OA19297A - Citrulline for treatment of sickle cell crisis. - Google Patents

Abstract

A method for administration of citrulline for the treatment of sickle cell disease and other complications of sickle cell disease thereof.

Description

[0041] The invention provides for a method by administration of citrulline, preferably via intravenous administration of citrulline, for the treatment of sickle cell anémia and complications thereof. In another mode, this invention provides for a method by administration of citrulline, preferably via intravenous administration of citrulline, for the treatment of sickle cell disease and complications thereof. In a preferred mode, citrulline is administered to a patient suffering from sickle cell crisis; more preferably, the patient to be treated is also exhibiting relative hypocitrullinemia.

[0042] In order that the invention herein described may be fully understood, the following detailed description is set forth. Various embodiments of the invention are described in detail and may be further illustrated by the provided examples. Additional viable variations of the embodiments can easily be envisioned.

Sickle Cell Disease Complications — Sickle cell crisis

[0043] The terms “sickle cell crisis” or “sickling crisis” may be used to describe several independent acute conditions occurring in patients with sickle cell disease (SCD). SCD results in anémia and crises that could be of many types including the vaso-occlusive crisis, aplastic crisis, séquestration crisis, hemolytic crisis, and others. Most épisodes of sickle cell crises last between five and seven days. Although infection, déhydration, and acidosis (ail of which favor sickling) can act as triggers, in most instances, no predisposing cause is identified. Kumar et al. (2009) Robbins and Cotran Pathologie Basis of Disease, Professional Edition: Expert Consult - Online (Robbins Pathology) Elsevier Health. Kindle Edition. Preferred modes of this invention include administration of citrulline to patients with SCD who are in one or another form of sickle cell crisis or who hâve been exposed to a predisposing cause or trigger for sickle cell crisis. [0044] The présent invention provides for spray dry préparations comprising citrulline for the treatment of sickle cell disease (SCD), preferably a complication of sickle cell anémia, including but not limited to sickle cell crisis, vaso-occlusive crisis, aplastic crisis, hemolytic crisis, dactylitis, acute chest syndrome, seizure, stroke, ischemia, transient ischémie attack, ischémie colitis, or a combination thereof.

Vaso-occlusive crisis

[0045] The vaso-occlusive crisis is caused by sickle-shaped red blood cells that obstruct capillaries and restrict blood flow to an organ resulting in ischemia, pain, necrosis, and often organ damage. The frequency, severity, and duration of these crises vary considerably. Painful crises are treated with hydration, analgésies, and blood transfusion; pain management requires opioid administration at regular intervals until the crisis has settled. For milder crises, a subgroup of patients manage on NSAIDs (such as diclofenac or naproxen). For more severe crises, most patients require inpatient management for intravenous opioids; patient-controlled analgesia devices are commonly used in this setting. Vaso-occlusive crisis involving organs such as the pénis, or lungs are considered an emergency and treated with red-blood cell transfusions. Incentive spirometry, a technique to encourage deep breathing to minimize the development of atelectasis, is recommended. Glassberg (2011) Emergency Medicine Practice 13 (8): 1—20; Olujohungbe & Bumett (2013) British Journal of Haematology 160(6): 754-65.

Splenic séquestration crisis

[0046] Because of its narrow vessels and function in clearing defective red blood cells, the spleen is frequently affected. Anie KA, Green J (2012). Anie, Ed. “Psychological thérapies for sickle cell disease and pain”. Cochrane Database of Systematic Reviews 2: CD001916. It is usually infarcted before the end of childhood in individuals suffering from sickle cell anémia. This spleen damage increases the risk of infection from encapsulated organisms; Pearson (1977) J Infect Dis. 136 Suppl: S25-30; Wong et al. (1992) Am J Hematol 39 (3): 176-82; préventive antibiotics and vaccinations are recommended for those lacking proper spleen function.

