**3. Pulmonary hypertension**

Pulmonary hypertension (PH) is an increase in blood pressure in the pulmonary artery, vein or capillaries, together known as the lung vasculature. In fetus life, PH is a normal state es‐ sential for survival. Since the placenta, not the lung, serves as the organ of gas exchange dur‐ ing embryonic development, most of the right ventricular output crosses from the ductus arteriosus to the aorta, and only 5–10% of the combined ventricular output is directed to the pulmonary vascular bed. Pulmonary vascular constriction plays a key role in maintaining high pulmonary vascular tone during fetal life. At the same time, the fetal lung and pulmo‐ nary vasculature must prepare for the dramatic adaptation to air breathing at the time of birth [40].

However, PH can continue even after the birth, called persistent pulmonary hypertension that develops when pulmonary vascular resistance remains elevated, resulting in right-toleft shunting of blood through fetal circulatory pathways. The pulmonary vascular resist‐ ance may remain elevated due to pulmonary hypoplasia and cause disease states, like congenital diaphragmatic hernia, as well as impaired development of the pulmonary arter‐ ies, resulting in the meconium aspiration syndrome, or failing adaption of the pulmonary vascular bed as occurs with perinatal asphyxia [41]. Moreover, PH has more than one etio‐ logical factor, thus it can be classified as idiopathic PH when there is no identifiable cause of this disease; familial PH with a previous disease history; and associated PH when an under‐ lying cause of PH such as connective tissue disease is present [42].

PH has been reported in patients with chronic hemolytic anemias, including sickle cell dis‐ ease, thalassemia, paroxysmal nocturnal hemoglobinuria, hereditary spherocytosis, malaria, among other disease states [43, 44]. The exact mechanism(s) involved in the development of PH in these patients is unclear. Hemolysis may result in a nitric oxide deficient state through free hemoglobin scavenging of nitric oxide and release of erythrocyte arginase, which limits L-arginine, a substrate for nitric oxide synthesis [45].

metabolic roles [24]. In view of that, compounds that increase AS activity and sustain tightly NO production avoiding an excess production will ensure adequate bioavailability for prop‐ er physiological functioning. Guerreiro and colleagues demonstrated that a *Bj*-PRO pro‐ motes activation of AS, assayed in the presence of the substrates ATP, citrulline, and aspartate, thus leading to NO production by endothelial cells [14]. More recently, we have demonstrated that other *Bj*-PROs induce NO production by activation of AS or kinin-B2 re‐ ceptors as well as by M1 muscarinic acetylcholine activation, thereby inducing vasodilata‐

The patent entitled "Proline-Rich Peptides, Pharmaceutical Composition, use of one or more peptides and method of treatment" was deposited to protect the use and application of *Bj*-PROs and analogous molecules [patent: BR2007/ 000003]. All applications contemplated by patent BR2007/ 000003 are consistent with the use of PROs as prototype molecules for the development of new drugs aiming to treat a range of pathological states related to deficien‐ cy in NO production and AS activity, e.g. lung hypertension, preeclampsia, essential hyper‐ tension, coagulopathies and citrullinemia. Some of these applications will be discussed

Pulmonary hypertension (PH) is an increase in blood pressure in the pulmonary artery, vein or capillaries, together known as the lung vasculature. In fetus life, PH is a normal state es‐ sential for survival. Since the placenta, not the lung, serves as the organ of gas exchange dur‐ ing embryonic development, most of the right ventricular output crosses from the ductus arteriosus to the aorta, and only 5–10% of the combined ventricular output is directed to the pulmonary vascular bed. Pulmonary vascular constriction plays a key role in maintaining high pulmonary vascular tone during fetal life. At the same time, the fetal lung and pulmo‐ nary vasculature must prepare for the dramatic adaptation to air breathing at the time of

