PL-3994: A Pharmacological and Clinical Development Review of a Natriuretic Peptide Receptor-A Agonist

1. Introduction to PL-3994

PL-3994 is an investigational therapeutic agent that has been the subject of research and development by Palatin Technologies for various potential medical indications.[1] At its core, PL-3994 is designed as an agonist of the natriuretic peptide receptor-A (NPR-A), a mechanism that underpins its exploration in diverse conditions ranging from respiratory to cardiovascular diseases. Initial information provided for this report, including its DrugBank identifier DB15355, highlighted its investigation in clinical trial NCT01304628 for mild to moderate asthma.[4] This trial serves as a key reference point in understanding its developmental trajectory.

Beyond the respiratory domain, the pharmacological characteristics of PL-3994 have prompted investigations into its utility for cardiovascular disorders, notably hypertension and heart failure.[1] The multi-indication pursuit for PL-3994 suggests a foundational belief in its broad therapeutic applicability, stemming from the pleiotropic effects mediated by NPR-A activation. NPR-A receptors are distributed across various tissues, including the airways, vasculature, and cardiac tissues. Agonism of these receptors stimulates the production of cyclic guanosine monophosphate (cGMP), which can mediate a range of physiological responses such as bronchodilation, vasodilation, and natriuresis/diuresis. It is likely that the developers aimed to harness these diverse effects for different pathological states. The initial focus on asthma, as indicated in the trial NCT01304628, may have represented an effort to identify a novel therapeutic niche for a bronchodilator operating via a distinct mechanism from existing treatments. However, the subsequent and concurrent exploration of cardiovascular indications aligns with the more established pharmacological roles of natriuretic peptide mimetics. The eventual "Withdrawn" status of the asthma trial [4] further hints at a potential strategic shift or deprioritization of this particular indication in favor of others or due to undisclosed challenges.

2. Chemical Profile and Classification

PL-3994 is identified by several key chemical and database identifiers, which are crucial for its unambiguous characterization.

• Name: PL-3994 [1]
• DrugBank ID: DB15355 [4]
• CAS Number: 952295-80-6 [4]
• Molecular Formula: C82​H127​N27​O20​S2​ [4]
• Molecular Weight: 1875.21 g/mol [4]
• Synonyms: PL 3994 [6]; Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH2 [5]

Regarding its classification, initial database entries, such as those in DrugBank, categorize PL-3994 broadly as a "Small Molecule".[4] However, a more detailed examination of its chemical structure and pharmacological descriptions reveals that PL-3994 is, in fact, a synthetic cyclic peptide or peptide mimetic.[1] Specifically, its chemical structure is Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH2.[5] This classification as a peptide is fundamental to understanding its properties and mechanism of action as a natriuretic peptide receptor-A (NPR-A) agonist.[5]

The discrepancy in classification likely arises from simplified categorization in broader databases. The detailed chemical structure and its function as an analog or mimetic of endogenous natriuretic peptides firmly place it within the peptide therapeutic class.[1] This distinction is critical because peptides generally exhibit different pharmacokinetic profiles—such as routes of administration (often parenteral, like the subcutaneous and intratracheal routes explored for PL-3994), metabolic stability, and cell permeability—compared to classical small organic molecules. Understanding PL-3994 as a peptide is essential for interpreting its designed resistance to neutral endopeptidase (NEP), a peptidase enzyme, and its specific agonist activity at peptide receptors.[1] Engineering NEP resistance is a common and vital strategy to overcome the inherently short half-lives of many natural therapeutic peptides.

Table 1: PL-3994 Key Identifiers and Physicochemical Properties

Property	Value	Source Snippet(s)
Name	PL-3994	5
DrugBank ID	DB15355	4
CAS Number	952295-80-6	4
Molecular Formula	C82​H127​N27​O20​S2​	4
Molecular Weight	1875.21 g/mol	4
Chemical Class	Synthetic cyclic peptide, Natriuretic Peptide Receptor-A (NPR-A) agonist	5
Full Peptide Name	Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH2	5
IUPAC Name	2--19-butyl-13,22-bis(3-carbamimidamidopropyl)-4--3-(4-hydroxyphenyl)-1-oxopropan-2-yl]carbamoyl]-37-(heptanoylamino)-7-(hydroxymethyl)-34-(1H-imidazol-4-ylmethyl)-28-...	7
Synonyms	PL 3994	6

