune modulating therapy in the respiratory insufficient patient

BackgroundThe FOLFOX regimen, i.e., folinic acid (FOL), fluorouracil (F) and oxaliplatin (OX), is a drug cocktail that is used to treat gastric and colorectal cancers. Despite the concomitant improvements in response rate, duration of response and patient survival, reports of serious toxic pulmonary side effects have progressively emerged.Case presentationWe describe a patient who was treated with FOLFOX as an adjuvant to a rectosigmoidal resection of a rectosigmoidal carcinoma and who developed respiratory insufficiency requiring mechanical ventilation. Computed tomography (CT) imaging and open lung biopsy findings were compatible with interstitial pneumonia (IP). She received multimodal combination treatment (acetylcysteine, corticosteroids, immune globulins and cyclophosphamide) and survived.We performed a systematic literature search and reviewed all 45 reported cases of FOLFOX-related lung toxicity and/or pulmonary fibrosis for their clinical characteristics and their outcomes related to therapy.ConclusionsWe found that for the 45 cases with available data, the median age was 70 years, and the male–female ratio was 3.5: 1. In the patients exhibiting only mild respiratory symptoms, discontinuation of the culprit drug (oxaliplatin) resulted in a 100% regression of the symptoms. However the prognosis of the respiratory insufficient patient proved to be grim: death occurred in 76.9% of the cases despite conventional treatment with corticosteroids. We therefore urge oncologists and critical care specialists not to limit their interventions to the discontinuation of chemotherapy, artificial ventilation, corticosteroids and glutathione replenishment and to consider the gradual introduction of additional immune-modulating agents whenever life-threatening respiratory symptoms in oxaliplatin-treated patients do not subside; all the more so considering the fact that our analysis showed that every patient who survived intubation and mechanical ventilation experienced a full clinical recovery.

British Society for Gene and Cell Therapy Annual Conference and Joint UK Regenerative Medicine Platform Meeting Royal Welsh College of Music & Drama Cardiff, Wales, United Kingdom Wednesday April 19-Friday April 21, 2017 Conference Abstracts.

British Society for Gene and Cell Therapy Annual Conference and Joint UK Regenerative Medicine Platform Meeting Royal Welsh College of Music & Drama Cardiff, Wales, United Kingdom Wednesday April 19-Friday April 21, 2017 Conference Abstracts.

Physiologically-based modelling in mice suggests an aggravated loss of clearance capacity after toxic liver damage

Diseases and toxins may lead to death of active liver tissue, resulting in a loss of total clearance capacity at the whole-body level. However, it remains difficult to study, whether the loss of metabolizing tissue is sufficient to explain loss of metabolic capacity of the liver or whether the surviving tissue undergoes an adaptive response to compensate the loss. To understand the cellular impact of toxic liver damage in an in vivo situation, we here used physiologically-based pharmacokinetic modelling to investigate pharmacokinetics of a specifically designed drug cocktail at three different sampling sites of the body in healthy mice and mice treated with carbon tetrachloride (CCl4). Liver zonation was explicitly quantified in the models through immunostaining of cytochrome P450s enzymes. Comparative analyses between the simulated decrease in clearance capacity and the experimentally measured loss in tissue volume indicated that CCl4-induced impairment of metabolic functions goes beyond the mere loss of metabolically active tissue. The here established integrative modelling strategy hence provides mechanistic insights into functional consequences of toxic liver damage in an in vivo situation, which would not have been accessible by conventional methods.

Effects of Metformin and Furosemide on Rosuvastatin Pharmacokinetics in Healthy Volunteers: Implications for Their Use as Probe Drugs in a Transporter Cocktail.