[0047] Splenic séquestration crises are acute, painful enlargements of the spleen, caused by intrasplenic trapping of red cells and resulting in a precipitous fall in hemoglobin levels with the potential for hypovolémie shock. Séquestration crises are considered an emergency. If not treated, patients may die within 1-2 hours due to circulatory failure. Management is supportive, sometimes with blood transfusion. These crises are transient, they typically continue for 3-4 hours and may last for as long as a day. Khatib et al. (2009) Pédiatrie Radiology 39(1): 17-22.

Acute chest syndrome

[0048] Acute chest syndrome (ACS) is defîned by at least two of the following signs or symptoms: chest pain, fever, pulmonary infiltrate or focal abnormality, respiratory symptoms, or hypoxemia. Glassberg (2011) Emergency Medicine Practice 13(8): 1-20. It is the second-most common complication and it accounts for about 25% of deaths in patients with SCD, majority of cases présent with vaso-occlusive crises then they develop ACS. Mekontso et al. (2008) Am. J,

Respir. Crit. Care Med. 177(6): 646—53; Paul et al. (2011) Eur. J. Haematol. 87(3): 191—207. Nevertheless, about 80% of patients hâve vaso-occlusive crises during ACS.

Aplastic crisis

[0049] Aplastic crises are acute worsening of the patient's baseline anémia, producing pale appearance, fast heart rate, and fatigue. This crisis is normally triggered by parvovirus B19, which directly affects production of red blood cells by invading the red cell precursors and multiplying in and destroying them. Kumar et al. (2009). Robbins and Cotran Pathologie Basis of Disease, Professional Edition: Expert Consult - Online (Robbins Pathology). Elsevier Health. Kindle Edition. Parvovirus infection almost completely prevents red blood cell production for two to three days. In normal individuals, this is of little conséquence, but the shortened red cell life of SCD patients results in an abrupt, life-threatening situation. Réticulocyte counts drop dramatically during the disease (causing reticulocytopenia), and the rapid turnover of red cells leads to the drop in hemoglobin. This crisis takes 4 days to one week to disappear. Most patients can be managed supportively; some need blood transfusion. Slavov et al. (2011) FEMS Immunology and Medical Microbiology 62(3): 247-62.

Hemolytic crisis

[0050] Haemolytic crises are acute accelerated drops in hemoglobin level. The red blood cells break down at a faster rate. This is particularly common in patients with coexistent G6PD deficiency. Balgir (2012) Cardiovascular & Hematological Agents in Médicinal Chemistry 10(1): 3-13. Management is supportive, sometimes with blood transfusions. Glassberg (2011) Emergency Medicine Practice 13(8): 1-20.

Other Complications of Sickle Cell Disease

[0051] One of the earliest clinical manifestations is dactylitis, presenting as early as six months of âge, and may occur in children with sickle cell trait. Jadavji & Prober (1985) Can Med Assoc J 132 (7): 814-5. The crisis can last up to a month. Worrall & Butera (1976) J Bone Joint Surg Am 58 (8): 1161-3. Another recognized type of sickle crisis, acute chest syndrome, is characterized by fever, chest pain, difficulty breathing, and pulmonary infiltrate on a chest X-ray. Given that pneumonia and sickling in the lung can both produce these symptoms, the patient is treated for both conditions. Miller (2011) Blood 117 (20): 5297-305. It can be triggered by painful crisis, respiratory infection, bone-marrow embolization, or possibly by atelectasis, opiate administration, or surgery. Hematopoietic ulcers may also occur. James et al. (2006). Andrews' Diseases of the Skin: clinical Dermatology. Saunders Elsevier. page 847.