3. Mechanism of Action and Molecular Targets

PL-3994 exerts its pharmacological effects primarily by acting as a potent and selective agonist of the Natriuretic Peptide Receptor-A (NPR-A), also known as Atrial Natriuretic Peptide Receptor 1 (NPR1).[1] Preclinical studies have demonstrated its high affinity for NPR-A across different species, with a reported Ki​ of 1 nM for human NPR-A (hNPR-A).[5]

The activation of NPR-A, which is a membrane-associated guanylyl cyclase, by PL-3994 leads to an increase in the intracellular concentration of the second messenger cyclic guanosine monophosphate (cGMP).[1] This was evidenced by concentration-dependent cGMP generation in cells expressing human, dog, or rat NPR-A, with EC50​ values of 2 nM, 3 nM, and 14 nM, respectively, for PL-3994.[5] Clinical studies also confirmed increases in plasma cGMP levels in hypertensive patients treated with PL-3994.[1]

In terms of receptor selectivity, PL-3994 exhibits affinity for the human natriuretic peptide receptor-C (hNPR-C), with a Ki​ of 7 nM. However, it did not stimulate cGMP generation via the human natriuretic peptide receptor-B (hNPR-B).[5] NPR-C is often regarded as a clearance receptor, involved in the removal of natriuretic peptides from circulation. Furthermore, screening against a panel of 75 diverse molecular targets showed that PL-3994, at a concentration of 1 µM, did not produce significant effects, indicating a good degree of selectivity for the natriuretic peptide system.[5]

A critical pharmacological characteristic of PL-3994 is its designed resistance to degradation by human Neutral Endopeptidase (hNEP), also known as neprilysin. In vitro studies showed that after a 2-hour incubation with hNEP, 92% of PL-3994 remained intact. This is in stark contrast to natural natriuretic peptide ligands such as Atrial Natriuretic Peptide (ANP) and C-type Natriuretic Peptide (CNP), which were almost completely metabolized (≤1% remaining) under the same conditions.[1] This NEP resistance is a cornerstone of its design, aiming to overcome the very short in vivo half-lives of endogenous natriuretic peptides, which limit their therapeutic utility.[1] By resisting this major degradation pathway, PL-3994 is engineered for a prolonged duration of action and more sustained therapeutic effects.[1] The combination of potent NPR-A agonism with NEP resistance is thus central to the therapeutic strategy for PL-3994, allowing for potentially more robust and sustained NPR-A pathway activation than achievable with natural peptides. The interaction with NPR-C, while showing binding, does not lead to cGMP generation via NPR-B, and its impact on overall clearance, when coupled with NEP resistance, appears to contribute to the extended half-life observed for PL-3994.[1]

Table 2: Preclinical Activity of PL-3994 at Natriuretic Peptide Receptors

Receptor Type	Species	Affinity (Ki​, nM)	Functional Activity (EC50​ for cGMP, nM)	Source Snippet(s)
NPR-A	Human	1	2	5
NPR-A	Dog	41	3	5
NPR-A	Rat	10	14	5
NPR-C	Human	7	No cGMP effect	5
NPR-B	Human	Not specified	No cGMP effect	5

4. Pharmacological Properties and Preclinical Findings

PL-3994 has demonstrated distinct pharmacological activities in preclinical models, relevant to its investigation in respiratory and cardiovascular diseases.

Respiratory System Effects (Relevant to Asthma):

The primary effect of PL-3994 in the respiratory system is bronchodilation, mediated by NPR-A agonism and subsequent cGMP elevation in airway smooth muscle.5

• In vitro studies showed that PL-3994 produced potent, concentration-dependent relaxation of pre-contracted guinea-pig trachea, with an IC50​ of 42.7 nM. It also elicited relaxation in pre-contracted human precision-cut lung slices (hPCLS).[5]
• In vivo, intratracheal administration of PL-3994 (1–1000 µg/kg) in anesthetized guinea pigs led to a dose-dependent inhibition of bronchoconstriction induced by aerosolized methacholine. Notably, this bronchodilatory effect, comparable to that of salbutamol at higher doses of PL-3994, was achieved without significant accompanying cardiovascular effects (e.g., changes in blood pressure or heart rate).[5]

Asthma is characterized by airway inflammation and bronchoconstriction.[5] PL-3994 offers a therapeutic mechanism distinct from existing bronchodilators, such as β2-agonists. This is potentially advantageous, as chronic use of β2-agonists can lead to tachyphylaxis (reduced effectiveness over time).[1] The local administration (intratracheal) achieving bronchodilation without systemic cardiovascular impact in preclinical models suggested a favorable therapeutic window for asthma.[5]

Cardiovascular System Effects (Relevant to Hypertension and Heart Failure):

PL-3994's NPR-A agonism also translates to significant cardiovascular effects when administered systemically.