BackgroundIn a recently described probe drug cocktail for clinically relevant drug transporters containing digoxin, furosemide, metformin and rosuvastatin, mutual interactions were essentially absent except for increases in the systemic exposure of rosuvastatin. To optimize the cocktail, we further examined the dose dependence of the effects of metformin and furosemide on rosuvastatin pharmacokinetics.MethodsThis was a randomized, open label, single center, six-treatment, six-period, six-sequence crossover trial. Eighteen healthy male subjects received 10 mg rosuvastatin as reference treatment and, as test treatments, 10 mg rosuvastatin combined with 10, 50 or 500 mg metformin (T1, T2 and T3) or with 1 or 5 mg furosemide (T4 and T5). Primary pharmacokinetic endpoints were rosuvastatin C max (maximum plasma concentration) and AUC0–tz (area under the plasma concentration–time curve from time zero to the last quantifiable concentration).ResultsThe relative bioavailability of rosuvastatin was essentially unchanged when administered with metformin in T1 and T2, but in T3 it increased to 152% for AUC0–tz (90% CI 135–171%) and 154% for C max (90% CI 132–180%). Coadministration with furosemide did not change rosuvastatin relative bioavailability in T4, but in T5 it increased slightly to 116% for AUC0–tz (90% CI 102–132%) and 118% for C max (90% CI 98–142%).ConclusionThe increased systemic exposure of rosuvastatin when administered as part of the proposed transporter cocktail is most likely attributable to metformin and only to a minor degree to furosemide. Reduction of the doses of metformin and furosemide is expected to eliminate the previously described interaction.EudraCT no. 2015-003052-46, ClinicalTrials.gov identifier NCT02574845.

Abstract 2073: A systematic investigation of the effect of scheduling of targeted combination therapies on response and dynamics of relapse in triple negative breast cancer cells

Abstract Cancer treatment typically involves administration of combination of targeted therapies, but initial response is often followed by disease relapse. The efficacy of a treatment regimen depends on the complex interplay between cancer growth dynamics, drug specificity and kinetics, treatment dose and its scheduling. In standard high-throughput drug screening assays, cells are treated with a single drug cocktail bolus and cell viability is assessed after 2-3 days, thus not considering treatment interactions and long-term effects. Recently we have reported a promising synergistic combination of crizotinib (ALK/MET inhibitor) and ABT-263 (BCL2/BCL-XL inhibitor) against triple negative breast cancer cells. To understand the effect of the sequence and combination doses of crizotnib and ABT-263, we designed a comprehensive experimental plan that involved a total of 567 treatment regimens by varying treatment duration with the first drug (1, 2, or 3 days), followed by drug withdrawal and recovery period (2, 5 or 10 days) and then by a second cycle of treatment and recovery periods over a 26-day period. Cell viability was assessed by the CellTiter-Glo luminescence assay. Interestingly, ABT-263 alone induced higher cytotoxicity than an equivalent dose of crizotinib, but the remaining viable cells recovered much faster after ABT-263 withdrawal than cells after crizotinib withdrawal. Furthermore, cells exposed to higher doses of ABT-263 eventually become less sensitive to crizotinib. Among sequential regimens, crizotinib followed by ABT-263 was significantly more effective than ABT-263 followed by crizotinib, and combinations that included lower doses of ABT-263 were most effective. Taken together, our results show a significant interaction between the two targeted therapies, and suggest that it may be possible to select treatment scheduling that can delay drug resistance and tumor relapse in vivo. Note: This abstract was not presented at the meeting. Citation Format: Gauri A. Patwardhan, Vikram B. Wali, Lajos Pusztai, Christos Hatzis. A systematic investigation of the effect of scheduling of targeted combination therapies on response and dynamics of relapse in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2073. doi:10.1158/1538-7445.AM2017-2073

Low Dose of Doxorubicin Potentiates the Effect of Temozolomide in Glioblastoma Cells

Glioblastoma (GBM) is an aggressive brain tumor with temozolomide (TMZ)-based chemotherapy as the main therapeutic strategy. Doxorubicin (DOX) is not used in gliomas due to its low bioavailability in the brain; however, new delivery strategies and low doses may be effective in the long term, especially as part of a drug cocktail. Our aim was to evaluate the chronic effects of low doses of DOX and TMZ in GBM. Human U87-ATCC cells and a primary GBM culture were chronically treated with TMZ (5μM) and DOX (1 and 10nM) alone or combined. DOX resulted in a reduction in the number of cells over a period of 35days and delayed the cell regrowth. In addition, DOX induced cell senescence and reduced tumor sphere formation and the proportion of NANOG- and OCT4-positive cells after 7days. Low doses of TMZ potentiated the effects of DOX on senescence and sphere formation. This combined response using low doses of DOX may pave the way for its use in glioma therapy, with new technologies to overcome its low blood-brain barrier permeability.

