C-Peptide: From Biosynthetic Byproduct to Potential Therapeutic Agent for Diabetic Complications

I. Introduction to C-Peptide

A. Historical Context and Evolving Understanding

C-peptide, or connecting peptide, was first identified as the segment linking the A- and B-chains within the proinsulin molecule.[1] For many years following its discovery, it was largely considered a biologically inert byproduct of insulin biosynthesis, with its primary clinical utility being a marker for endogenous insulin production and pancreatic β-cell function.[3] This perception stemmed from early studies that failed to demonstrate direct insulin-like effects on glucose metabolism.[4] The measurement of C-peptide levels became, and remains, a valuable tool in distinguishing between type 1 and type 2 diabetes mellitus (T1DM and T2DM), assessing residual β-cell function in insulin-treated patients, and evaluating states of hypoglycemia.[3]

However, over the past few decades, a paradigm shift has occurred. A growing body of evidence from preclinical and clinical studies has revealed that C-peptide is, in fact, a biologically active peptide hormone with its own specific cellular targets and physiological effects.[4] This revised understanding was partly fueled by observations that T1DM patients who retained some level of endogenous C-peptide production experienced a lower incidence and severity of chronic microvascular complications, such as neuropathy, nephropathy, and retinopathy, compared to those with no detectable C-peptide.[4] This suggested a protective role for the peptide, independent of insulin's glycemic control. The historical undervaluation of C-peptide's intrinsic bioactivity likely delayed concerted research into its therapeutic potential for these debilitating diabetic complications. The initial focus on insulin as the sole active product of proinsulin processing, coupled with C-peptide's lack of direct glucose-lowering effects, contributed to this oversight. The subsequent recognition of its protective roles signifies a crucial evolution in our understanding of diabetes pathophysiology, suggesting that T1DM might be better characterized as a dual hormone deficiency syndrome.

B. Nomenclature and Basic Identifiers

The standard nomenclature for this molecule is C-peptide. It is also commonly referred to as Proinsulin C-peptide, Insulin C-peptide, or Connecting Peptide.[1] For research and database purposes, it is assigned the DrugBank ID: DB16187.[1]

Two primary CAS (Chemical Abstracts Service) Numbers are associated with C-peptide:

• 59112-80-0: This CAS number is often linked to C-peptide in a general sense, or specifically to proinsulin C-peptide.[1]
• 33017-11-7: This CAS number is frequently used to identify human C-peptide specifically.[1] The presence of multiple CAS numbers may reflect different sources, purities, salt forms, or the specific context (e.g., human proinsulin C-peptide vs. mature human C-peptide) in various databases and commercial preparations. This can be a source of minor confusion and necessitates careful documentation in research to ensure clarity regarding the exact molecular entity being studied.

C. Classification: Correcting the "Small Molecule" Misconception

Contrary to the "Small Molecule" classification provided in the initial query and sometimes found in high-level database summaries (e.g., DrugBank's general classification for DB16187 [20]), C-peptide is unequivocally a peptide hormone. In humans, it is typically composed of 31 amino acids.[1] Peptides are distinguished from small molecules by their larger size, higher molecular weight, and more complex structure, being polymers of amino acids.[23] Small molecules are generally defined as organic compounds with a molecular weight of less than 900 Daltons. Human C-peptide has a molecular weight of approximately 3020 Daltons [1], placing it firmly in the peptide category. DrugBank's more detailed classification for DB16187 correctly includes "Amino Acids, Peptides, and Proteins," "Peptide Hormones," and "Peptides" [20], aligning with its biochemical nature.

Accurate classification is fundamental. Peptides, unlike typical small molecules, face distinct challenges in pharmaceutical development, including susceptibility to enzymatic degradation, poor oral bioavailability, and potential immunogenicity, all of which influence formulation strategies and routes of administration.[25] Misclassification could lead to incorrect assumptions about its drug-like properties or the most appropriate development pathways.

D. Scope of the Report

This report aims to provide a comprehensive scientific review of C-peptide. It will cover its physicochemical properties, biosynthesis, and diverse physiological roles. A detailed examination of its pharmacodynamics, including mechanisms of action and receptor interactions, and its pharmacokinetic profile will be presented. The report will extensively discuss the clinical development of C-peptide as a therapeutic agent, with a particular focus on its investigation for diabetic peripheral neuropathy, including clinical trial NCT00278980. Finally, it will evaluate its overall therapeutic potential, current challenges in its development (such as formulation and stability), safety profile, regulatory status, and future research directions.

II. Physicochemical Properties and Molecular Structure

A. Amino Acid Sequence and Structure

Human C-peptide is a single, linear polypeptide chain consisting of 31 amino acids.1 The specific amino acid sequence for human C-peptide is:

H-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-OH.

In single-letter code, this is represented as: EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ.1

Unlike many other biologically active peptides or proteins, C-peptide does not possess a well-defined, ordered tertiary structure under physiological conditions; it is considered a relatively flexible and predominantly random coil peptide.[6] This inherent flexibility might contribute to its susceptibility to proteolytic degradation in vivo, which could partly explain its relatively short biological half-life and the consequent research efforts towards developing more stable analogues.

A notable characteristic of C-peptide is the considerable variability in its amino acid sequence across different species. This is in stark contrast to the highly conserved molecular structure of insulin.[6] This species-specific sequence variation is significant because it implies that the binding interactions and biological effects of C-peptide might also differ between species. Consequently, results from animal models using non-human C-peptide or studying endogenous animal C-peptide may not always be directly translatable to the human physiological or therapeutic context unless human C-peptide or a very closely related analogue is used.

B. Molecular Formula and Weight

For the 31-amino acid human C-peptide, the molecular formula is consistently reported as C129​H211​N35​O48​.[1] The corresponding average molecular weight is approximately 3020.3 g/mol (or Daltons).[1] Some sources list alternative formulas and weights, such as C112​H179​N35​O46​ with a molecular weight of 2751.83 g/mol [16]; these likely refer to different peptide fragments, C-peptides from other species, or represent errors in databases.

