Structure-activity relationship study on a simple cationic peptide motif for cellular delivery of antisense peptide nucleic acid.

Improving cellular uptake and biodistribution remains one of the major obstacles for a successful and broad application of peptide nucleic acids (PNAs) as antisense therapeutics. Recently, we reported the identification and functional characterization of an antisense PNA, which redirects splicing of murine CD40 pre-mRNA. In this context, it was discovered that a simple octa(l-lysine) peptide covalently linked to the PNA is capable of promoting free uptake of the conjugate into BCL1 cells as well as primary murine macrophages. On the basis of this peptide motif, the present study aimed at identifying the structural features, which define effective peptide carriers for cellular delivery of PNA. While the structure-activity relationship study revealed some clear correlations, only a few modifications actually led to an overall improvement as compared to the parent octa(l-lysine) conjugate. In a preliminary PK/tissue distribution study in healthy mice, the parent conjugate exhibited relatively broad tissue distribution and only modest elimination via excretion within the time frame of the study.

Regulation of retinal vascular endothelial growth factor and receptors in rabbits exposed to hyperoxia.

PURPOSE: To evaluate the molecular responses of vascular endothelial growth factor (VEGF) and its receptors to dexamethasone (Dex) and celecoxib (Cel) during hyperoxia and during hyperoxia followed by recovery in room air, in newborn rabbit retinas. METHODS: Newborn rabbits at 3 days postnatal age (n = 96) received room air or oxygen (80%-100%) for 4 days, during which they were administered saline (Sal), Dex, vehicle (Veh), or Cel (n = 12/treatment group). Six animals from each group were killed immediately after hyperoxia (or room air) and the remainder exposed to room air for 5 days. Retinal mRNA expression of VEGF(121), VEGF(165), VEGF receptor-1 (VEGFR-1, or Flt-1), and VEGFR-2 (or KDR/Flk-1) was determined. RESULTS: Hyperoxia resulted in increased retinal expression of mRNA of the VEGF splice variants in the groups treated with Sal, Dex, and Veh, whereas a decrease in VEGF(121) was noted in the Cel-treated group. In contrast, retinal Flt-1 receptor mRNA was markedly increased in the Cel-treated group only, whereas retinal VEGFR-2 (KDR/Flk-1) receptor mRNA was suppressed in all the treatment groups. Hyperoxia followed by recovery in room air resulted in a minimal decrease in expression of retinal Flt-1 mRNA in the Sal and Dex groups. Cel treatment abolished its expression. CONCLUSIONS: The findings of increased retinal expression of VEGF mRNA in the newborn rabbit in response to hyperoxia are most likely due to species differences. Selective targeting of VEGF(121) and Flt-1 mRNA by Cel may represent one regulatory pathway for their anti-inflammatory effects. Further studies are needed to evaluate the therapeutic benefits of cyclooxygenase (COX)-2 inhibitors for the treatment and/or prevention of diseases associated with neovascularization.

Differential regulation of prostacyclin and thromboxane by dexamethasone and celecoxib during oxidative stress in newborn rabbits.

To compare the effects of dexamethasone (Dex) and celecoxib (Cel) on F-isoprostane, prostacyclin (PGI2), and thromboxane A2 (TxA2) following hyperoxia, and hyperoxia followed by recovery in room air (RA), newborn rabbits were exposed to hyperoxia (80-100% oxygen) for 4 days, during which they were treated with saline (Sal, i.m.), Dex (i.m.), vehicle (Veh, PO), or Cel (PO, n = 12 per group). Six animals in each group were sacrificed immediately following hyperoxia, and the remainder allowed to recover in RA for 5 days. The control litters were treated simultaneously in RA with all conditions other than atmospheric oxygen being identical. Blood samples were assayed for 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha), 6-keto prostaglandin F1alpha (6-ketoPGF1alpha), and TxB2. Dex and Cel decreased 8-epi-PGF2alpha during hyperoxia and the recovery period. Dex increased 6-ketoPGF2alpha following hyperoxia, while similar increments were noted during recovery with Cel. Although TxB2 was decreased only during the recovery period, TxB2/6-ketoPGF1alpha ratio was lower during hyperoxia and recovery in both treated groups. The effect of Cel on 8-epi-PGF2. and TxA2/PGI2 ratio confirm the formation of a COX-derived F2-isoprostane that is possibly linked to TxA2 receptors. Further studies are required to examine whether Cel can be used as a therapeutic alternative to Dex for oxygen-induced injury in the newborn.

