Angiotensin-converting enzyme (ACE); the IC50 value for inhibiting rabbit small intestinal brush border membrane ACE was 0.02 μM [2]

Angiotensin-converting enzyme (ACE) inhibitor ramipril (HOE-498) has an IC50 of 5 nM[1]. However, in endothelial cells expressing an S1270A ACE mutant or in ACE-deficient cells, ramipril (HOE-498) is unable to activate JNK or boost the nuclear accumulation of c-Jun. Instead, it increases the activity of ACE-associated CK2 and the phosphorylation of ACE Ser1270 in cultured endothelial cells. Long-term Ramipril use raises ACE expression in mouse lung in vivo and primary cultures of human endothelial cells, a phenomenon that can be avoided by pretreating with the JNK inhibitor SP600125[2].

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Ramipril (HOE-498) exhibited inhibitory activity against angiotensin-converting enzyme (ACE) isolated from rabbit small intestinal brush border membrane. In the enzyme activity assay using Hippuryl-His-Leu as the substrate, Ramipril (HOE-498) dose-dependently reduced the ACE-mediated hydrolysis of the substrate. The half-maximal inhibitory concentration (IC50) of Ramipril (HOE-498) for rabbit small intestinal brush border membrane ACE was determined to be 0.02 μM [2]

In contrast to the apoptosis effect in vitro, chronic in vivo administration of Ramipril (HOE-498) to rats at a dosage that has similar hypotensive effects in vitro HUVECs considerably lowers the rate of LPS-induced apoptosis relative to the other ACE inhibitors[3 [4].

Preparation of rabbit small intestinal brush border membrane: The small intestine of rabbits was excised, and the mucosa was scraped off. The mucosal tissue was homogenized in a suitable buffer, followed by differential centrifugation steps (including low-speed centrifugation to remove debris and high-speed centrifugation to pellet the brush border membrane). The resulting membrane pellet was resuspended in buffer to obtain the ACE-containing enzyme source.
- ACE activity assay procedure: The reaction mixture contained the prepared rabbit small intestinal brush border membrane enzyme solution, the substrate Hippuryl-His-Leu (dissolved in buffer), and different concentrations of Ramipril (HOE-498). The mixture was incubated at 37°C for a specified period. After incubation, trichloroacetic acid was added to terminate the reaction. The mixture was then centrifuged to remove precipitated proteins. The absorbance of the supernatant was measured at 228 nm to determine the amount of hippuric acid released (a product of substrate hydrolysis by ACE). The ACE activity was calculated based on the absorbance value, and the inhibitory rate of Ramipril (HOE-498) at each concentration was computed. The IC50 value was derived from the dose-response curve [2]

Dissolved in distilled water by using gum arabic (10% w/v); 0.03-10 mg/kg; Oral gavage
Male spontaneously hypertensive rats

