Jolanta B. Zawilska

Prodrugs: A challenge for the drug development

It is estimated that about 10% of the drugs approved worldwide can be classified as prodrugs. Prodrugs, which have no or poor biological activity, are chemically modified versions of a pharmacologically active agent, which must undergo transformation in vivo to release the active drug. They are designed in order to improve the physicochemical, biopharmaceutical and/or pharmacokinetic properties of pharmacologically potent compounds. This article describes the basic functional groups that are amenable to prodrug design, and highlights the major applications of the prodrug strategy, including the ability to improve oral absorption and aqueous solubility, increase lipophilicity, enhance active transport, as well as achieve site-selective delivery. Special emphasis is given to the role of the prodrug concept in the design of new anticancer therapies, including antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT).

Regulatory mechanisms in melatonin biosynthesis in retina

The vertebrate retina produces melatonin in a light-dependent rhythmic fashion, synchronized with, but independent from the rhythm of the hormone formation in the pineal gland. This review summarizes the current status of our knowledge on regulatory mechanisms involved in controlling the retinal melatonin biosynthesis. Special emphasis is given to the role and mode of action of dopamine and GABA, two established retinal neurotransmitters, as well as that of second messengers (cyclic AMP, calcium ions). Comparisons are made between lower vertebrates and mammals.

Rhythmic changes in metabolism of dopamine in the chick retina: the importance of light versus biological clock

Rhythmic changes in dopamine (DA) content and metabolism were studied in retinas of chicks that were adapted to three different lighting conditions: 12‐h light : 12‐h dark (LD), constant darkness (DD) and continuous light (LL). Retinas of chicks kept under LD conditions exhibited light–dark‐dependent variations in the steady‐state level of DA and the two metabolites of DA, i.e. 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA). Concentrations of DA, DOPAC and HVA were high in light hours and low in dark hours of the LD illumination cycle. In retinas of chicks kept under DD, the content of DA, DOPAC and HVA oscillated in a rhythmic manner for 2 days, with higher values during the subjective light phase than during the subjective dark phase. The amplitudes of the observed oscillations markedly and progressively declined compared with the amplitudes recorded under the LD cycle. In retinas of chicks kept under LL conditions, levels of DA, DOPAC and HVA were similar to those found during the light phase of the LD cycle. Changes in the retinal contents of DA and HVA did not exhibit pronounced daily oscillations, while on the first day of LL the retinal concentrations of DOPAC were significantly higher during the subjective light phase than during the subjective dark phase. Acute exposure of chicks to light during the dark phase of the LD cycle markedly increased DA and DOPAC content in the retina. In contrast, light deprivation during the day decreased the retinal concentrations of DA and DOPAC. It is suggested that of the two regulatory factors controlling the level and metabolism of DA in the retina of chick, i.e. light and biological clock, environmental lighting conditions seem to be of major importance, with light conveying a stimulatory signal for the retinal dopaminergic cells.

Chick retina and pineal gland differentially respond to constant light and darkness: in vivo studies on serotoninN-acetyltransferase (NAT) activity and melatonin content

Oscillations in serotonin N-acetyltransferase (NAT) activity and melatonin content were investigated in retina and pineal gland of chicks kept for 5 days in constant darkness (DD) or continuous light (LL). Under DD the rhythm of the pineal melatonin biosynthesis resembled that found under diurnal illumination (LD), whereas in the retina DD resulted in significant elevations of NAT activity and melatonin level during subjective light. A low-amplitude rhythm of retinal NAT activity continued under LL with a period close to 24 h. In the pineal gland, light exposure suppressed the level of NAT activity and melatonin content (an effect being substantially weaker than that observed in retina), delayed the first peak of NAT activity by 3 h, and prolonged the rhythms period to about 26 h. Our data suggest the existence of some differences in the activity of circadian oscillators that regulate the melatonin generating system in the retina and pineal gland of chick.

Orexins Protect Neuronal Cell Cultures Against Hypoxic Stress: an Involvement of Akt Signaling

Orexins A and B are peptides produced mainly by hypothalamic neurons that project to numerous brain structures. We have previously demonstrated that rat cortical neurons express both types of orexin receptors, and their activation by orexins initiates different intracellular signals. The present study aimed to determine the effect of orexins on the Akt kinase activation in the rat neuronal cultures and the significance of that response in neurons subjected to hypoxic stress. We report the first evidence that orexins A and B stimulated Akt in cortical neurons in a concentration- and time-dependent manner. Orexin B more potently than orexin A increased Akt phosphorylation, but the maximal effect of both peptides on the kinase activation was very similar. Next, cultured cortical neurons were challenged with cobalt chloride, an inducer of reactive oxygen species and hypoxia-mediated signaling pathways. Under conditions of chemical hypoxia, orexins potently increased neuronal viability and protected cortical neurons against oxidative stress. Our results also indicate that Akt kinase plays an important role in the pro-survival effects of orexins in neurons, which implies a possible mechanism of the orexin-induced neuroprotection.

PACAP in Avians: Origin, Occurrence, and Receptors - Pharmacological and Functional Considerations

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel member of the secretin/glucagon/vasoactive intestinal peptide (VIP) superfamily. In vertebrates, including avians, it occurs in two forms: PACAP(38) and PACAP(27). PACAP structure is well conserved during evolution, being identical in mammals, and showing one amino acid dfifference in avians (chick, turkey). PACAP is widely distributed in the central nervous system and peripheral tissues and displays a pleiotropic activity, including functions as a hypophysiotropic hormone, neuromodulator, and neurotrophic factor. PACAP exerts its biological actions through three types of receptors designated PAC(1), VPAC(1) and VPAC(1). This review (1) presents the current knowledge on PACAP origin, distribution and function, (2) compares the avian findings with those found in mammals, and (3) describes receptor-linked mechanisms in avians, including recent data on receptor-related signal transduction pathways, with a special emphasis on receptor pharmacology and function.