Folate-targeted polymersomes loaded with both paclitaxel and doxorubicin for the combination chemotherapy of hepatocellular carcinoma.

Combination chemotherapy is a promising method of improving cancer treatment, but the distinct pharmacokinetics of combined drugs and non-specific drug distribution slow down the development in the clinic. In our study, novel folate-targeted co-delivery polymersomes (Co-PS) were successfully developed to encapsulate a hydrophobic-hydrophilic chemotherapeutic drug pair (paclitaxel and doxorubicin) into the different compartments of the vesicle. In vivo studies revealed that the combination chemotherapy of polymersomes to BEL-7404 xenograft tumor via intravenous injection was superior to free drug cocktail treatment, and the FA-targeted Co-PS exhibited significantly higher tumor growth inhibition than non-targeted Co-PS group. Therefore, the newly developed FA-targeted co-delivery polymersomes hold great promise for simultaneous delivery of multiple chemotherapeutics and would have great potential in tumor-targeting and combination chemotherapy.

In vivo Phenotyping Methods: Cytochrome P450 Probes with Emphasis on the Cocktail Approach.

Differences in drug response among patients are common. Most major drugs are effective in only 25 to 60 percent of the patients, in part due to the CYP enzymes, whose activity vary up to 50-fold between individuals for some index metabolic reactions. Several factors affect CYP activity, among which genetic polymorphisms have been studied as the major cause for long time. Age, gender, disease states, and environmental influences such as smoking, concomitant drug treatment or exposure to environmental chemicals are also important. This article reviews the available literature on multiple phenotypes assessment as an important tool to predict possible therapeutic failures or toxic reactions to conventional drug doses during patient evaluation. Probe drugs can be used in various combinations allowing for the in vivo assessment of multiple pathways of drug metabolism in a single experiment, configuring a new tool known as phenotyping "cocktails". There are several drug cocktails with different advantages and disadvantages. Most of them have sufficient clinical evidence and data validation to support their use in clinical setting as a surrogate for the risk of adverse reaction in the course of therapy, leading to a better balance between efficacy and safety. Probes characteristics and metabolic ratio measurements are important in the evaluation of phenotyping cocktails as near-future applications.

Investigation of Drug Cocktail Effects on Cancer Cell-Spheroids Using a Microfluidic Drug-Screening Assay

Development of drugs based on potential anti-cancer chemotherapeutic agents has been hindered by its necessary tedious procedures and failure in the clinical trials because of unbearable toxicity and extremely low clinical efficacy. One of the technical challenges is the mismatch between laboratory settings and human body environments for the cancer cells responding upon treatments of the anti-cancer agents. This major limitation urges for applying more reliable platforms for evaluating drugs with a higher throughput and cell aggregates in a more natural configuration. Here, we adopt a microfluidic device integrated with a differential micromixer and multiple microwell-containing channels (50 microwells per channel) for parallel screening of suspending cell spheroids treated by drugs with different combinations. We optimize the culture conditions of the surfactant-coated microwells in order to facilitate the spheroid formation of the breast cancer cell line (MDA-MB-231). We propose a new drug cocktail combined with three known chemotherapeutic agents (paclitaxel, epirubicin, and aspirin) for the drug screening of the cancer cell-spheroids. Our results exhibit the differential responses between planar cell layers in traditional culture wells and cell-spheroids grown in our microfluidic device, in terms of the apoptotic rates under treatments of the drug cocktails with different concentrations. These results reveal a distinct drug resistance between planar cell layers and cell-spheroids. Together, this work offers important guidelines on applying the cell-spheroid microfluidic cultures for development of more efficacious anticancer drugs.

Automatic optimization of drug cocktails on an integrated microfluidic system.

Drug cocktails have been popular for a variety of therapies of complicated diseases. Nevertheless, it is a tediously challenging task to optimize formulations, especially using traditional methods. Hence, an automatic system capable of precise dispensing multiple drugs is of great need. Herein, a new integrated microfluidic system combined with a two-axis traverse module was developed to dispense and mix a small amount of drug combination precisely and automatically. This on-chip dispensing process could be performed with a precise and accurate manner when compared to the manual operations. The efficacy of both single and multiple drugs could be examined through the developed microfluidic system with extremely low variation of drug formulations. Analysis of cell viabilities for normal and tumor cells was also performed to verify potential drug combinations. It is envisioned that this automatic system, which is flexible to combine with standard cell analysis methods and novel drug formulation algorithm, could provide precise and high-throughput drug cocktail formulations and expedite the drug screening processes.