Absorption, Distribution and Excretion
Absorption is at least 50-60%. Food reduces the rate of absorption in the gastrointestinal tract but does not affect the extent of absorption. The absolute bioavailability of ramipril and ramiprilat after oral administration is 28% and 44%, respectively (compared to intravenous administration). The serum concentration of ramiprilat remains unchanged after opening the capsule and dissolving the contents in water, apple juice, or applesauce. Approximately 60% of the dose is excreted unchanged (<2%) in the urine as ramipril and its metabolites after oral administration. Approximately 40% of the dose is present in the feces, including unabsorbed drug and drug and metabolites excreted via bile. Urinary excretion may be reduced in patients with impaired renal function. Renal clearance of ramipril and ramiprilat has been reported to be 7.2 mL/min/1.73 m², respectively. According to reports, the mean renal clearance of ramipril and ramiprilat in healthy elderly patients with normal renal function was 10.7 mL/min and 126.8 mL/min, respectively. Furthermore, the Cmax value of ramiprilat was approximately 20% higher in this population. Although impaired renal function did not appear to affect the pharmacokinetics of ramipril, plasma concentrations and half-lives of ramiprilat increased. Ramipril concentrations were initially elevated in patients with hepatic failure, and the tmax value of ramiprilat was prolonged due to decreased drug metabolism. However, the steady-state concentration of ramiprilat in patients with hepatic failure was the same as in healthy patients. /Breast Milk/ Ramipril and its metabolites were not detected in breast milk after a single oral dose of 10 mg ramipril. Peak plasma concentration (Cmax) is reached within 1 hour after oral administration of ramipril. Absorption is at least 50% to 60% and is not affected by food in the gastrointestinal tract, although the absorption rate may be reduced. Ramipril exhibits a three-phase decline in plasma concentration (initial rapid decline, apparent elimination phase, and terminal elimination phase). The initial rapid decline represents drug distribution to larger peripheral compartments, followed by binding to plasma and tissue angiotensin-converting enzyme (ACE), with a half-life of 2 to 4 hours. Due to ramipril's potent binding affinity to ACE and its slow dissociation from the enzyme, its elimination process occurs in two phases. The apparent elimination phase corresponds to the clearance of free ramipril, with a half-life of 9 to 18 hours. The terminal elimination phase has a longer half-life (>50 hours) and may represent the binding/dissociation kinetics of the ramipril/ACE complex, without leading to drug accumulation. After multiple daily doses of 5 to 10 mg ramipril, the half-life of ramipril within the therapeutic concentration range is 13 to 17 hours. Plasma concentrations of both ramipril and ramipril increase with increasing dose, but not strictly in a dose-proportional relationship. However, within the dose range of 2.5 mg to 20 mg, the 24-hour AUC of ramipril is dose-dependent. The absolute bioavailability of ramipril (5 mg orally) and ramipril (44%) compared to the same intravenous dose is 28% and 44%, respectively. For more complete data on the absorption, distribution, and excretion of ramipril (7 metabolites), please visit the HSDB record page. Metabolism/Metabolites 75% of the total metabolism of ramipril is carried out by the liver. Of this, 25% is metabolized by the liver via hepatic esterases to produce the active metabolite ramipril. 100% is metabolized by the kidneys to convert ramipril to ramipril. Other metabolites, such as diketopiperazine ester, diketopiperazine acid, and glucuronides of ramipril and ramipril, are inactive.
Cleavage of the ester group (primarily in the liver) converts ramipril to its active diacid metabolite, ramiprilat. Peak plasma concentrations of ramiprilat are reached 2 to 4 hours after administration. The serum protein binding rate of ramipril is approximately 73%, while that of ramiprilat is approximately 56%; in vitro studies have shown that these percentages are concentration-independent within the concentration range of 0.01 μg/mL to 10 mcg/mL.
Following oral administration, ramipril is rapidly converted to ramiprilat in dogs via deesterification. The bioavailability of ramiprilat after daily administration of 0.25 mg/kg ramipril is approximately 6.7%.
Ramipril is a prodrug with virtually no pharmacological activity until it is hydrolyzed to ramiprilat in the liver. Ramipril is almost completely metabolized to ramiprilat, which has approximately six times the angiotensin-converting enzyme (ACE) inhibitory activity of ramipril. Additionally, ramipril is metabolized to diketopiperazine ester, diketopiperazine acid, and glucuronide of both ramipril and ramiprilat; these metabolites are inactive.
Biological Half-Life
The plasma concentration of ramiprilat exhibits a three-phase decline. The initial rapid decline represents drug distribution to tissues, with a half-life of 2–4 hours. The apparent elimination phase has a half-life of 9–18 hours, which is considered to represent the clearance of free drug. The terminal elimination phase has a half-life > 50 hours, which is considered to represent clearance due to the slow dissociation of the drug after binding to ACE. The half-life of ramiprilat after multiple daily (MDD) administration is dose-dependent, with a half-life of 13-17 hours for 5-10 mg MDD and 27-36 hours for 2.5 mg MDD. Plasma concentrations of ramiprilat (a metabolite of ramiprilat) exhibit a three-phase decline (initial rapid decline, apparent elimination phase, and terminal elimination phase). The initial rapid decline represents drug distribution to larger peripheral compartments, followed by binding to ACE in plasma and tissues, with a half-life of 2 to 4 hours. Due to the potent binding of ramiprilat to ACE and its slow dissociation from the enzyme, its elimination process occurs in two phases. The apparent elimination phase corresponds to the clearance of free ramiprilat, with a half-life of 9 to 18 hours. The prolonged half-life (>50 hours) of the terminal elimination phase likely represents the binding/dissociation kinetics of the ramiprilat/ACE complex. It does not lead to drug accumulation. After multiple daily doses of 5 mg to 10 mg ramipril, the half-life of ramipril concentration within the therapeutic range is 13 to 17 hours.