C. Solubility and Stability

Lyophilized C-peptide is generally soluble in sterile aqueous solutions, with recommendations to reconstitute it in high-purity water (e.g., 18MΩ-cm H2​O) at concentrations not less than 100 µg/ml, after which it can be further diluted.[12]

The stability of C-peptide varies with its state:

• Lyophilized Form: Stable at room temperature for short durations (e.g., up to 3 weeks) but requires storage under desiccated conditions at temperatures below -18°C for long-term preservation.[12]
• Reconstituted Solution: Should be stored at 4°C for short-term use (2-7 days). For longer-term storage of the solution, temperatures below -18°C are recommended. The addition of a carrier protein, such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA), is often advised to enhance stability and prevent loss during long-term storage of reconstituted peptide solutions.[12] This practice suggests that C-peptide might be prone to adsorption to container surfaces or aggregation, especially at low concentrations, which could affect its effective concentration and biological activity if not properly managed.
• In Vivo Stability: Native C-peptide exhibits limited stability in the physiological environment, characterized by a short biological half-life of approximately 30 minutes.[1] This rapid clearance is a significant factor driving the development of long-acting formulations for therapeutic purposes.

These physicochemical properties—solubility and stability—are critical determinants for successful formulation development, ensuring the peptide maintains its integrity and biological activity during storage, handling, and administration in both experimental and therapeutic contexts.

D. Formulation and Manufacturing Considerations

C-peptide for research and potential therapeutic use is typically supplied as a lyophilized (freeze-dried) powder.[2] Synthetic C-peptide, produced through chemical peptide synthesis methods such as Solid-Phase Peptide Synthesis (SPPS) or, less commonly for longer peptides, Liquid-Phase Peptide Synthesis (LPPS), is the standard for such applications.[12] Following synthesis, rigorous purification steps, commonly employing High-Performance Liquid Chromatography (HPLC) coupled with Mass Spectrometry (MS) for characterization, are necessary to achieve high purity levels, often exceeding 95%.[2] The purification process may result in the peptide being complexed with counterions, such as trifluoroacetate (TFA), which should be noted as they can potentially influence the peptide's properties or biological effects in sensitive assays.[2] The manufacturing process, final purity, and the nature of any counterions are important considerations as they can impact the peptide's biological activity, stability, and potential immunogenicity.

Table 1: Physicochemical Properties of Human C-Peptide

Property	Value	Source(s)
Common Name	C-peptide, Connecting Peptide	1
Amino Acid Sequence (Human)	EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ	1
Number of Amino Acids	31	1
Molecular Formula (Human)	C129​H211​N35​O48​	1
Average Molecular Weight	Approx. 3020.3 Da	1
CAS Number (Human specific)	33017-11-7	1
CAS Number (General)	59112-80-0	1
DrugBank ID	DB16187	1
Solubility	Soluble in aqueous solutions; lyophilized form reconstituted in sterile water	12
Isoelectric Point (Predicted)	Acidic (XLogP3 from PubChem suggests -12.6, indicative of net negative charge at physiological pH)	1 (via PubChem)
In Vivo Half-life (Native)	Approx. 20-30 minutes	1
Storage (Lyophilized)	Long-term at ≤ -18°C, desiccated	12
Storage (Reconstituted)	Short-term (2-7 days) at 4°C; long-term at ≤ -18°C (carrier protein recommended)	12
Structure	Flexible, predominantly random coil	6

III. Biosynthesis and Endogenous Physiological Roles

A. Proinsulin Processing and Co-secretion with Insulin

C-peptide originates as an integral part of the proinsulin molecule, serving as a "connecting peptide" that links the insulin A-chain to its B-chain.[1] This connection is not merely structural; C-peptide plays an essential role during insulin biosynthesis by facilitating the correct folding of proinsulin and the proper formation of the inter- and intra-chain disulfide bridges that are critical for the mature insulin structure and function.[4] This intricate folding process occurs within the endoplasmic reticulum of pancreatic β-cells.

Following correct folding, proinsulin is transported to the Golgi apparatus and then packaged into secretory granules. Within these granules, proinsulin undergoes proteolytic cleavage by specific enzymes, primarily prohormone convertases PC1/3 and PC2, followed by the action of carboxypeptidase E.[1] This enzymatic processing excises the C-peptide segment, resulting in the formation of the mature, two-chain insulin molecule and free C-peptide. Both insulin and C-peptide are then stored within these secretory granules and are co-secreted from the β-cells in equimolar amounts in response to various stimuli, most notably elevated blood glucose levels.[1] This equimolar secretion is a cornerstone of C-peptide's utility as a clinical biomarker.

B. C-Peptide as a Biomarker of β-Cell Function

The co-secretion of C-peptide and insulin in equimolar ratios makes C-peptide an exceptionally valuable and widely utilized biomarker for assessing endogenous insulin production and overall pancreatic β-cell secretory reserve.[1] Several factors contribute to its superiority over direct measurement of peripheral insulin levels for this purpose:

1. Longer Half-Life: C-peptide has a significantly longer plasma half-life (approximately 20-30 minutes) compared to insulin (approximately 3-5 minutes).[1] This longer persistence in circulation provides a more stable and integrated measure of insulin secretion over time.
2. Hepatic Extraction: Unlike insulin, about 50% of which is extracted and degraded by the liver during its first pass, C-peptide undergoes negligible hepatic clearance.[3] This means that peripheral C-peptide levels more accurately reflect pancreatic secretion rates. The differential hepatic clearance of insulin versus C-peptide is a key physiological divergence that underpins C-peptide's utility as a more stable biomarker. This distinct handling by the liver may also suggest that C-peptide evolved to serve functions requiring more sustained systemic presence, unlike insulin's primary role in acute glucose regulation.
3. Exogenous Insulin Interference: In patients receiving insulin therapy, measuring C-peptide allows for the assessment of residual endogenous insulin production because C-peptide assays do not cross-react with exogenous insulin.[3]
4. Clearance Variability: C-peptide clearance is generally less variable than that of insulin.[3]