Does D4 dopamine receptor mediate the inhibitory effect of light on melatonin biosynthesis in chick retina

The dopamine (DA) receptor mediating the inhibitory effect of light on melatonin formation in the chick retina was characterized pharmacologically. Nighttime serotonin N-acetyltransferase (NAT) activity was significantly decreased by either light exposure or by intraocular (i.oc.) administration of DA and quinpirole (a predominant D3/D4 DA receptor agonist). Several D2-like DA receptor antagonists, i.e. clozapine, haloperidol, spiroperidol, sulpiride, (+)-UH-232 and YM-09151-2, given i.oc. to light-adapted chicks markedly elevated retinal NAT activity. In contrast, raclopride (a D2/D3 DA receptor antagonist) and remoxipride (a D2-selective DA receptor antagonist) were ineffective. Spiroperidol, clozapine, haloperidol and sulpiride significantly increased melatonin content in the light-exposed retina, but had no effect on the activity of hydroxyindole-O-methyltransferase. None of D2-like DA receptor blockers tested modified the nighttime NAT activity in the chick retina. Our results indicate that the light-evoked inhibition of the nocturnal increase in melatonin biosynthesis in chick retina may involve stimulation of the D4 subtype DA receptor.

Clozapine and other neuroleptic drugs antagonize the light-evoked suppression of melatonin biosynthesis in chick retina: involvement of the D4-like dopamine receptor

The subtype of dopamine receptor mediating the suppressive effect of light on melatonin biosynthesis in chick retina was characterized pharmacologically. Acute exposure of animals to light during the dark phase of the light-dark cycle dramatically decreased melatonin levels and activity of serotonin N-acetyltransferase (NAT; a key regulatory enzyme in melatonin biosynthetic pathway). Various antagonists of dopamine receptors were tested for their ability to block this action of light on the retinal melatonin formation. Intraocular (i. oc.) pretreatment of chicks with neuroleptic drugs — blockers of the D2-family of dopamine receptors, i.e., clotiapine, clozapine (an atypical neuroleptic with high affinity for a D4-subtype dopamine receptor), haloperidol, spiroperidol, sulpiride, and YM-09151-2, significantly antagonized the light-evoked suppression of the nighttime NAT activity of the chick retina in a dosedependent manner. In contrast, remoxipride (a D2-selective dopamine antagonist), raclopride and (+)-UH-232 (D2/D3-dopamine receptor antagonists), as well as SCH 23390, a blocker of the D1-family of dopamine receptors, were ineffective. Clozapine, haloperidol, spiroperidol and sulpiride also potently antagonized the suppressive action of light on melatonin content of the chick retina. It is suggested that the dopamine receptor mediating the inhibitory effect of light stimulation on the nighttime melatonin biosynthesis in the retina of chick represents a D4-like subtype.

Dopamine receptor regulating serotonin N-acetyltransferase activity in chick retina represents a D4-like subtype: Pharmacological characterization

The dopamine (DA) receptor regulating serotonin N-acetyltransferase (NAT) activity in chick retina was characterized pharmacologically. Intraocular (i.o.) administration of DA significantly decreased the nighttime NAT activity of chick retina. The effect of DA was antagonized by blockers of the D2 family of DA receptors, spiroperidol, YM-09151-2 and clozapine, and it was not affected by SCH23390, a selective antagonist of D1 DA receptor. Several agonists of D2 family of DA receptors given i.o. suppressed the nighttime NAT activity of the chick retina with quinpirole (D3/D4 receptor selective) and bromocriptine (D2/D3 receptor selective) being the most and the least potent drugs, respectively. The rank-order potency of antagonists of D2 family of DA receptors to block the inhibitory effect of quinpirole on the enzyme activity (with clozapine being relatively very potent, and (+)-butaclamol, raclopride and remoxipride-ineffective) match the characteristics of the D4 DA receptor. Moreover, although sulpiride effectively prevented the quinpirole-induced decline in the nighttime NAT activity of the chick retina, there was no marked stereoselectivity in its action. It is suggested that DA receptor regulating NAT activity in chick retina represents a D4-like subtype.

PACAP-induced formation of cyclic AMP in the chicken brain: regional variations and the effect of melatonin.

We have studied the effects of pituitary adenylate cyclase-activating polypeptide (PACAP27 and PACAP38) on cyclic AMP formation in chick brain, and the action of melatonin upon the PACAP-evoked effects. PACAP stimulated cyclic AMP production in the hypothalamus>cerebral cortex>pineal gland>optic lobes. In the hypothalamus and cerebral cortex, the rank-order of both PACAP forms and VIP in evoking the cyclic AMP response was: PACAP38 approximately PACAP27>>VIP, suggesting the presence in the tested tissues of PAC1 receptors. Melatonin suppressed (IC50=19.8 nM) the PACAP27 (0.1 microM)-induced cyclic AMP response in the hypothalamus, but not in the cerebral cortex. Melatonin also suppressed the hypothalamal cyclic AMP synthesis stimulated by forskolin, but not that evoked by histamine or isoprenaline. Our observations show that PACAP is capable of potently stimulating cyclic AMP formation in some regions of the chick brain, and suggest that the hypothalamus may be a site for a functional interaction between PACAP and the pineal hormone melatonin.