Toxicity Summary
Identification and Uses: Ramipril is a prodrug with minimal pharmacological activity before being hydrolyzed in the liver to ramiprilat. It is an angiotensin-converting enzyme (ACE) inhibitor indicated for the treatment of hypertension. It is also used in stable patients who develop congestive heart failure within days of an acute myocardial infarction. Human Studies: Rare ACE inhibitor-related clinical syndromes may occur, initially presenting as cholestatic jaundice. This syndrome may progress to fulminant hepatic necrosis and can be fatal. Patients taking ACE inhibitors, including ramipril, should immediately discontinue the drug and be monitored appropriately if jaundice or significantly elevated liver enzymes occur. Anaphylactic Reactions, including anaphylactic shock and angioedema (including laryngeal or tongue edema), can be fatal. Angioedema of the head and neck involving the tongue, glottis, or larynx can lead to airway obstruction. If stridor or angioedema of the face, tongue, or glottis occurs, ramipril should be immediately discontinued and appropriate treatment (e.g., epinephrine) initiated. Use of drugs acting on the renin-angiotensin system in the mid-to-late stages of pregnancy can reduce fetal renal function and increase fetal and neonatal morbidity and mortality. The resulting oligohydramnios may be associated with fetal pulmonary hypoplasia and skeletal malformations. Potential neonatal adverse reactions include craniosynostosis, anuria, hypotension, renal failure, and death. Ramipril should be discontinued as soon as pregnancy is confirmed. Mutagenic activity against unplanned DNA synthesis was not detected in human cell lines. Animal studies: No tumorigenic effects were observed in rats administered ramipril by gavage for up to 24 months (up to 500 mg/kg/day) or mice administered ramipril by gavage for up to 18 months (up to 1000 mg/kg/day). A rat study showed no adverse effects on fertility even at doses up to 500 mg/kg/day. Organogenesis and functional development of the rat kidneys continue after birth. Using a littermate design, the sensitivity of rat kidneys to angiotensin-converting enzyme inhibitors was characterized at the third week after birth. No treatment-related effects were observed in rats administered the drug on day 21 (PND 21). Following administration on day 14 (PND 14), a dose-related increase in mean serum urea nitrogen and/or creatinine levels was observed on day 17 (PND 17), but these levels returned to normal by day 28 (PND 28). On day 17 (PND 17), macroscopic and microscopic changes in the kidneys included renal tubular hypoplasia, renal papillary edema, cortical tubular dilatation, hydronephrosis (renal pelvis dilatation), and tubular basophilia; these changes remained significant and more severe on day 14 (PND 28). No mutagenic activity was detected in the Ames test for bacteria, the mouse micronucleus test, or the positive gene mutation assay using Chinese hamster ovary cell lines.

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Hepatotoxicity
As with other ACE inhibitors, ramipril is associated with a low incidence of elevated serum transaminases (
Probability score: C (likely a rare cause of clinically significant liver injury)).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
Since there is no information on the use of ramipril during lactation, alternative medications are recommended, especially for breastfed newborns or preterm infants.
◉ Effects on breastfed infants
No relevant published information found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information found as of the revision date. Date.