• In a Phase 2a clinical trial involving patients with controlled hypertension, subcutaneously administered PL-3994 resulted in dose-related decreases in blood pressure, increases in plasma cGMP levels, and increases in urine volume and sodium excretion (natriuretic and diuretic effects).[1] These observations are consistent with the known physiological roles of NPR-A activation.
• For heart failure, preclinical research conducted at Temple University using a conditional cardiomyocyte-restricted Corin knock-out mouse model provided compelling data. Corin is a cardiac serine protease responsible for converting pro-natriuretic peptides (like pro-ANP and pro-BNP) into their biologically active forms. Loss-of-function mutations in the corin gene, which are relatively prevalent in certain populations (e.g., approximately 13% of Black Americans), can lead to deficient natriuretic peptide signaling and are implicated in the pathogenesis of heart failure and hypertension.[2] In this model of impaired endogenous natriuretic peptide activation, treatment with PL-3994 significantly reduced cardiac hypertrophy and attenuated the activation of pro-fibrotic and inflammatory genes.[2] This suggests that PL-3994, as a direct NPR-A agonist, can bypass the need for corin processing and effectively restore or enhance natriuretic peptide signaling in conditions of corin deficiency or heart failure. The anti-hypertrophic and anti-fibrotic effects are recognized consequences of sustained NPR-A agonism and cGMP elevation in cardiac tissue.

Pharmacokinetics (PK):

A key distinguishing feature of PL-3994 is its improved pharmacokinetic profile compared to endogenous natriuretic peptides.

• PL-3994 exhibits an extended half-life.[1] This is primarily attributed to its engineered resistance to degradation by Neutral Endopeptidase (NEP) and potentially reduced affinity for natriuretic peptide clearance receptors (NPR-C).[1] As NEP is a major enzyme responsible for the inactivation of natriuretic peptides, PL-3994's resistance to this enzyme is crucial for prolonging its presence and activity in the body.
• Early Phase 1 studies in humans indicated that biological effects, such as smooth muscle relaxation, were observed for up to eight hours following single administrations of PL-3994.[1] This prolonged duration of action is a direct consequence of its enhanced metabolic stability.

The route of administration influences the observed pharmacodynamic effects. While intratracheal delivery for asthma aimed for localized airway effects with minimal systemic cardiovascular impact [5], systemic administration (subcutaneous or intravenous) for cardiovascular indications leverages these very systemic effects on blood pressure and fluid balance.[1]

5. Clinical Development Program

PL-3994 has been evaluated in clinical trials for several indications, including asthma, hypertension, and heart failure with preserved ejection fraction (HFpEF).[3] The development program has seen varied outcomes and statuses across these trials.

Table 3: Summary of PL-3994 Clinical Trials

NCT Number	Phase	Indication	Status (Date if available)	Key Design/Intervention	Reported Outcomes/Endpoints	Participants	Source Snippet(s)
NCT01304628	2a	Mild to Moderate Asthma	Withdrawn	SC PL-3994, double-blind, multi-center, escalating dose, placebo-controlled, cross-over	Evaluate safety, efficacy, tolerability	1	4
NCT00686803	2a	Controlled Hypertension	Completed	SC PL-3994, single-center, single ascending dose, placebo-controlled, randomized, double-blind	Safety, tolerability, PK, PD (dose-related BP decrease, cGMP increase, increased urine volume & sodium excretion)	-	1
NCT04318145	2	Heart Failure with Preserved Ejection Fraction (HFpEF)	Unknown Status (AdisInsight: Ongoing Nov 2022)	IV PL-3994, multi-center, unblinded, single-arm study to evaluate sex differences in acute effects on myocardial cGMP enhancement and hemodynamics	-	1	3
Not Applicable	1	Healthy Volunteers	Completed	Single SC doses	Safety, tolerability, PK (significantly longer half-life than endogenous NPs, biological effects up to 8 hours)	-	1

