A model-based approach for assessing in vivo combination therapy interactions

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach compares in vivo tumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.

p53-independent apoptosis induced by paclitaxel through an indirect mechanism

The efficacy of chemotherapeutic agents may be determined by a number of different factors, including the genotype of the tumor cell. The p53 tumor suppressor gene frequently is mutated in human tumors, and this may contribute to chemotherapeutic resistance. We tested the requirement for wild-type p53 in the response of tumor cells to treatment with paclitaxel (trade name Taxol), an antineoplastic agent that stabilizes cellular microtubules. Although paclitaxel is broadly effective against human tumor xenografts in mice, including some known to carry p53 mutations, we found that p53-containing mouse tumor cells were significantly more sensitive to direct treatment with this drug than were p53-deficient tumor cells. In an attempt to reconcile this apparent discrepancy, we examined the requirement for p53 in the cytotoxic effects of tumor necrosis factor α (TNF-α), a cytokine released from murine macrophages upon paclitaxel treatment. Conditioned medium from paclitaxel-treated macrophages was capable of inducing p53-independent apoptosis when applied to transformed mouse embryonic fibroblasts and was inhibitable by antibodies against TNF-α. Furthermore, in response to direct treatment with TNF-α, both wild-type and p53-deficient tumor cells underwent apoptosis to similar extents and with similar kinetics. Our results suggest that the efficacy of paclitaxel in vivo may be due not only to its microtubule-stabilizing activity, but its ability to activate local release of an apoptosis-inducing cytokine.

The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid

The γ-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (α1–6, β1–3, γ1–3, δ1, and ɛ1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the β subunit subtype present within the receptor. Receptors containing β2- or β3-, but not β1 subunits, are highly sensitive to the agent. Here, chimeric β1/β2 subunits coexpressed in Xenopus laevis oocytes with human α6 and γ2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the β3 subunit to Ser (the homologous residue in β1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the β1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk−) cells stably expressing the α6β3γ2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.

Diagnostic ultrasound activation of contrast agent gas bodies induces capillary rupture in mice

Interaction of diagnostic ultrasound with gas bodies produces a useful contrast effect in medical images, but the same interaction also represents a mechanism for bioeffects. Anesthetized hairless mice were scanned by using a 2.5-MHz transducer (610-ns pulses with 3.6-kHz repetition frequency and 61-Hz frame rate) after injection of Optison and Evans blue dye. Petechial hemorrhages (PHs) in intestine and abdominal muscle were counted 15 min after exposure to characterize capillary rupture, and Evans blue extravasation was evaluated in samples of muscle tissue. For 5 ml⋅kg-1 contrast agent and exposure to 10 alternating 10-s on and off periods, PH counts in muscle were approximately proportional to the square of peak negative pressure amplitude and were statistically significant above 0.64 MPa. PH counts in intestine and Evans blue extravasation into muscle tissue were significant above 1.0 MPa. The PH effect in muscle was proportional to contrast dose and was statistically significant for the lowest dose of 0.05 ml⋅kg-1. The effects decreased nearly to sham levels if the exposure was delayed 5 min. The PH effect in abdominal muscle was significant and statistically indistinguishable for uninterrupted 100-s exposure, 10-s exposure, 100 scans repeated at 1 Hz, and even for a single scan. The results confirms a previous report of PH induction by diagnostic ultrasound with contrast agent in mammalian skeletal muscle [Skyba, D. M., Price, R. J., Linka, A. Z., Skalak, T. C. & Kaul, S. (1998) Circulation 98, 290–293].