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Protein binding
The protein binding rate of ramipril is approximately 73%, and that of ramiprilat is approximately 56%. Protein binding is concentration-independent within the concentration range of 0.1 μg/mL to 10 μg/mL.
Interactions
In elderly patients, patients with low blood volume (including those receiving diuretics), or patients with impaired renal function, concomitant use of nonsteroidal anti-inflammatory drugs (NSAIDs, including selective cyclooxygenase-2 (COX-2) inhibitors) and angiotensin-converting enzyme (ACE) inhibitors (including ramipril) may lead to worsening of renal function and even acute renal failure. These effects are usually reversible. Patients receiving ramipril and NSAIDs should have their renal function monitored regularly. The antihypertensive effect of ACE inhibitors (including ramipril) may be suppressed. NSAIDs may weaken their effect.
Elevated serum lithium levels and symptoms of lithium toxicity have been reported in patients taking angiotensin-converting enzyme inhibitors while receiving lithium therapy; therefore, frequent monitoring of serum lithium levels is recommended. The risk of lithium toxicity may increase if diuretics are used concurrently.
Concomitant use of ramipril with other drugs that can increase serum potassium levels may lead to hyperkalemia. Serum potassium levels should be monitored in such patients. Patients taking diuretics, especially those who have recently started taking diuretics, may sometimes experience an excessive drop in blood pressure after starting ramipril. The likelihood of ramipril-induced hypotension can be minimized by reducing or discontinuing the diuretic or increasing salt intake before starting ramipril. If this is not possible, the starting dose should be reduced.

[1]. Combined blockade of AT1-receptors and ACE synergistically potentiates antihypertensive effects in SHR. J Hypertens, 2004. 22(3): p. 611-8.

[2]. Stevens, B.R., M.I. Phillips, and A. Fernandez, Ramipril inhibition of rabbit (Oryctolagus cuniculus) small intestinal brush border membrane angiotensin converting enzyme. Comp Biochem Physiol C, 1988. 91(2): p. 493-7.

[3]. Angiotensin-converting enzyme is involved in outside-in signaling in endothelial cells. Circ Res, 2004. 94(1): p. 60-7.

[4]. Differences in the effect of angiotensin-converting enzyme inhibitors on the rate of endothelial cell apoptosis: in vitro and in vivo studies. Cardiovasc Drugs Ther, 2007. 21(6): p. 423-9.

Therapeutic Uses
Angiotensin-converting enzyme inhibitor; antihypertensive drug. ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes a summary of the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure being studied); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for patient health information) and PubMed (for citations and abstracts of academic articles in the medical field). Ramipril is listed in the database. Ramipril capsules are indicated for the treatment of hypertension to lower blood pressure. Lowering blood pressure reduces the risk of fatal and non-fatal cardiovascular events, primarily stroke and myocardial infarction. These benefits have been observed in controlled trials of antihypertensive drugs across multiple pharmacological classes, including this drug. ...Ramipril capsules can be used alone or in combination with thiazide diuretics. /US product label contains/
Ramipril capsules are indicated for stable patients who develop clinical symptoms of congestive heart failure within days of an acute myocardial infarction. Use of ramipril capsules in these patients has been shown to reduce the risk of death (primarily cardiovascular death) and to reduce the risk of heart failure-related hospitalizations and progression to severe/refractory heart failure. /US product label contains/
For more complete data on the therapeutic uses of ramipril (8 types), please visit the HSDB record page.

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Drug Warning
/Black Box Warning/ Ramipril should be discontinued as soon as pregnancy is discovered. Drugs that act directly on the renin-angiotensin system may cause damage or even death to the developing fetus.
Use of drugs that act on the renin-angiotensin system in the second and third trimesters of pregnancy can reduce fetal renal function and increase fetal and neonatal morbidity and mortality. Oligohydramnios resulting from this condition may be associated with fetal lung malformation and skeletal deformities. Potential neonatal adverse reactions include craniosynostosis, anuria, hypotension, renal failure, and death. Ramipril should be discontinued as soon as pregnancy is confirmed. These adverse consequences are usually associated with use of this medication in the second or third trimester. Most epidemiological studies investigating fetal malformations following early pregnancy use of antihypertensive drugs have not differentiated between drugs affecting the renin-angiotensin system and other antihypertensive drugs. Proper management of maternal hypertension during pregnancy is crucial for optimizing maternal and infant outcomes. Use of angiotensin-converting enzyme (ACE) inhibitors in the second or third trimester can lead to fetal and neonatal morbidity and mortality. Use of ACE inhibitors in early pregnancy also increases the risk of severe congenital malformations in the fetus. Once pregnancy is confirmed, medication should be discontinued as soon as possible unless continued use is deemed life-saving. Almost all pregnant women can successfully switch to other therapies until the end of pregnancy. Angiotensin-converting enzyme (ACE) inhibitors /
Rare angiotensin-converting enzyme (ACE) inhibitor-related clinical syndromes may occur, initially presenting as cholestatic jaundice; it may progress to fulminant hepatic necrosis and can be fatal. Patients taking ACE inhibitors (including ramipril) should discontinue the drug and be monitored appropriately if jaundice or significantly elevated liver enzymes occur.
For more complete data on drug warnings for ramipril (18 in total), please visit the HSDB records page.
Pharmacodynamics