Clinically, C-peptide measurements are integral to the diagnosis and management of diabetes. They help differentiate T1DM (characterized by very low or undetectable C-peptide due to β-cell destruction) from T2DM (where C-peptide levels can be normal, high due to insulin resistance, or low in late stages due to β-cell exhaustion).[3] It is also used to monitor β-cell function over time in T2DM, assess the "honeymoon phase" or remission in T1DM, and evaluate the cause of hypoglycemia (e.g., factitious hypoglycemia vs. insulinoma).[3] Normal fasting C-peptide levels in healthy individuals are typically in the range of 0.3–0.6 nmol/L (or 0.5-2.0 ng/mL), rising to 1–3 nmol/L postprandially.[3] Levels below 0.2 nmol/L are often indicative of severe insulin deficiency, characteristic of T1DM.[3] The variable C-peptide levels observed in T2DM highlight the heterogeneity of this condition, reflecting differing degrees of insulin resistance and β-cell dysfunction. This complexity makes the therapeutic application of C-peptide in T2DM less straightforward than in T1DM, where a clear deficiency state exists.

C. Established Biological Activities Beyond Insulin Synthesis

The initial view of C-peptide as biologically inert has been definitively overturned. It is now recognized as a physiologically active peptide hormone that interacts with various cellular targets and elicits a range of biological effects.[4] These activities are particularly relevant in the context of diabetes, where C-peptide deficiency (in T1DM) or altered levels (in T2DM) may contribute to the pathogenesis of chronic complications.

Key biological activities attributed to C-peptide include:

• Prevention or amelioration of diabetic microvascular complications: Extensive research suggests C-peptide can exert protective effects against diabetic neuropathy, nephropathy, and retinopathy.[4] The fact that T1DM is effectively a dual hormone deficiency (lacking both insulin and C-peptide) [28] has profound implications. Standard insulin replacement therapy only addresses one part of this deficiency, potentially leaving patients vulnerable to complications linked to C-peptide absence. This provides a strong rationale for investigating C-peptide co-administration.
• Anti-inflammatory effects: C-peptide has been shown to modulate inflammatory responses.[13]
• Antioxidant properties: It can reduce oxidative stress by mitigating the production of reactive oxygen species (ROS).[13]
• Anti-apoptotic actions: C-peptide can protect cells from programmed cell death.[13]
• Modulation of blood flow and endothelial function: It influences vascular tone and improves endothelial cell function, often through nitric oxide (NO)-dependent mechanisms.[4]
• Influence on neural activity: C-peptide has demonstrated beneficial effects on nerve conduction and structure.[4]

These intrinsic biological activities form the scientific basis for investigating C-peptide as a potential therapeutic agent, particularly for mitigating the long-term complications associated with T1DM, where its deficiency is absolute.

IV. Pharmacodynamics: Mechanism of Action

The understanding of C-peptide's pharmacodynamics has evolved significantly, revealing specific cellular interactions and signaling cascades that underpin its diverse physiological effects.

A. Cellular Targets and Receptor Interactions

C-peptide exerts its biological effects by binding specifically to the surfaces of various cell types, including endothelial cells, renal tubular cells, fibroblasts, and neurons.[4] This binding is saturable, occurring at nanomolar concentrations, which are within the physiological range observed postprandially.[31] Importantly, this binding is stereospecific and is not competed by insulin, proinsulin, insulin-like growth factor I (IGF-I), or IGF-II, indicating a distinct recognition site.[31]

The primary hypothesis for C-peptide's receptor interaction points towards a G protein-coupled receptor (GPCR).[31] This is supported by evidence that pertussis toxin, a known modifier of certain G proteins (specifically Gi/o​ proteins), abolishes C-peptide binding and its downstream effects in some systems.[48] More recently, the orphan GPCR, GPR146, has been identified as a putative receptor for C-peptide, or at least a component of its signaling complex.[6] Studies have shown that knockdown of GPR146 can block C-peptide-induced cellular responses, such as cFos expression.[49] However, the role of GPR146 as the definitive and sole C-peptide receptor is still under active investigation and is not universally confirmed across all tissues or effects. Some literature continues to state that C-peptide has "no known receptor" [46], reflecting the ongoing complexity and perhaps the possibility of multiple receptor interactions or non-traditional binding mechanisms. This uncertainty surrounding the receptor remains a significant knowledge gap and a challenge for targeted drug development. The difficulty in definitively identifying a single, high-affinity receptor might suggest that C-peptide interacts with a receptor complex or multiple distinct receptors, or that its membrane interactions are more nuanced than a simple ligand-receptor model, possibly explaining its pleiotropic effects.

B. Key Intracellular Signaling Pathways Activated by C-Peptide

Upon binding to its putative receptor(s), C-peptide initiates a variety of intracellular signaling cascades:

• Calcium (Ca2+)-dependent pathways: An early and consistent finding is that C-peptide binding leads to a rise in intracellular Ca2+ concentrations.[35]
• Mitogen-Activated Protein Kinase (MAPK) pathway: Activation of the MAPK pathway, particularly extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 MAPK, is frequently reported.[14] This pathway is crucial for regulating cell proliferation, differentiation, survival, and inflammatory responses.
• Phosphoinositide 3-Kinase (PI3K)/Akt pathway: C-peptide can activate the PI3K-Akt signaling cascade, which is known to play a vital role in promoting cell survival, growth, and metabolism, and is also involved in eNOS activation.[14]
• Endothelial Nitric Oxide Synthase (eNOS) activation: A key effect of C-peptide is the stimulation of eNOS activity and, in some cases, its transcription, leading to increased production of nitric oxide (NO).[4] NO is a critical vasodilator and plays roles in anti-inflammatory processes and inhibiting platelet aggregation.
• Na+,K+-ATPase stimulation: C-peptide has been shown to activate the Na+,K+-ATPase (sodium-potassium pump) in various tissues, including nerves and renal tubules.[4] This enzyme is essential for maintaining cellular ion gradients, membrane potential, and, consequently, normal cell function, particularly in excitable tissues like nerves.
• AMP-activated protein kinase α (AMPKα) activation: C-peptide can activate AMPKα, an energy-sensing enzyme. This activation has been linked to the suppression of NADPH oxidase activity, reduction of reactive oxygen species (ROS) generation, and protection against endothelial cell apoptosis.[13]
• Nuclear Factor kappa B (NF-κB) signaling: C-peptide can modulate inflammatory responses by suppressing the NF-κB signaling pathway, a major transcriptional regulator of pro-inflammatory genes. This can lead to reduced leukocyte adhesion and overall inflammation.[13]
• Regulation of transcription factors: Beyond NF-κB, C-peptide influences other transcription factors, such as downregulating p53 (a tumor suppressor involved in apoptosis) and activating factors important for anti-inflammatory and cellular protective responses.[13] Some studies also suggest that C-peptide can be internalized and translocate to the nucleus, where it may directly influence gene expression, for example, by affecting histone acetylation.[40] This indicates mechanisms extending beyond classical cell-surface receptor signaling, potentially contributing to more sustained, long-term cellular effects.

The activation of these diverse signaling pathways explains the pleiotropic effects of C-peptide, suggesting it acts as a general homeostatic regulator for vascular and neural tissues, particularly under conditions of metabolic stress such as those found in diabetes. Its influence on fundamental cellular processes like ion transport, redox balance, inflammation, cell survival, and vascular tone underscores its potential importance in maintaining tissue health.

C. Molecular Basis for Therapeutic Effects

The engagement of these signaling pathways provides a molecular basis for the observed therapeutic effects of C-peptide in the context of diabetic complications:

• Anti-inflammatory effects: Achieved through the suppression of NF-κB activity and reduction of leukocyte adhesion to the endothelium.[13]
• Antioxidant effects: Mediated by the reduction of ROS generation (e.g., via AMPKα activation and NADPH oxidase suppression) and inhibition of pro-oxidant enzymes like p66shc.[13]
• Anti-apoptotic effects: Resulting from the regulation of apoptotic mediators like p53, caspases (e.g., reducing caspase-3 activity), and Bcl-2 family proteins (promoting anti-apoptotic Bcl-2), as well as inhibiting mitochondrial dysfunction.[13]
• Improved Microcirculation and Endothelial Function: Primarily through the stimulation of eNOS, leading to increased NO bioavailability, vasodilation, and improved blood flow in various tissues.[4]
• Neuroprotection: Attributed to a combination of improved endoneurial blood flow, restoration of Na+,K+-ATPase activity in nerve cells (which helps maintain nerve membrane potential and conduction), and potentially the stimulation of neurotrophic factors.[4]
• Renoprotection: Involves multiple actions including the reduction of glomerular hyperfiltration (by modulating afferent and efferent arteriolar tone), diminution of urinary albumin excretion, and prevention of glomerular hypertrophy and mesangial matrix expansion. These effects are linked to eNOS stimulation and potential interference with pro-fibrotic pathways involving TGF-β1 and TNFα.[4]

Table 2: Overview of C-Peptide Activated Signaling Pathways and Their Key Physiological/Pathophysiological Consequences

Signaling Pathway Activated by C-Peptide	Key Molecular Effect(s)	Implicated Physiological/Therapeutic Outcome(s)	Selected Source(s)
eNOS/NO	Increased Nitric Oxide (NO) production	Vasodilation, improved endothelial function, increased tissue blood flow (nerve, kidney, muscle)	4
Na+,K+-ATPase	Increased enzyme activity, restoration of ion gradients	Improved nerve conduction, normal cellular function in renal tubules	4
MAPK (ERK1/2, p38)	Phosphorylation and activation of downstream targets	Cell survival, growth, differentiation, modulation of inflammatory responses, neurotrophic effects	14
PI3K/Akt	Activation of Akt kinase	Cell survival, anti-apoptotic effects, eNOS activation	14
AMPKα	Activation of AMPKα, suppression of NADPH oxidase	Reduced ROS production, antioxidant effects, anti-apoptotic effects in endothelial cells	13
NF-κB	Suppression of NF-κB activation/translocation	Anti-inflammatory effects, reduced leukocyte adhesion	13
Ca2+ signaling	Increased intracellular Ca2+	Trigger for multiple downstream pathways (e.g., PKC, calmodulin-dependent enzymes)	35
p53 regulation	Downregulation of p53	Anti-apoptotic effects	13

V. Pharmacokinetics

Understanding the pharmacokinetic profile of C-peptide—its absorption, distribution, metabolism, and excretion (ADME)—is crucial for its development as a therapeutic agent.

A. Absorption and Bioavailability

Endogenously produced C-peptide is secreted directly from pancreatic β-cells into the portal circulation along with insulin.[28] When administered therapeutically, C-peptide has been given via intravenous (IV) infusion or subcutaneous (SC) injection in clinical trials.[4] As is typical for most peptide drugs, the oral bioavailability of C-peptide is expected to be very low. This is due to its susceptibility to enzymatic degradation in the gastrointestinal tract and poor permeability across the intestinal mucosa.[26] This "peptide problem" inherently limits its convenience for chronic conditions and is a major driver for the development of alternative delivery systems or more stable analogues.