Mode of Action

[0052] Without being bound to a particular theory, the inventors point out that the pathophysiological hallmark of sickle cell disease (SCD) is vaso-occlusion in or of the small blood vessels. During times of deoxygenation, transformation and polymerization of hemoglobin S molécules occurs so that the doughnut shaped red blood cells (rbcs) assume a sickle shape resulting in a highly viscous and semi-solid gel that leads to microvascular obstruction (vasoocclusion). Ferrone Microcirculation (2004) 11:115-128; Samuel et al. Nature 345: 833-835. Over the last several years it has become apparent that vaso-occlusion is a highly complex phenomenon with multiple factors that play important rôles. Endothélial activation occurs as shown by increased expression of adhesion molécules and also increased expression of P-selectin by endothélial cells in mouse models of SCD. Solovey et al. N Engl J Med (1997) 337: 1584— 1590. This promûtes adhesion of deformed sickle red blood cells to the endothélium enabling further polymerization and trapping of sickle red-blood cells in the microcirculation with increased production of inflammatory cytokines. Hebbel et al. N Engl J Med (1980) 302: 992— 995. Furthermore, endothélial activation propagates progressive recruitment and adhesion of leukocytes allowing greater interaction between sickle red-blood cells and white blood cells that further contributes to the process of vaso-occlusion. Finnegan et al. Am J Hematol (2007) 82: 266-275. Several studies hâve also shown that there is increased platelet activation and elevated markers of thrombosis indicating that SCD is a hypercoagulable State. Hsu et al. Blood (2007) 109: 3088-3098. In addition to the above factors, perturbations in the arginine pathway with hemolysis associated nitric oxide (NO) déplétion hâve been found to play a central rôle in the pathogenesis of vaso-occlusion in SCD.

[0053] NO is a powerful vasodilator and plays a fundamental rôle in maintaining normal vasomotor tone. NO, a soluble gas continuously synthesized in endothélial cells by the NO synthase enzyme Systems, régulâtes basal vascular tone and endothélial function. These vital homeostatic processes may be impaired in SCD and contribute to the pathogenesis of SCD complications such as Vaso-Occlusive crisis (VOC), leg ulcers and priapism due to disturbed NO production in the endothélium contributing to endothélial dysfunction and local vasoconstrictors exceeding vasodilators. Hence, NO plays a significant rôle in the pathophysiology of SCD.

[0054] NO is synthesized from arginine by a family of enzymes called the NO synthases (NOS). NOS catalyze the oxidation of arginine to NO and L-citrulline, and L-citrulline in tum can be recycled to arginine by the combined action of arginosuccinate synthase (ASS) and arginosuccinate lyase (ASL), which is expressed in ail cell types. Arginine may also be catabolized by arginase to produce ornithine to fuel the urea cycle.

[0055] As a conséquence of hemolysis and breakdown of the red blood cell membrane in SCD, reactive oxygen species are generated that may react with NO. During hemolysis, arginase from érythrocyte membranes is released into the circulation, and dégradés Arginine, leading to further réduction in NO bioavailability. The limited intravascular hemolysis that occurs in SCD results in release of hemoglobin into plasma where it scavenges NO. Hsu et al. Blood (2007) 109: 3088-3098; Taylor et al. PLos One (2008) 3: e2095. Lactate dehydrogenase, a marker of intravascular hemolysis was found to be elevated in patients with SCD and is correlated with accelerated NO consumption. To different extents, ail these factors contribute to the global déplétion of NO associated with SCD.

[0056] Multiple studies hâve evaluated NO levels and precursors in SCD. Plasma arginine levels appear to vary with âge and the presence of VOC. Morris et al. J Pediatr Hematol Oncol (2000) 22: 515-520. Arginine levels are normal in steady-state children with SCD, but significantly lower in children experiencing a VOC. Levels were normal at présentation of these patients, but decreased during hospitalization in those children with VOC, suggesting a relationship between the arginine-nitric oxide pathway and VOC in SCD. Adults with SCD hâve been shown to hâve low arginine levels during steady-state which significantly decreases further during a VOC. Oral arginine given to normal Controls, SCD patients, and SCD patients hospitalized with a VOC increased NO formation by 68% in normal Controls, whereas steady-state SCD patients demonstrated a paradoxical decrease in NO that was not overcome by higher doses, suggesting that arginine is metabolized differently in SCD than in healthy Controls. In a randomized, doubleblind placebo controlled clinical study the benefit of arginine in reducing parentéral opioid use in children with VOC and also lowered pain scores was demonstrated. Morris et al. Haematologica (2013) 98: 1375-1382. However, citrulline is a more potent NO booster than arginine for several reasons.