An Introduction to Terminology and Methodology of Chemical Synergy-Perspectives from Across Disciplines.

The idea of synergistic interactions between drugs and chemicals has been an important issue in the biomedical world for over a century. As complex diseases, especially cancer, are being treated with various drug cocktails, understanding the interactions among these drugs is increasingly vital to ensuring successful treatment regimens. However, the idea of synergy is not limited to only the biomedical realm and these ideas have developed across many different disciplines, as well. In this review, we first discuss the various terminology surrounding the idea of synergy, providing a comprehensive list of terms defined across numerous disciplines. We then review the most common methodology for detection and quantification of synergy, including the two most prominent reference models for describing additive interactions: Loewe Additivity and Bliss Independence. We also discuss advantages and limitations to each method, with a focus on the Chou-Talalay Combination Index method. Finally, we describe how methods development and terminology have developed among disciplines outside of biomedicine and pharmacology, to synthesize the literature for readers.

High-value hi-tech product introduction in emerging countries

PurposeThis paper aims to study how multinational enterprises (MNEs) can best integrate legitimacy concerns into their new product-launching strategy to successfully introduce high-value hi-tech innovations in emerging countries.Design/methodology/approachTheoretical constructs on the role and process of legitimacy construction for the introduction of a new product are built upon the existing literature. Then they are validated and refined through the formulation and analysis of case studies of the launch of genetically modified cotton seeds by Monsanto in India and a HIV/AIDS drug cocktail by Merck in Brazil.FindingsLegitimacy construction can serve MNEs to face challenges successfully while launching high-value hi-tech products in emerging countries. Challenges to MNEs are likely to be founded on a combination of four types of uncertainties: technological, commercial, organizational and societal. Expected challengers are public agencies and actors representing civil society. An MNE can prepare itself through legitimacy construction along three dimensions: redesign of technology, revision of marketing strategy and non-market investments. To implement the aforesaid, MNEs can engage in outreach in the form of strategic patience, market transaction, business collaboration, compromise and/or confrontation with diverse carefully chosen stakeholders.Research limitations/implicationsThe authors limited ourselves to tracing only the formal interactions of MNEs, while it is well-known that many informal and backdoor activities can also accompany their growth in emerging economies.Practical implicationsLegitimacy construction can help MNEs face challenges successfully while launching high-value hi-tech products in emerging countries. This calls for an evaluation of the systemic uncertainties followed by the formulation of a strategy for legitimacy construction and implementation through outreach to diverse systemic actors. Strategic patience can yield positive returns. Market transactions can serve as economic anchors. Collaboration can be pursued with parties who can share the costs of legitimization construction and/or reduce technological and marketing uncertainties. Confrontation should be the last choice. Compromise is the most probable but not the only outreach strategy possible after a confrontation.Social implicationsLegitimacy implies product acceptance not only from the targeted consumer but also other societal stakeholders concerned with the safety and equity of the consumption in the emerging country, especially when regulations are not well-defined and/or implemented. The two kinds of societal stakeholders which are likely to monitor MNEs are public agencies and civil society groups. Public agencies will be concerned about the quantity, quality, technology or price of the innovation to be introduced. Civil society and NGOs may help the MNE act as citizen watchdogs for the environment and vulnerable communities.Originality/valueTheoretical constructs have been developed in this paper on the sources of challenges in new product introduction, the types of challengers and the components of the firm’s legitimacy construction strategy and its implementation through an outreach strategy.