B. Distribution

Once in the systemic circulation, C-peptide is distributed throughout the body. Its specific binding to various cell types, including endothelial cells, renal tubular cells, and neurons, indicates that it distributes to these tissues to exert its biological effects.[4] Detailed information on its volume of distribution is not extensively provided in the available materials for C-peptide specifically. However, for peptides in general, the volume of distribution is often limited, primarily encompassing the extracellular fluid volume, due to their hydrophilic nature and size, which restricts passive diffusion across cell membranes.[26]

C. Metabolism and Clearance

The primary route of metabolism and clearance for C-peptide is via the kidneys.[3] This involves glomerular filtration followed by subsequent proteolysis within the renal tubules, a common fate for many peptides.[26] Unlike insulin, C-peptide undergoes negligible hepatic clearance.[3] This renal-centric clearance means that impaired kidney function, a common complication in diabetes, can significantly affect C-peptide levels. In patients with diabetic nephropathy or other causes of renal insufficiency, the clearance of C-peptide is reduced, leading to elevated plasma concentrations if there is ongoing endogenous production, or a prolonged half-life if administered exogenously.[8] This interplay is important both when C-peptide is used as a biomarker of β-cell function in patients with kidney disease and when considering it as a therapeutic agent, as dosage adjustments may be necessary.

D. Half-life

The biological half-life of native human C-peptide in individuals with normal renal function is approximately 20-30 minutes.[1] Some reports suggest it may be slightly longer in individuals with diabetes, around 40 minutes.[30] This is significantly longer than the plasma half-life of insulin, which is typically only 3-5 minutes.[1] The marked difference in half-life between C-peptide and insulin, despite their equimolar secretion, suggests a potential temporal dissociation in their physiological roles. While insulin acts rapidly to manage acute glucose fluctuations, the more sustained presence of C-peptide might allow it to exert longer-term modulatory effects on cellular functions relevant to tissue maintenance and protection against chronic stress. However, from a therapeutic standpoint, a 30-minute half-life is still considered short and necessitates frequent administration (multiple daily injections) or continuous infusion to maintain stable, physiologically relevant plasma concentrations. This pharmacokinetic limitation has been a major impetus for the development of long-acting C-peptide formulations.

VI. Clinical Development and Therapeutic Investigations

C-peptide has been investigated for its therapeutic potential in various diabetic complications, driven by its observed biological activities and the consequences of its deficiency in T1DM.

A. C-Peptide in Diabetic Peripheral Neuropathy (DPN)

Diabetic peripheral neuropathy is a common and debilitating complication of diabetes, and C-peptide replacement has been a key area of investigation.

1. Clinical Trial NCT00278980: Effect of C-peptide on Diabetic Peripheral Neuropathy

This specific clinical trial, identified by the user (NCT00278980), was a Phase 2 study evaluating the effect of C-peptide on diabetic peripheral neuropathy.1 The trial is listed as completed.54 While the direct sponsor details from ClinicalTrials.gov were not accessible in the provided materials 55, research publications often detail sponsorship, which frequently involves pharmaceutical companies or collaborative research groups.

A key publication by Ekberg et al. (2007) in Diabetes Care appears to report the results of either NCT00278980 or a very closely aligned study, given the matching indication and trial phase.[50] This study involved 139 T1DM patients (mean age 44.2 years, mean diabetes duration 30.6 years), 86% of whom had clinical neurological impairment at baseline. The intervention consisted of C-peptide replacement therapy for 6 months.

• Efficacy Results (from Ekberg et al., 2007 [50]):
• Sensory Nerve Conduction Velocity (SCV): SCV showed improvement in the C-peptide treated groups compared to baseline (P<0.007). A greater number of patients treated with C-peptide demonstrated an SCV peak potential improvement of >1.0 m/s compared to those receiving placebo (P<0.03). In a subgroup of patients who were least severely affected at baseline (SCV <2.5 SD below normal, n=70), SCV improved by 1.0 m/s (P<0.014 vs. placebo).
• Neuropathy Impairment Score (NIA): The NIA score improved within the C-peptide-treated groups (P<0.011).
• Vibration Perception Threshold (VPT): VPT also showed improvement in the C-peptide-treated groups (P<0.002).
• Glycemic Control: HbA1c levels, which were 7.6±0.1% at baseline, decreased slightly but similarly in both C-peptide- and placebo-treated patients during the study, indicating that the observed neurological improvements were not solely due to changes in glycemic control.
• Conclusion: The authors concluded that C-peptide treatment for 6 months improves sensory nerve function in patients with early-stage T1DM neuropathy.
• Safety and Tolerability: Specific adverse event data for NCT00278980 would be detailed in the full study publication or the ClinicalTrials.gov record. Generally, C-peptide replacement is reported to be well-tolerated.[39]

The findings from NCT00278980 (or its associated publication) provide direct evidence supporting a beneficial role for C-peptide in DPN, although the improvements appear more pronounced in patients with less severe neuropathy at the outset. This suggests a potential therapeutic window for C-peptide intervention.

2. Other Relevant Preclinical and Clinical Studies in Neuropathy

Numerous preclinical studies in animal models of T1DM (e.g., streptozotocin-induced diabetic rats) have demonstrated that C-peptide administration can improve nerve conduction velocity, increase endoneurial blood flow (often via eNOS stimulation), restore Na+,K+-ATPase activity in nerves, and ameliorate structural abnormalities such as paranodal swelling, axonal atrophy, and demyelination.4

Early clinical studies in T1DM patients with subclinical neuropathy also showed promising results; for instance, 3 months of C-peptide replacement led to a substantial improvement in SCV by 2.7 m/s.4 Furthermore, beneficial effects on autonomic nerve function, including improvements in heart rate variability and erectile dysfunction, have been reported in T1DM patients receiving C-peptide.4

B. C-Peptide in Other Diabetic Complications

1. Diabetic Nephropathy

C-peptide has shown renoprotective effects in both animal models and human studies.