[0057] Arginine is subject to extensive élimination by arginases présent both in the gut and liver. Morris JNutr (2004) 134: 2743S-2747S; Morris JNutr (2007) 137: 1602S-1609S. Arginine may act as a common substrate for both NOS and arginase, and arginase may reduce NO production by competing with NOS for arginine. Unlike arginine, citulline is not subject to presystemic breakdown in the gut. The majority of citrulline released by the intestine is metabolized within the kidney where it is converted to arginine, known as the intestinal-renal axis, representing ~15% of de novo arginine production. Curis et al. Amino Acids (2005) 29: 177-205. Citrulline may therefore serve as an arginine precursor. As noted above, citrulline can also be converted to arginine via ASS and ASL. While both arginine and citrulline support NO synthesis in a variety of tissues, including the vascular endothélium, both NOS and arginase use arginine as a common substrate, and arginase may reduce NO production by competing with NOS for arginine, which makes citrulline a more effective NO precursor than arginine.

[0058] Endothélial cell activation plays a critical rôle in the vasculopathy associated with SCD. The significantly lower arginine levels during a VOC likely leads to an imbalance in the NO metabolism in the endothélium that contributes to the perfusion defects in the microvasculature in SCD. In an investigation in murine endothélial cells to détermine whether arginosuccinate synthase, the rate limiting enzyme for arginine régénération from citrulline, plays a pivotai rôle in supplying arginine to endothélial (eNOS), but not inducible (iNOS) for NO production, it was found that extracellular arginine was the only arginine supply for NO production by iNOS while due to the presence of arginosuccinate synthase in endothélial cells, citrulline was the major supply for intracellular arginine and endothélial NO production. Shen et al. Biochem Pharmacol (2005) 69: 97-104. The presence of arginosuccinate synthase in endothélial cells appears to play a critical rôle and offers the opportunity to use citrulline supplémentation to increase intracellular endothélial arginine availability for NO product, which may be expected to help ameliorate the microcirculatory occlusion seen in SCD. Moreover, enhanced arginase-induced arginine consumption is believed to play an intégral rôle in the pathogenesis of sickle cell complications, and in a more recent study, citrulline supplémentation increased NO production and improved microcirculatory flow during conditions with acute arginase-induced arginine deficiency, while Arginine did not increase NO production. Wijands et al. Nutrients (2015) 7: 5217-5238.

[0059] Inhaled nitric oxide (iNO) has been shown to affect mouse models of sickle cell disease. However, a randomized, placebo-controlled study with iNO showed no effect on time-toresolution of vaso-occlusive crisis. Gladwin et al. JAMA (2011) 305(9): 893-902. The présent inventors speculate that the lack of effect in this study was due to the short half-life of NO in tissue generally and the short path length of diffusion for NO in lung tissue. On the other hand, endogenously-produced NO needs only to diffuse from vascular endothélial cells to nearby vascular smooth muscle cells.

[0060] A study giving citrulline orally twice daily in daily doses of approximately 0.1 g/kg in a pilot phase II clinical trial during steady States in four homozygous sickle cell disease subjects and one sickle cell hemoglobin C disease patient [âges 10-18] has been published. There was dramatic improvements in symptoms of well-being, raised plasma arginine levels, and réductions in high total leukocyte and high segmented neutrophil counts toward or to within normal limits. Continued citrulline supplémentation in compilant subjects continued to lessen symptomatology, to maintain plasma arginine concentrations greater than control levels, and to maintain nearly normal total leukocyte and neutrophil counts. Side effects or toxicity from citrulline were not experienced. Waugh et al. N Natl Med Assoc. (2001) 93: 363-371.