Detailed Clinical Trial Information:

• NCT01304628 (Asthma): This Phase 2a trial was designed to evaluate the safety, efficacy, and tolerability of subcutaneously administered PL-3994 in patients with mild to moderate asthma using a cross-over design.[6] However, the trial status is consistently reported as Withdrawn.[4] The specific reasons for withdrawal are not detailed in the available information, with some databases noting "just information to hide" as a placeholder.[4] This withdrawal strongly suggests a halt or deprioritization of PL-3994 development for asthma.
• NCT00686803 (Hypertension): This Phase 2a study assessed single ascending subcutaneous doses of PL-3994 in subjects with controlled hypertension.[6] The trial is reported as Completed.[4] Findings indicated that PL-3994 produced dose-related reductions in blood pressure, increases in plasma cGMP, and enhanced urine volume and sodium excretion, all consistent with its mechanism as an NPR-A agonist. Importantly, no serious or severe adverse events were reported in this study.[1] The positive pharmacokinetic and pharmacodynamic signals from this trial likely provided support for investigating PL-3994 in other cardiovascular conditions like heart failure.
• NCT04318145 (Heart Failure with Preserved Ejection Fraction - HFpEF): This Phase 2 trial was designed as a multi-center, unblinded, single-arm study to evaluate sex differences in the acute effects of intravenously administered PL-3994 on myocardial cGMP enhancement and hemodynamics in patients with HFpEF.[3] The status of this trial is less clear; while some databases list it as "Unknown Status" [4], AdisInsight reported it as "still in phase II trials for Heart failure in USA (IV, Infusion)" as of November 2022, and noted that Palatin Technologies had planned this Phase 2a trial for the fourth quarter of 2020.[3] The lack of recent updates from Palatin on this program (discussed further in Section 7) raises questions about its current activity.
• Phase 1 Studies (Healthy Volunteers): Completed Phase 1 studies in healthy volunteers demonstrated that PL-3994 has a significantly longer half-life than endogenous natriuretic peptides, with biological effects attributable to smooth muscle relaxation observed for up to eight hours following single administrations. These early studies also reported no serious or severe adverse events.[1]

The clinical development path of PL-3994 shows an evolution in focus. While initially explored for asthma, this indication appears to have been abandoned. The positive results in hypertension likely encouraged further exploration in heart failure, a more complex cardiovascular disease. However, the current momentum of the heart failure program is uncertain. This trajectory may reflect a strategic realignment by Palatin Technologies towards indications where natriuretic peptides have a more established biological rationale or where the systemic effects of PL-3994 are better leveraged.

6. Safety and Tolerability Profile

The available information from early-phase clinical trials and preclinical studies suggests that PL-3994 was generally well-tolerated.

• In Phase 1 studies conducted in healthy volunteers and the Phase 2a trial (NCT00686803) in patients with controlled hypertension, PL-3994 was reported to have no serious or severe adverse events.[1] The observed pharmacodynamic effects, such as dose-related decreases in blood pressure in the hypertension trial, are consistent with the known actions of NPR-A agonists and were anticipated.[1]
• Preclinical studies evaluating PL-3994 for asthma indications also provided positive safety signals. Specifically, when administered intratracheally to guinea pigs to assess bronchodilator effects, PL-3994 did not cause significant effects on cardiovascular parameters such as mean arterial blood pressure or heart rate.[5] This finding is particularly relevant for an asthma therapeutic, as it suggests the potential for achieving local airway effects without inducing undesirable systemic cardiovascular side effects, which can be a concern with some systemically acting vasodilators.

Overall, the early safety database for PL-3994 appeared favorable, supporting its progression into further clinical investigation for various indications. The drug's resistance to NEP and potentially optimized interaction with clearance receptors were hypothesized to contribute to a favorable profile by allowing for sustained therapeutic action with potentially fewer issues related to rapid clearance or high peak concentrations of metabolites.[5]

However, it is important to note that the "Withdrawn" status of the Phase 2a asthma trial (NCT01304628) and the "Unknown Status" of the Phase 2 HFpEF trial (NCT04318145) leave open questions regarding the safety, tolerability, or efficacy findings from these later or more specific patient population studies. The provided information does not clarify whether any adverse findings contributed to these trial outcomes or if the decisions were based on other factors, such as insufficient efficacy or strategic considerations.