• Mechanisms: It acts by reducing glomerular hyperfiltration through modulation of afferent (constriction) and efferent (dilation) arteriolar tone, inhibiting tubular reabsorption, diminishing urinary albumin excretion, and preventing glomerular hypertrophy and mesangial matrix expansion. These effects are thought to be mediated, in part, by stimulation of renal eNOS and Na+,K+-ATPase activity, and potentially by interfering with pro-fibrotic signaling pathways involving TGF-β1 and TNFα.[4]
• Clinical Evidence: Short-term (up to 3 months) C-peptide replacement in T1DM patients has been documented to reduce glomerular hyperfiltration and urinary albumin excretion.[6] Moreover, long-term data from patients receiving pancreas or islet transplants, which restore endogenous C-peptide production, show significant amelioration of glomerular structural abnormalities.[41]

2. Diabetic Retinopathy

The role of C-peptide in diabetic retinopathy is also an area of active research.

• Mechanisms: C-peptide may avert retinopathy by suppressing intracellular ROS accumulation, reducing stress fiber formation in endothelial cells, preserving endothelial cell integrity, and limiting vascular endothelial growth factor (VEGF)-triggered microvascular permeability.[14]
• Clinical/Preclinical Evidence: Animal studies have indicated that C-peptide replacement can improve signs of retinopathy.[34] In human studies involving T2DM patients, a complex, non-linear relationship has been observed between fasting C-peptide (FCP) levels and the risk of diabetic retinopathy. C-peptide appears to be protective below a certain FCP threshold, but this protective effect may diminish or become uncertain at higher FCP levels.[43] This highlights the nuanced role of C-peptide, particularly in T2DM where endogenous levels can be elevated due to insulin resistance.

3. Cardiovascular Complications

C-peptide may also influence cardiovascular health in diabetes.

• It has been shown to improve microvascular blood flow in the myocardium and skeletal muscle.[5]
• An anti-atherogenic role has been suggested in T1DM and early T2DM, attributed to its ability to protect endothelial cells and inhibit vascular smooth muscle cell (VSMC) proliferation.[13]
• However, paradoxically, in late T2DM characterized by sustained high C-peptide levels (due to insulin resistance), C-peptide itself has been implicated as potentially promoting atherosclerosis.[13] This dual role underscores the complexity of C-peptide's effects, which appear to be context-dependent (deficiency vs. excess) and potentially differ between T1DM and various stages of T2DM.

C. C-Peptide Replacement Therapy: Overall Outcomes and Challenges

While preclinical studies and early clinical trials have shown promise for C-peptide replacement therapy in improving nerve function, kidney function, and potentially retinopathy in C-peptide deficient states (primarily T1DM) [4], the translation to widespread clinical use has faced significant challenges.

• Key Challenges and Unsatisfactory Clinical Trial Results:
• The short biological half-life of native C-peptide (around 30 minutes) necessitates frequent dosing or continuous infusion to maintain therapeutic levels, which is impractical for chronic treatment.[28]
• Some later-stage or larger clinical trials, including those with modified C-peptide formulations, have yielded "unsatisfactory" or mixed results, failing to consistently meet primary endpoints for all parameters evaluated or show benefits significantly superior to placebo.[34] This discrepancy between promising early data and later-stage trial outcomes may be due to factors such as patient selection (e.g., stage or severity of complications), the choice of clinical endpoints (NCV, for instance, can exhibit high variability and placebo responses [52]), difficulties in determining optimal dosing regimens, and the inherent complexity of translating C-peptide's pleiotropic effects into singular, robust clinical improvements.
• The lack of a definitively identified high-affinity receptor continues to complicate targeted drug design and a full mechanistic understanding of C-peptide's actions.[6]
• Demonstrating robust, long-term clinical benefits in large-scale, adequately powered clinical trials has proven difficult.[32]
• Standardization of C-peptide assays for both biomarker use and therapeutic monitoring, as well as defining the optimal therapeutic range for C-peptide, remain ongoing challenges.[3]
• Development of Long-Acting C-Peptide Analogues: To address the short half-life of native C-peptide, efforts have focused on developing long-acting formulations:
• PEG-C-peptide: This involves covalently attaching polyethylene glycol (PEG) to the C-peptide molecule, which significantly prolongs its biological half-life (to approximately 6-7 days).[29]
• A notable Phase 2 trial (NCT01681290) evaluated once-weekly subcutaneous administration of PEG-C-peptide in 250 T1DM patients with DPN over 52 weeks. The trial demonstrated a marked improvement in VPT compared with placebo. However, it did not achieve significant improvements in the primary endpoint of SNCV or in the modified Toronto Clinical Neuropathy Score (mTCNS). The PEG-C-peptide was reported to be well-tolerated.[51] These mixed results highlight that simply extending the half-life may not be sufficient to overcome all efficacy hurdles for all relevant endpoints.
• Nanosphere Encapsulation/Delivery Systems: Research has also explored the use of polypeptide nanospheres for C-peptide immobilization or encapsulation as a strategy to achieve prolonged stability and sustained release.[29]

The therapeutic focus for C-peptide appears to be more on preventing the progression of complications or treating early-stage disease, rather than reversing advanced, established structural damage.[45] Its known mechanisms, such as improving blood flow, reducing inflammation, and supporting cellular function, are more aligned with preventative or early interventional strategies. This suggests that the therapeutic window for C-peptide might be earlier in the course of diabetic complications.