Translational learning from clinical studies predicts drug pharmacokinetics across patient populations

Early indication of late-stage failure of novel candidate drugs could be facilitated by continuous integration, assessment, and transfer of knowledge acquired along pharmaceutical development programs. We here present a translational systems pharmacology workflow that combines drug cocktail probing in a specifically designed clinical study, physiologically based pharmacokinetic modeling, and Bayesian statistics to identify and transfer (patho-)physiological and drug-specific knowledge across distinct patient populations. Our work builds on two clinical investigations, one with 103 healthy volunteers and one with 79 diseased patients from which we systematically derived physiological information from pharmacokinetic data for a reference probe drug (midazolam) at the single-patient level. Taking into account the acquired knowledge describing (patho-)physiological alterations in the patient cohort allowed the successful prediction of the population pharmacokinetics of a second, candidate probe drug (torsemide) in the patient population. In addition, we identified significant relations of the acquired physiological processes to patient metadata from liver biopsies. The presented prototypical systems pharmacology approach is a proof of concept for model-based translation across different stages of pharmaceutical development programs. Applied consistently, it has the potential to systematically improve predictivity of pharmacokinetic simulations by incorporating the results of clinical trials and translating them to subsequent studies.

Chemically Modified Human Serum Albumin Potently Blocks Entry of Ebola Pseudoviruses and Viruslike Particles.

Ebola virus (EBOV), the causative pathogen of the deadly Ebola virus disease (EVD), can be transmitted via contact with EVD patients, including sexual contact with EVD survivors. At present, no licensed vaccine or therapeutic is available. In this study, we compared eight anhydride-modified proteins for their entry-inhibitory activity against the pseudovirus (PsV) carrying the envelope glycoprotein (GP) of the EBOV Zaire or Sudan species (Zaire PsV and Sudan PsV, respectively). We found that 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) was the most effective in inhibiting the entry of both Zaire PsV and Sudan PsV, with the 50% effective concentration being at the nanomolar level and with HP-HSA being more potent than EBOV-neutralizing antibody MIL77-2 (4G7, a component antibody of the ZMapp drug cocktail). The combination of HP-HSA and MIL77-2 exhibited a synergistic effect. HP-HSA had no obvious in vitro or in vivo toxicity. The EBOV PsV entry-inhibitory activity of HP-HSA remained intact after storage at 45°C for 8 weeks, suggesting that HP-HSA has the potential for worldwide use, including tropical regions in African countries, as either a therapeutic to treat EBOV infection or a prophylactic microbicide to prevent the sexual transmission of EBOV.

Effects of MicroRNA-34a on the Pharmacokinetics of Cytochrome P450 Probe Drugs in Mice.

MicroRNAs (miRNAs or miRs), including miR-34a, have been shown to regulate nuclear receptor, drug-metabolizing enzyme, and transporter gene expression in various cell model systems. However, to what degree miRNAs affect pharmacokinetics (PK) at the systemic level remains unknown. In addition, miR-34a replacement therapy represents a new cancer treatment strategy, although it is unknown whether miR-34a therapeutic agents could elicit any drug-drug interactions. To address this question, we refined a practical single-mouse PK approach and investigated the effects of a bioengineered miR-34a agent on the PK of several cytochrome P450 probe drugs (midazolam, dextromethorphan, phenacetin, diclofenac, and chlorzoxazone) administered as a cocktail. This approach involves manual serial blood microsampling from a single mouse and requires a sensitive liquid chromatography-tandem mass spectrometry assay, which was able to illustrate the sharp changes in midazolam PK by ketoconazole and pregnenolone 16α-carbonitrile as well as phenacetin PK by α-naphthoflavone and 3-methylcholanthrene. Surprisingly, 3-methylcholanthrene also decreased systemic exposure to midazolam, whereas both pregnenolone 16α-carbonitrile and 3-methylcholanthrene largely reduced the exposure to dextromethorphan, diclofenac, and chlorzoxazone. Finally, the biologic miR-34a agent had no significant effects on the PK of cocktail drugs but caused a marginal (45%-48%) increase in systemic exposure to midazolam, phenacetin, and dextromethorphan in mice. In vitro validation of these data suggested that miR-34a slightly attenuated intrinsic clearance of dextromethorphan. These findings from single-mouse PK and corresponding mouse liver microsome models suggest that miR-34a might have minor or no effects on the PK of coadministered cytochrome P450-metabolized drugs.

Visualization of mycobacterial membrane dynamics in live cells.