Citrulline

[0061] Arginine is the substrate for nitric oxide production by nitric oxide synthetase. Because the majority ofcirculating arginine is from urea cycle synthesis and not dietary sources, citrulline availability is critical to maintaining adéquate arginine supply for nitric oxide production. Citrulline is the first intermediate in the urea cycle after the rate-limiting enzyme carbamyl phosphate synthetase I. Citrulline also crosses mitochondrial and cellular membranes easily and therefore can be transported to other organs in the body. In the vascular endothélium (including the pulmonary endothélium), citrulline can then be converted into arginine and subsequently into nitric oxide. Consequently, modifying systemic citrulline to enhance endogenous NO production is a much more reliable method of delivering NO to tissue than iNO. [0062] Oral administration of citrulline obligates the citrulline to pass through the gut and then the liver which removes the citrulline. This is due to the absorption of the citrulline from the gut and transport directly to the liver by the hepatic portai vein in the first pass through the patient’s System. For example, the inventors found that only 2 out of 3 patients met the target level of citrulline when administered orally in an ICU (Intensive Care Unit). This would require multiple dosing regimen to achieve a therapeutic plasma level of citrulline. Thus, it is préférable for citrulline therapy to administer the active agent in a manner which bypasses the gut.

Relative Hypocitrullinemia

[0063] Relative hypocitrullinemia is a condition in which a subject suffering from the condition has reduced plasma citrulline as compared to a subject not suffering from the condition. Patients with relative hypocitrullinemia may hâve a plasma citrulline level below 100 pmol/L, for example below 37, 40, 50, 60, 70, 80, 90, or 100 μιηοΙ/L. For example, a patient with relative hypocitrullinemia may hâve a plasma citrulline level is below 80—100 μπιοΙ/L citrulline. These same patients do not suffer from statistically significant deficiencies in nitric oxide or arginine at a steady State. However, hypocitrullinemic patients may be much less responsive to a need for increased NO to stimulate vasodilation short term.

Methods of use

[0064] Administration of citrulline results in an increase in plasma citrulline levels. Smith teaches that oral citrulline may be administered safely to increase and maintain plasma citrulline levels. Smith, et al. (2006) J Thorac Cardiovasc Surg 132: 58-65, Figure 3. Barr teaches that intravenous citrulline may be administered safely to increase and maintain plasma citrulline levels. Barr, et al. (2007) J Thorac Cardiovasc Surg 134: 319-326, Figure 5. Accordingly, the administration of citrulline may be used to overcome relative hypocitrullinemia by increasing the plasma citrulline levels.

Citrulline Formulations

[0065] Citrulline (2-amino-5-(carbamoylamino)pentanoic acid) [C6H13N3O3] is an amino acid. Citrulline solution for IV administration may be manufactured by methods known in the art. See, e.g., Kakimoto, et al. (1971) Appl Microbiol 22(6): 992-999.

[0066] The effective amount of citrulline may be about 100-300 μmol/L of plasma. The citrulline may be formulated to achieve a concentration of about 100, 125, 150, 175, 200, 225, 250, 275, or 300 μιηοΙ/L.

Citrulline Dosages

[0067] The citrulline may be provided in dose unit form. For example, the citrulline may be provided in a container containing 300 mg stérile citrulline formulated for injection. This may be reconstituted for use using 6 mL stérile water and further diluted with approximately 5.9 mL stérile NaCl solution 0.9% Ph. Eur. to a total volume of 12 mL and a concentration of 300 mg/12 mL (i.e., 25 mg/mL). The citrulline may be formulated for injection at a concentration of 10—40 mg/mL, for example 10, 15, 20, 25, 30, 35, or 40 mg/mL. The citrulline may be provided as a drug product at 500 mg stérile citrulline for injection in 10 mL of stérile water. This may be used to infuse patients using sodium chloride 0.9% Ph. Eur.

[0068] The effective amount of citrulline may be about 10-300 mg/kg. The effective amount of citrulline may be about 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/kg. In a preferred mode, the effective amount of citrulline may be about 150 mg/kg.