However, PH can continue even after the birth, called persistent pulmonary hypertension that develops when pulmonary vascular resistance remains elevated, resulting in right-toleft shunting of blood through fetal circulatory pathways. The pulmonary vascular resist‐ ance may remain elevated due to pulmonary hypoplasia and cause disease states, like congenital diaphragmatic hernia, as well as impaired development of the pulmonary arter‐ ies, resulting in the meconium aspiration syndrome, or failing adaption of the pulmonary vascular bed as occurs with perinatal asphyxia [41]. Moreover, PH has more than one etio‐ logical factor, thus it can be classified as idiopathic PH when there is no identifiable cause of this disease; familial PH with a previous disease history; and associated PH when an under‐

PH has been reported in patients with chronic hemolytic anemias, including sickle cell dis‐ ease, thalassemia, paroxysmal nocturnal hemoglobinuria, hereditary spherocytosis, malaria, among other disease states [43, 44]. The exact mechanism(s) involved in the development of

lying cause of PH such as connective tissue disease is present [42].

tion *in vivo* [16, 17].

**3. Pulmonary hypertension**

below.

462 Drug Discovery

birth [40].

Nitric oxide is synthesized from terminal nitrogen of L-arginine by NOS. All three NOS iso‐ forms are expressed in the lung and are distinguished by regulation of their activities, as well as by specific sites and developmental patterns of expression [46]. The isoform eNOS is expressed in vascular endothelial cells and is believed to be the predominant source of NO production in pulmonary circulation [40]. This hypothesis is corroborated by the fact that NO inhalation in premature newborns with severe respiratory failure due to PH provides improvement of symptoms, accompanied by marked increase in oxygenation [34].

Although large well-designed studies paved the way to Food and Drug Administration (FDA) approval of therapeutic NO inhalation, it is equally important to note that inhaled NO did not reduce the mortality, length of hospitalization, or the risk of significant neurode‐ velopmental impairment associated with persistent PH in newborn children [40]. It is known that at excessive levels NO can react with reactive oxygen species (ROS) such as su‐ peroxide and hydrogen peroxide. Such increase in ROS was observed in the smooth muscle and adventitia of pulmonary arteries from lambs with chronic intrauterine PH [47, 48], forming peroxynitrite, an anion with deleterious tissue-oxidant effects [49].

Inhaled NO is usually delivered with high concentrations of oxygen. Whereas hyperoxic ventilation continues to be a mainstay in the treatment of PH, little is known about the side effects of oxygen supply together with NO. The extreme hyperoxia routinely used in PH management may in fact be toxic to the developing lung due to ROS formation [39, 50, 51]. Superoxide may react with arachidonic acid to increase concentrations of isoprostanes and may also combine with NO to form peroxynitrite [52] with possible induction of vaso‐ constriction, cytotoxicity, and damage to surfactant proteins and lipids. Moreover, peroxy‐ nitrite has been shown to directly induce NOS uncoupling. New data indicate that even brief (30 min) periods of exposure to 100% O2 are sufficient to increase reactivity of pulmo‐ nary vessels in healthy lambs [53, 54], to diminish the response of the pulmonary vascula‐ ture to endogenous and exogenous nitric oxide [54], and to increase the activity of cGMPspecific phosphodiesterases [51]. Inhaled NO would theoretically benefit patients with chronic primary or secondary pulmonary hypertension, but its therapeutic application in this setting has been limited by the risk of causing rebound pulmonary hypertension, if it is inadvertently discontinued, and the lack of practical home-based continuous delivery devices.

Therefore, we have proposed an alternative approach for controlled induction of NO pro‐ duction. We believe that cytosolic L-arginine provides a major NO donor. Arginine con‐ centrations subject to metabolic fine-tuning controls will assure that the amino acid is kept in a homeostatic concentration range. These effects could be achieved by the action of *Bj*-PRO on AS, a target unexplored by the pharmaceutical industry. Compounds inducing an increase in AS expression and activity are promising for the treatment of diseases related with deficient NO production.