Ramipril is an ACE inhibitor, similar to benazepril, fosinopril, and quinapril. It is an inactive prodrug that is converted to ramiprilat in the liver (the main site of activation) and kidneys. Ramiprilat exerts its antihypertensive effect by antagonizing the renin-angiotensin-aldosterone system (RAAS). The RAAS is a mechanism for maintaining homeostasis and is responsible for regulating hemodynamics, water, and electrolyte balance. When the sympathetic nervous system is excited or renal blood pressure or blood flow decreases, renin is released from the granulosa cells of the renal juxtaglomeruli. In the blood, renin cleaves circulating angiotensinogen into angiotensin II (AT1), which is then cleaved into angiotensin II (AT2) by angiotensin-converting enzyme (ACE). AT2 raises blood pressure through several mechanisms. First, it stimulates the adrenal cortex to secrete aldosterone. Aldosterone reaches the distal convoluted tubule (DCT) and collecting duct of the nephron, promoting sodium and water reabsorption by increasing the number of sodium channels and sodium-potassium ATPases on the cell membrane. Second, angiotensin II (ATII) stimulates the posterior pituitary gland to secrete vasopressin (also known as antidiuretic hormone or ADH). ADH further promotes renal water reabsorption by inserting aquaporin 2 (AQP2) channels into the apical membrane of distal convoluted tubule and collecting duct cells. Third, ATII raises blood pressure by directly constricting arterial blood vessels. Stimulation of type I ATII receptors on vascular smooth muscle cells triggers a series of events that ultimately lead to muscle cell contraction and vasoconstriction. In addition to these main effects, ATII also induces thirst response by stimulating hypothalamic neurons. Angiotensin-converting enzyme (ACE) inhibitors inhibit the rapid conversion of angiotensin I (ATI) to ATII and antagonize the increase in blood pressure caused by the renin-angiotensin-aldosterone system (RAAS). Angiotensin-converting enzyme (also known as kininase II) is also involved in the enzymatic inactivation of bradykinin (a vasodilator). Inhibition of bradykinin inactivation increases bradykinin levels and maintains the effect of ramipril by increasing vasodilation and lowering blood pressure. Ramipril (HOE-498) is an angiotensin-converting enzyme inhibitor (ACEI) class of drugs that exert its biological effects by inhibiting the activity of ACE[2]

C23H32N2O5

416.51

416.231

87333-19-5

Ramipril-d5;1132661-86-9;Ramipril-d3;2673269-81-1

5362129

White to off-white solid powder

1.2±0.1 g/cm3

616.2±55.0 °C at 760 mmHg

106-108°C

326.4±31.5 °C

0.0±1.9 mmHg at 25°C

1.556

3.41

2

6

10

30

619

5

CCOC(=O)[C@H](CCC1=CC=CC=C1)N[C@@H](C)C(=O)N2[C@H]3CCC[C@H]3C[C@H]2C(=O)O

HDACQVRGBOVJII-JBDAPHQKSA-N

InChI=1S/C23H32N2O5/c1-3-30-23(29)18(13-12-16-8-5-4-6-9-16)24-15(2)21(26)25-19-11-7-10-17(19)14-20(25)22(27)28/h4-6,8-9,15,17-20,24H,3,7,10-14H2,1-2H3,(H,27,28)/t15-,17-,18-,19-,20-/m0/s1

(2S,3aS,6aS)-1-[(2S)-2-[[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl]amino]propanoyl]-3,3a,4,5,6,6a-hexahydro-2H-cyclopenta[b]pyrrole-2-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 3.25 mg/mL (7.80 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3.25 mg/mL (7.80 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 3.25 mg/mL (7.80 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% Propylene glycol: 30 mg/mL

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Solubility in Formulation 5: 20 mg/mL (48.02 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)