Table 3: Summary of Key Clinical Trials Investigating C-Peptide and Analogues for Diabetic Complications

Trial Identifier	Phase	Indication	C-Peptide Formulation	Dosage & Route	Duration	Key Efficacy Outcomes	Key Safety Findings	Overall Conclusion/Status	Source(s)
NCT00278980 (or similar reported by Ekberg et al., 2007)	2	T1DM with DPN	Native Human C-peptide	SC (specific dose not detailed in summary)	6 months	Improved SCV (esp. in less severe DPN), NIA, and VPT vs. baseline/placebo.	Generally well-tolerated (inferred).	C-peptide improves sensory nerve function in early-stage T1DM neuropathy. Completed.	50
NCT01681290	2	T1DM with mild to moderate DPN	PEG-C-peptide (long-acting)	0.8 mg or 2.4 mg SC weekly	52 weeks	Marked improvement in VPT vs. placebo. No significant improvement in SNCV or mTCNS vs. placebo.	Well-tolerated; AEs similar to placebo. Low immunogenicity.	Did not meet primary SNCV endpoint, but improved VPT. Completed.	51
Early Human Studies (e.g., Johansson et al., Wahren et al. - various short-term)	1/2	T1DM with neuropathy or nephropathy	Native Human C-peptide	IV infusion or SC injections	Days to 3 months	Improved NCV, GFR, albumin excretion, blood flow.	Well-tolerated.	Suggests beneficial physiological effects. Completed.	4

VII. Safety and Tolerability of C-Peptide Therapy

The safety profile of C-peptide is a critical consideration for its potential therapeutic use, particularly for chronic conditions like diabetic complications.

A. Adverse Event Profile from Clinical Trials

• Native C-peptide: In numerous short-term studies involving physiological replacement doses in T1DM patients, native human C-peptide has generally been reported as well-tolerated.[4] Significant adverse effects directly attributable to C-peptide itself at these replacement levels have not been a prominent feature in these trials. This favorable safety profile is a significant advantage, suggesting that the primary obstacles to its clinical use are related to efficacy and delivery rather than inherent toxicity at physiological replacement doses.
• PEG-C-peptide: The Phase 2 trial (NCT01681290) of long-acting PEG-C-peptide provided more extensive safety data over a 52-week period.[51]
• The incidence of treatment-related adverse events was low (11.3–16.4%) and similar across the placebo, low-dose PEG-C-peptide, and high-dose PEG-C-peptide groups.
• The most commonly reported adverse events were nasopharyngitis, upper respiratory tract infection, and nausea. These were generally mild to moderate.
• Non-severe hypoglycemic events occurred with similar frequency across all treatment groups (66–74%), which is expected in an insulin-treated T1DM population. Severe hypoglycemic events (requiring assistance) were rare (0–4%).
• No notable laboratory abnormalities or clinically significant changes in vital signs were observed over time that were attributed to PEG-C-peptide. Minor electrocardiogram changes (PR interval shifts) were similar across groups.
• Context from other peptide therapeutics: While not direct C-peptide therapy, related peptide drugs provide context. For example, teplizumab, an anti-CD3 monoclonal antibody that can preserve endogenous C-peptide production, has common adverse effects like lymphopenia, rash, and headache.[15] GLP-1 receptor agonists like tirzepatide are often associated with gastrointestinal symptoms.[59] The safety profile of C-peptide itself appears more benign compared to some of these more complex biological or pharmacological interventions.

B. Immunogenicity Potential

• Native Human C-peptide: As an endogenous molecule, native human C-peptide is expected to have very low immunogenicity when administered to humans, especially in a replacement context where the body is already familiar with the peptide (though its production is absent or severely reduced in T1DM).
• Modified Forms (e.g., PEG-C-peptide): The introduction of modifications, such as PEGylation, carries a theoretical risk of inducing an immune response. In the NCT01681290 trial of PEG-C-peptide, most subjects (over 90%) had negative results for anti–PEG–C-peptide antibodies, suggesting low immunogenicity for this specific formulation over the 52-week treatment period.[51] While this is encouraging, the potential for PEG itself to be immunogenic in a subset of patients is a general consideration for all PEGylated biologics, as anti-PEG antibodies could affect drug clearance or, rarely, cause hypersensitivity reactions. Long-term or broader population use might reveal more instances.
• Compounded Peptides: It is important to distinguish between regulated, investigational C-peptide products developed under an Investigational New Drug (IND) application and non-standardized compounded peptides. The FDA has raised general concerns about certain compounded peptides regarding potential risks of immunogenicity, impurities, and lack of adequate safety data.[60] These concerns do not directly apply to C-peptide products undergoing rigorous clinical development but highlight the importance of stringent manufacturing and quality control for any peptide therapeutic.

Overall, the available data suggest that C-peptide therapy, particularly with native or carefully modified forms, has a favorable safety and tolerability profile.

VIII. Regulatory Landscape and Future Directions

Despite decades of research highlighting its biological activity and potential therapeutic benefits, C-peptide has not yet achieved regulatory approval as a standalone therapeutic agent for diabetic complications from major authorities like the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA).[11]

A. Current Regulatory Status (FDA, EMA) for Therapeutic Use

The lack of approval stems primarily from the challenge of consistently demonstrating robust, clinically meaningful efficacy in large, well-controlled late-stage clinical trials, rather than significant safety concerns. The pharmacokinetics of native C-peptide, particularly its short half-life, has also posed a considerable hurdle.

Interestingly, the FDA has expressed support for the measurement of C-peptide as a "reasonably likely surrogate endpoint" (RLSE) to support Accelerated Approval submissions for therapies aimed at preserving β-cell function in T1DM.[33] This acknowledgment of C-peptide's link to clinical benefit as a biomarker is significant. If preserving endogenous C-peptide is considered sufficiently indicative of clinical benefit to warrant accelerated approval for certain immunotherapies, it logically follows that replacing C-peptide in states of complete deficiency (as in established T1DM) should also be viewed as potentially beneficial, provided that direct therapeutic efficacy can be conclusively demonstrated. This regulatory acceptance for C-peptide as a biomarker might subtly lower the conceptual barrier for its eventual acceptance as a therapeutic, contingent upon compelling trial data.