Mycobacteria are endowed with a highly impermeable mycomembrane that confers intrinsic resistance to many antibiotics. Several unique mycomembrane glycolipids have been isolated and structurally characterized, but the underlying organization and dynamics of glycolipids within the cell envelope remain poorly understood. We report here a study of mycomembrane dynamics that was enabled by trehalose–fluorophore conjugates capable of labeling trehalose glycolipids in live actinomycetes. We identified fluorescein–trehalose analogues that are metabolically incorporated into the trehalose mycolates of representative Mycobacterium, Corynebacterium, Nocardia, and Rhodococcus species. Using these probes, we studied the mobilities of labeled glycolipids by time-lapse microscopy and fluorescence recovery after photobleaching experiments and found that mycomembrane fluidity varies widely across species and correlates with mycolic acid structure. Finally, we discovered that treatment of mycobacteria with ethambutol, a front-line tuberculosis (TB) drug, significantly increases mycomembrane fluidity. These findings enhance our understanding of mycobacterial cell envelope structure and dynamics and have implications for development of TB drug cocktails.

Predicting transporter-mediated drug interactions: Commentary on: "Pharmacokinetic evaluation of a drug transporter cocktail consisting of digoxin, furosemide, metformin and rosuvastatin" and "Validation of a microdose probe drug cocktail for clinical drug interaction assessments for drug transporters and CYP3A".

Transporters, expressed in various tissues, govern the absorption, distribution, metabolism, and excretion of drugs, and consequently their inherent safety and efficacy profiles. Drugs may interact with a transporter as a substrate and/or an inhibitor. Understanding transporter-mediated drug-drug interactions (DDIs), in addition to enzyme-mediated DDIs, is an integral part of risk assessment in drug development and regulatory review because the concomitant use of more than one medication in patients is common.

Cocktail of chemical compounds robustly promoting cell reprogramming protects liver against acute injury

Tissue damage induces cells into reprogramming-like cellular state, which contributes to tissue regeneration. However, whether factors promoting the cell reprogramming favor tissue regeneration remains elusive. Here we identified combination of small chemical compounds including drug cocktails robustly promoting in vitro cell reprogramming. We then administrated the drug cocktails to mice with acute liver injuries induced by partial hepatectomy or toxic treatment. Our results demonstrated that the drug cocktails which promoted cell reprogramming in vitro improved liver regeneration and hepatic function in vivo after acute injuries. The underlying mechanism could be that expression of pluripotent genes activated after injury is further upregulated by drug cocktails. Thus our study offers proof-of-concept evidence that cocktail of clinical compounds improving cell reprogramming favors tissue recovery after acute damages, which is an attractive strategy for regenerative purpose.

Characteristics of Ivabradine-Sensitive Currents in Mouse Sinoatrial Node Myocytes

Cardiac pacemaking is driven by spontaneous action potentials (APs) in sinoatrial node myocytes (SAMs). The funny current (If) is thought play a role in the generation of pacemaker activity in SAMs, but the degree to which it contributes is incompletely understood. In this study, we used AP clamp experiments and the If blocker, ivabradine, to investigate the role of If in pacemaking. Spontaneous APs were recorded from acutely-isolated SAMs from mice, and ∼10 s of the AP recording was incorporated into a voltage clamp command. This AP clamp protocol elicited both inward and outward ivabradine-sensitive currents in SAMs. If was defined as the ivabradine-sensitive current remaining in the presence of a pharmacological cocktail of sodium, calcium and potassium channel blockers. Measurable inward ivabradine-sensitive current was evident during diastole in 57% of all cells and was generally larger in cells with the most negative diastolic voltages. Although we found that 30 μM ivabradine also blocked both L- and T-type calcium currents in SAMs, the majority of the ivabradine-sensitive inward current in the AP clamp experiments appeared to be If because control experiments showed that the drug cocktail blocked ∼85% of the calcium currents. Ivabradine also blocked a substantial outward current during the AP upstroke and repolarization phases. This outward ivabradine-sensitive current likely includes a contribution of If, which results from its relatively slow deactivation. However some of the outward ivabradine-sensitive current may also reflect off-target block of potassium currents by ivabradine. We are currently assaying the contribution of potassium currents to the outward ivabradine-sensitive current. Our results suggest that If is active not only during diastole, but also throughout the AP upstroke and repolarization phases of the mouse SAM AP.