7. Current Development Status and Future Outlook

The current development status of PL-3994 by Palatin Technologies appears to be inactive or significantly deprioritized. This assessment is based on the absence of PL-3994 in recent company communications detailing their pipeline and anticipated corporate milestones. Press releases from Palatin Technologies in January 2025 and April 2025 extensively outline their focus on melanocortin receptor agonist programs for indications such as obesity, dry eye disease, ulcerative colitis, and diabetic kidney disease, with no mention of ongoing or planned activities for PL-3994.[11]

This contrasts with earlier reports. For instance:

• An SEC filing by Palatin in 2017 indicated that PL-3994 was in development for heart failure, with Phase 2A clinical trials anticipated to commence later that calendar year.[13]
• AdisInsight reported in November 2022 that PL-3994 was still in Phase II trials for heart failure in the USA (via IV infusion), referencing trial NCT04318145.[3]
• Synapse Patsnap currently lists the asthma and hypertension indications for PL-3994 as "Inactive Indication," while heart failure with normal ejection fraction is listed as Phase 2.[6] The asthma trial NCT01304628 is marked as "Withdrawn" [4], and the HFpEF trial NCT04318145 has an "Unknown Status" in ClinicalTrials.gov as per these database entries.[4]

Synthesizing these pieces of information, it is evident that while PL-3994 showed initial promise and progressed to Phase 2 clinical trials for multiple indications, its active development by Palatin Technologies has likely ceased or been suspended. The disconnect between the earlier active investigation period and its current omission from the company's prioritized pipeline is significant. This suggests that PL-3994 may have encountered substantial hurdles, which could range from unpublicized negative or inconclusive trial results, manufacturing challenges, unfavorable competitive landscaping, to a strategic decision by Palatin to allocate resources to its melanocortin platform, which now appears to be their primary focus.

The journey of PL-3994 highlights the inherent risks and complexities of drug development. Even with a rational design aimed at improving upon endogenous molecules (e.g., NEP resistance for enhanced half-life) and positive early-phase data, progression to market is not guaranteed. Factors beyond initial efficacy and safety, including the cost of goods, formulation challenges for peptide therapeutics, the evolving treatment landscape for targeted indications, and overall corporate strategy, play crucial roles in determining the fate of an investigational drug.

8. Conclusion

PL-3994 is a synthetic cyclic peptide engineered as a potent and selective natriuretic peptide receptor-A (NPR-A) agonist. Its key design feature is resistance to degradation by neutral endopeptidase (NEP), intended to provide a longer duration of action compared to naturally occurring natriuretic peptides. This pharmacological profile underpinned its investigation for therapeutic use in conditions where NPR-A activation is beneficial.

Preclinical studies demonstrated promising bronchodilator effects, suggesting utility in asthma, and also indicated potential in heart failure through reduction of cardiac hypertrophy and fibrosis. Early-phase clinical trials in healthy volunteers and patients with controlled hypertension showed that PL-3994 was generally well-tolerated, increased cGMP levels, and exerted expected pharmacodynamic effects such as blood pressure reduction and natriuresis/diuresis.

PL-3994's clinical development included a Phase 2a trial for asthma (NCT01304628), which was subsequently withdrawn. A Phase 2a trial in hypertension (NCT00686803) was completed with positive initial findings. A Phase 2 trial for heart failure with preserved ejection fraction (NCT04318145) was initiated, but its current status is uncertain and likely inactive.

Despite its interesting pharmacological attributes and some positive early data, PL-3994 appears to be no longer an active part of Palatin Technologies' prioritized development pipeline. The absence of PL-3994 in recent company communications on future milestones strongly suggests that its development for the explored indications has been deprioritized or discontinued. The specific reasons for this are not fully elucidated in the available information but reflect the multifaceted challenges inherent in advancing novel therapeutics through the stages of clinical development.

While PL-3994 itself may not reach the market, the research conducted has contributed to the understanding of NEP-resistant natriuretic peptide mimetics. The insights gained from its design, preclinical characterization, and early human studies could still inform future efforts in the development of therapeutics targeting the natriuretic peptide system. The trajectory of PL-3994 serves as a case study in the complexities of pharmaceutical R&D, where initial scientific promise must navigate numerous scientific, strategic, and financial hurdles to achieve clinical and commercial success.

Works cited

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