Purpose.: To characterize melphalan pharmacokinetics after superselective ophthalmic artery infusion (SSOAI) in animals and children with retinoblastoma. Methods.: Vitreous and plasma samples of five Landrace pigs were obtained over a 4-hour period after SSOAI of melphalan (7 mg). Melphalan cytotoxicity was evaluated in retinoblastoma cell lines with and without topotecan. Plasma samples were obtained from 17 retinoblastoma patients after SSOAI of 3 to 6 mg of melphalan to one (n = 14) or two eyes (n = 3). Correlation between plasma pharmacokinetics and age, dosage, and systemic toxicity was studied in patients. Results.: In animals, melphalan peak vitreous levels were greater than its IC50 and resulted in 3-fold vitreous-to-plasma exposure. In patients, a large variability in pharmacokinetic parameters was observed and it was explained mainly by body weight (P < 0.05). A significantly higher systemic area under the curve was obtained in children receiving more than 0.48 mg/kg for bilateral tandem infusions (P < 0.05). These children had 50% probability of grades 3–4 neutropenia. Plasma concentrations after 2 and 4 hours of SSOAI were significantly higher in these children (P < 0.05). A synergistic cytotoxic effect of melphalan and topotecan was evident in cell lines. Conclusions.: Potentially active levels of melphalan after SSOAI were achieved in the vitreous of animals. Low systemic exposure was found in animals and children. Doses greater than 0.48 mg/kg, given for bilateral tandem infusions, were associated with significantly higher plasma levels and increased risk of neutropenia. Synergistic in vitro cytotoxicity between melphalan and topotecan favors combination treatment.

Many effects believed to be because of angiotensin II (Ang II) are attributable to the action of endothelin (ET)-1, which is released/produced by Ang II. We investigated whether Ang II elicits its positive inotropic effect (PIE) by the action of endogenous ET-1, in addition to the role played by reactive oxygen species (ROS) in this mechanism. Cat cardiomyocytes were used for: (1) sarcomere shortening measurements; (2) ROS measurements by epifluorescence; (3) immunohistochemical staining for preproET-1, BigET-1, and ET-1; and (4) measurement of preproET-1 mRNA by RT-PCR. Cells were exposed to 1 nmol/L Ang II for 15 minutes. This low concentration of Ang II increases sarcomere shortening by 29.2±3.7% ( P <0.05). This PIE was abrogated by Na ⁺ /H ⁺ exchanger or Na ⁺ /Ca ²⁺ exchanger reverse mode inhibition. The production of ROS increased in response to Ang II treatment (ΔROS respect to control: 68±15 fluorescence units; P <0.05). The Ang II–induced PIE and ROS production were blocked by the Ang II type 1 receptor blocker losartan, the nonselective ET-1 receptor blocker TAK044, the selective ET _A receptor blocker BQ-123, or the ROS scavenger N-(2-mercapto-propionyl)glycine. Exogenous ET-1 (0.4 nmol/L) induced a similar PIE and increase in ROS production to those caused by Ang II. Immunostaining for preproET-1, BigET-1, and ET-1 was positive in cardiomyocytes. The preproET-1 mRNA abundance increased from 100±4.6% in control to 241.9±39.9% in Ang II–treated cells ( P <0.05). We conclude that the PIE after exposure to 1 nmol/L Ang II is due to endogenous ET-1 acting through the ET _A receptor and triggering ROS production, Na ⁺ /H ⁺ exchanger stimulation, and Na ⁺ /Ca ²⁺ exchanger reverse mode activation.

Desmopressin (dDAVP) is a well-known peptide analog of the antidiuretic hormone vasopressin, used to prevent excessive bleeding during surgical procedures. dDAVP increases hemostatic mediators, such as the von Willebrand factor (vWF), recently considered a key element in resistance to metastasis. Studies in mouse models and veterinary trials in dogs with locally-advanced mammary tumors demonstrated that high doses of perioperative dDAVP inhibited lymph node and early blood-borne metastasis and significantly prolonged survival. We conducted a phase II dose-escalation trial in patients with breast cancer, administering a lyophilized formulation of dDAVP by intravenous infusion in saline, 30–60 min before and 24 h after surgical resection. Primary endpoints were safety and tolerability, as well as selection of the best dose for cancer surgery. Secondary endpoints included surgical bleeding, plasma levels of vWF, and circulating tumor cells (CTCs) as measured by quantitative PCR of cytokeratin-19 transcripts. Only 2 of a total of 20 patients experienced reversible adverse events, including hyponatremia (grade 4) and hypersensitivity reaction (grade 2). Reactions were adequately managed by slowing the infusion rate. A reduced intraoperative bleeding was noted with increasing doses of dDAVP. Treatment was associated with higher vWF plasma levels and a postoperative drop in CTC counts. At the highest dose level evaluated (2 μg/kg) dDAVP appeared safe when administered in two slow infusions of 1 μg/kg, before and after surgery. Clinical trials to establish the effectiveness of adjunctive perioperative dDAVP therapy are warranted. This trial is registered on www.clinicaltrials.gov (NCT01606072).

The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca²⁺-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca²⁺-activated small-conductance type 2 (SK2)K⁺ channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABA_B(1a,2) receptors [GABA_B(1a,2)Rs] that downregulate the amount of ACh released at the OC–hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABA_BRs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABA_B1–GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABA_B1a isoform selectively inhibits release at efferent cholinergic synapses.

Disseminated retinoblastoma is the major cause of mortality associated with this tumor worldwide.¹ Retinoblastoma may disseminate to the central nervous system (CNS) through direct optic nerve extension and/or by hematogenous dissemination.² Bone marrow is the most common metastatic site, followed by bones, liver, and other less common organs.^2,3 Although widely recognized from the clinical point of view, little is known about the kinetics of the dissemination of retinoblastoma.⁴