Dose response of RU486 in a novel rabbit model of noninfectious preterm birth: comparative efficacy of 3 routes of administration.

OBJECTIVE: The purpose of this study was to examine whether the pregnant rabbit model can be used as a viable model for the study of non-infection-mediated preterm birth. STUDY DESIGN: Timed pregnant New Zealand rabbits were injected with a single dose of RU486 on day 22 of gestation. Three doses (50 mg, 75 mg, and 100 mg) were administered intramuscularly, intraperitoneally, or subcutaneously. The rabbits were monitored for preterm delivery. Progesterone, cortisol, and cytokine levels were examined before the induction and after delivery. Uterine and cervical progesterone, cortisol, and cytokine levels were determined after delivery. RESULTS: RU486 resulted in 100% preterm delivery in all doses and modes of administration, compared with 0% of controls. Intramuscular administration appeared to generate the most favorable preterm delivery time. Rabbits that received 100 mg RU486 intramuscularly showed significantly decreased serum progesterone levels and uterine progesterone levels, compared with 100 mg subcutaneously and intraperitoneally. CONCLUSION: RU486 that was administered intramuscularly appears to be a potent and effective method for inducing preterm birth. This model of hormonally mediated preterm birth might serve as a useful model for the investigation of the possible mechanisms of preterm labor.

Effect of the antenatal administration of celecoxib during the second and third trimesters of pregnancy on prostaglandin, cytokine, and nitric oxide levels in rabbits.

OBJECTIVE: The purpose of this study was to examine the effects of celecoxib on prostaglandin, cytokine, and nitric oxide synthesis in the pregnant rabbit. STUDY DESIGN: Pregnant rabbits received celecoxib from 13 to 20 days (celecoxib-A), from 13 to 28 days (celecoxib-B), or vehicle from 13 to 28 days by gavage. Blood and tissue were assayed for prostaglandin, cytokine, and nitric oxide oxidation products. RESULTS: Preterm delivery occurred in 4 of 11 controls, 0 of 9 in celecoxib-A, and 0 of 8 in celecoxib-B. Plasma prostaglandin F(2alpha) was reduced in both treated groups at 20 days and at delivery in celecoxib-B. Plasma thromboxane B(2) was suppressed in celecoxib-B at 20 days and delivery. Cervical prostaglandin E(2) was increased; uterine and cervical plasma thromboxane B(2) declined in celecoxib-B. Celecoxib administration suppressed plasma nitric oxide oxidation products at delivery and cervical nitric oxide oxidation products in celecoxib-B. Uterine and cervical interleukin-1beta and interleukin-6 were decreased, and uterine tumor necrosis factor-alpha increased in celecoxib-B. CONCLUSION: Further studies are required to evaluate the therapeutic benefits of cyclo-oxygenase-2 inhibitors in the setting of preterm parturition.

Regulation of matrix metalloproteinases (type IV collagenases) and their inhibitors in the virgin, timed pregnant, and postpartum rat uterus and cervix by prostaglandin E(2)-cyclic adenosine monophosphate.

OBJECTIVE: Our purpose was to examine whether the type IV collagenases (metalloproteinase [MMP]-2 and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) are regulated by a prostaglandin E(2) (PGE(2))-cyclic adenosine monophosphate (cAMP) mechanism in nonpregnant virgin, preterm and term pregnant and postpartum rats. STUDY DESIGN: Sprague-Dawley rats were infused with either saline solution or PGE(2) over 24 hours or were noninfused. Plasma and tissue were analyzed for cAMP, MMP-2 and MMP-9, and TIMP-1 and TIMP-2. RESULTS: PGE(2) evoked elevations in plasma and tissue levels of cAMP and MMP-2 in the preterm and term pregnant rats. MMP-9 levels were elevated in the preterm plasma and uterus, whereas in the term pregnant and postpartum rats, MMP-9 levels were increased in the cervix. CONCLUSIONS: MMP levels in the pregnant and postpartum rat uterus and cervix are regulated in part by a PGE(2)-cAMP mechanism. Specifically, MMP-9 may be involved in preterm labor, cervical maturity, and involution of the postpartum uterus.