[0069] Alternatively, citrulline may be administered orally at a dosage of about 5-15 g/kg, i.e., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 g/kg of citrulline. In a preferred mode, the oral dosage of citrulline may be about 9 g/kg of citrulline.

[0070] The target level for plasma citrulline may be maintained at about 37 pmol/L to 2.5 mM. For example, the plasma citrulline level of the patient may be maintained above 37, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, or 300 pmol/L. The plasma citrulline level of the patient may be maintained above 37, 100, or 200 pmol/L. The methods described herein may be used to maintain a patient’s citrulline plasma level between about 37 μιηοΙ/L to 200 μmol/L, 100 pmol/L to 1 mM/L, 150 pmol/L to 500 pmol/L citrulline. In a preferred mode, the plasma citrulline level may be maintained at about 100 μηιοΙ/L.

[0071] In some instances, it is désirable to deliver a unit dose, such as doses of 50 mg, more preferably 100 mg, and more preferably 160 mg of citrulline. The above described doses of 50 mg, 100 mg, 160 mg, 250 mg, and 300 mg may be delivered in a dose.

[0072] The following dose ranges relate to the goal of obtaining and maintaining a plasma citrulline level of 100 μΜ/L. The following dosing regimen may be used: an intravenous bolus dose of 20 mg/kg over 5 minutes and then continuons administration at a rate of 7 mg/kg/hour for the next 23 hours or an intravenous bolus does of 20 mg/kg followed by 9 mg/kg/hour for 23 hours or a lower continuous dose of 5 mg/kg/hour for 23 hours.

[0073] Although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it should be understood that certain changes and modifications may be practiced within the scope of the appended claims. Modifications of the above-described modes for carrying out the invention that would be understood in view of the foregoing disclosure or made apparent with routine practice or implémentation of the invention to persons of skill in surgery, biochemistry, medicine, physiology, and/or related fields are intended to be within the scope of the following claims.

[0074] Ail publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications mentioned in this spécification are indicative of the level of skill of those skilled in the art to which this invention pertains. Ail such publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent, patent application publication, or patent application was specifically and individually indicated to be incorporated by reference.

[0075] The examples contained herein are offered by way of illustration and not by any way of limitation.

EXAMPLES

EXAMPLE 1

CITRULLINE AND SICKLE CELL CRISIS

[0076] Background: Hemolysis associated nitric oxide (NO) déplétion plays a central rôle in the pathogenesis of vaso-occlusion in sickle cell disease (SCD). Citrulline is an effective NO booster, even during conditions of inflammation and acute arginase-induced arginine deficiency, characteristic of SCD. This study demonstrates the safety and pharmacokinetic profile of intravenous citrulline in patients with sickle cell disease and provides guidance for an appropriate dosage range.

[0077] Methods: Each cohort of participants received an intravenous bolus of 20 mg/kg of citrulline over five minutes with dose incréments of 10 mg/kg until a target plasma citrulline level of 80 to 100 pmol/L. FIG. 1. Plasma samples were collected at certain time points for pharmacokinetic studies. Adverse events were followed according to the NCI Common Terminology Criteria for Adverse Events (CTCAE).

[0078] Results: In the first cohort of four participants, the intravenous bolus infusion of 20 mg/kg of citrulline yielded a mean peak level of 259 pmol/L and trough level in the range of 2040 pmol/L 4 hours after the infusion. FIG. 2. Ail subjects had a signifïcant rise in their arginine level within one hour of receiving the bolus intravenous citrulline (mean incrément of 182%). FIG. 3. One subject transiently dropped the diastolic blood pressure by >20% within 30 minutes of study drug with no intervention needed. There were no other reported side effects. Further analysis using a simulated dosing scheme indicates that a 20 mg/kg bolus dose of intravenous citrulline followed by a continuous infusion of 7 mg/kg/hour is needed to maintain the target citrulline concentration. See Fig. 4.

Table 1 Pharmacokinetic parameters for individual patients

	Rapp	Krein	M	CL
Patient	mmol/hr/kg	1/hr	L/kg	L/hr/kg
1	12.9	1.01	0.70	0.70
2	8.3	1.34	0.38	0.51
3	14.0	0.89	0.46	0.41
4	9.3	1.01	0.46	0.46
Average	11.1	1.06	0.50	0.52
SD	2.8	0.19	0.14	0.13

[0079] Conclusion: Bolus intravenous citrulline is safe and well tolerated in patients with SCD but has a rapid clearance. Continuous dose intravenous citrulline in SCD, including its effect on NO production, appears useful in treating sickle cell pain crisis.

[0080] While the foregoing invention has been described in connection with this preferred embodiment, it is not to be limited thereby but is to be limited solely by the scope of the claims which follow.

Claims (18)

1. Citrulline for use intravenously in an effective amount in a method of treating a complication of sickle cell disease in a patient.

2. The citrulline for use intravenously in an effective amount of claim 1, wherein the complication is sickle cell crisis, vaso-occlusive crisis, acute chest syndrome, aplastic crisis, hemolytic crisis, dactylitis, seizure, stroke, ischemia, transient ischémie attack, ischémie colitis, priapism, séquestration crisis, or a combination thereof.

3. The citrulline for use intravenously in an effective amount of claim 1, wherein the effective amount of citrulline is 100-1,000 mg/kg.

4. The citrulline for use intravenously in an effective amount of claim 1, wherein the effective amount of citrulline is 100-1,000 mg in dose unit form.

5. The citrulline for use intravenously in an effective amount of claim 1, wherein the method comprises an intravenous bolus of 5-50 mg/kg of citrulline.

6. The citrulline for use intravenously in an effective amount of claim 1, wherein the method comprises an intravenous bolus of 20 mg/kg of citrulline over 1-60 minutes.

7. The citrulline for use intravenously in an effective amount of claim 5, wherein the method comprises a second intravenous bolus of 5-50 mg/kg of citrulline over 1-10 minutes, said second intravenous bolus being administered an hour after the first intravenous bolus of citrulline.

8. The citrulline for use intravenously in an effective amount of claim 1, wherein the method comprises a continuous infusion of citrulline at 1-10 mg/kg/hour.

9. The citrulline for use intravenously in an effective amount of claim 8, wherein the continuous infusion is for 1-24 hours.

10. The citrulline for use intravenously in an effective amount of claim 1, wherein administration ofthe citrulline raisesthe patient's plasma citrulline level above 100 pmol/L.

11. pThe citrulline for use intravenously in an effective amount of claim 1, wherein administration ofthe citrulline raisesthe patient's plasma citrulline level for up to 48 hours.

12. The citrulline for use intravenously in an effective amount of claim 1, wherein the patient is at risk for acute lung injury.

13. The citrulline for use intravenously in an effective amount of claim 1, wherein the effective amount of citrulline is an amount sufficient to prevent or reduce uncoupling of the eNOS dimer.

14. The citrulline for use intravenously in an effective amount of claim 1, wherein the patient has relative hypocitrullinemia.

15. The citrulline for use intravenously in an effective amount of claim 1, wherein administration ofthe citrulline maintains the patient's plasma citrulline level above 50 pmol/L.

16. The citrulline for use intravenously in an effective amount of claim 1, wherein administration ofthe citrulline maintains the patient's plasma citrulline level above 75 pmol/L.

17. The citrulline for use intravenously in an effective amount of claim 1, wherein administration ofthe citrulline maintains the patient's plasma citrulline level above 150 pmol/L.

18. The citrulline for use intravenously in an effective amount of claim 2, wherein the complication is sickle cell crisis, vaso-occlusive crisis, acute chest syndrome, aplastic crisis, hemolytic crisis, dactylitis, seizure, priapism, splenic séquestration crisis, or a combination thereof.