General FDA concerns regarding certain compounded peptide preparations, citing risks of immunogenicity, impurities, and limited safety information [60], are distinct from the rigorous regulatory pathway followed by investigational C-peptide products developed under an IND. The EMA also has established guidelines for therapeutic peptides and advanced therapy medicinal products (ATMPs), but no specific approval for C-peptide as a treatment for diabetic complications is currently noted.[62]

B. Potential for Orphan Drug Designation

Orphan Drug Designation (ODD) could theoretically be pursued for C-peptide therapy if it were developed for a specific, narrowly defined rare diabetic complication or a subset of T1DM patients that meets the prevalence criteria for orphan diseases (e.g., affecting fewer than 200,000 people in the U.S. or not more than 5 in 10,000 in the EU). T1DM itself might qualify in some jurisdictions, or a severe, distinct complication within T1DM could be targeted. ODD provides significant incentives for drug development, including market exclusivity, tax credits, and regulatory assistance.[64] To qualify, a drug must generally target a condition for which no satisfactory treatment exists, or if one does, the new drug must offer a significant benefit. This pathway could facilitate further development of C-peptide by mitigating some financial and regulatory hurdles.

C. Unmet Needs and Opportunities for C-Peptide in Clinical Practice

There remains a substantial unmet medical need for effective therapies that can prevent the onset or halt the progression of chronic diabetic complications, particularly in T1DM where patients face a lifetime of managing the disease and its sequelae.[5] C-peptide's potential to address multiple complications—neuropathy, nephropathy, and retinopathy—through its diverse mechanisms of action makes it an attractive candidate.[4] A key opportunity lies in its potential use as an adjunct to insulin therapy in T1DM, aiming for a more comprehensive physiological replacement that addresses both insulin and C-peptide deficiency.[5]

D. Recommendations for Future Research

To overcome current limitations and fully realize the therapeutic potential of C-peptide, future research should focus on several key areas:

1. Receptor Identification and Mechanism Clarification: Definitive identification and characterization of C-peptide's specific receptor(s) (e.g., further validation and exploration of GPR146's role) and elucidation of the full spectrum of its molecular mechanisms are paramount.[6]
2. Robust Clinical Trials: Large-scale, long-term, randomized controlled trials with clearly defined, clinically meaningful endpoints are essential to definitively establish efficacy and safety for specific diabetic complications.[32] These trials may need more refined strategies for patient stratification (e.g., early-stage complications, specific patient phenotypes) and endpoint selection.
3. Improved Formulations and Delivery Systems: Continued development and testing of stable, long-acting C-peptide formulations (beyond current PEGylated versions) or innovative delivery systems (e.g., nanocarriers, oral peptide technologies if feasible) are crucial to address the pharmacokinetic challenges.[12]
4. Optimal Dosing and Patient Populations: Further investigation is needed to determine optimal dosing regimens, identify patient populations most likely to benefit (e.g., based on duration of diabetes, severity of C-peptide deficiency, stage of complications), and define the optimal therapeutic window.[40]
5. Role in T2DM: The complex and potentially dual role of C-peptide in T2DM (protective in deficiency, potentially detrimental in states of excess/insulin resistance) warrants further investigation to determine if specific subsets of T2DM patients (e.g., late-stage with β-cell failure) might benefit from C-peptide therapy.[10]
6. Assay Standardization: Continued efforts towards the international standardization of C-peptide assays are needed for reliable comparison of data across studies and for precise therapeutic monitoring if C-peptide therapy becomes a reality.[3]
7. Combination Therapies: The future of C-peptide therapy might lie in combination with other strategies, such as advanced insulin delivery systems, or emerging cell-based therapies aimed at β-cell regeneration that could also be engineered to co-secrete C-peptide (e.g., genetically engineered Sertoli cells [38]), thereby more closely mimicking natural physiology.
8. Application of Novel Peptide Technologies: Recent advancements in peptide engineering, chemical modifications to enhance stability and permeability, and novel delivery mechanisms could be applied to C-peptide to overcome its inherent limitations.[66]

IX. Conclusion

A. Summary of C-peptide's Journey

C-peptide has undergone a remarkable scientific journey, evolving from its initial characterization as an inert byproduct of insulin biosynthesis, primarily valued as a biomarker of pancreatic β-cell function, to being recognized as a bioactive peptide hormone with a spectrum of distinct physiological roles. Decades of research have illuminated its capacity to interact with specific cellular targets and modulate multiple intracellular signaling pathways, particularly in vascular endothelial cells, renal cells, and neurons. These actions translate into potentially protective effects against the chronic microvascular complications of diabetes mellitus, such as neuropathy, nephropathy, and retinopathy, especially in the context of T1DM where C-peptide is deficient.

B. Overall Assessment of Therapeutic Promise

The preclinical rationale for C-peptide replacement therapy in T1DM is strong, supported by numerous animal studies demonstrating beneficial effects on nerve function, kidney integrity, and vascular health. Early-phase clinical trials have provided encouraging, albeit sometimes modest, signals of efficacy, particularly in improving sensory nerve function in DPN (as suggested by trials like NCT00278980) and reducing glomerular hyperfiltration in early diabetic nephropathy. Its generally favorable safety profile, especially for native C-peptide, further supports its therapeutic potential.

However, the translation of this promise into an approved clinical therapy has been fraught with challenges. The most significant of these is the short biological half-life of native C-peptide, necessitating frequent administration. While efforts to develop long-acting analogues like PEG-C-peptide have addressed this pharmacokinetic limitation to some extent, they have not consistently delivered robust efficacy across all desired clinical endpoints in larger trials. The lack of a definitively characterized high-affinity receptor has also complicated a full understanding of its mechanisms and the rational design of highly targeted mimetics.

C. Hurdles to Clinical Application and Future Outlook

The primary hurdle to the widespread clinical application of C-peptide therapy is the gap between promising basic science and early clinical findings, and the consistent demonstration of significant, clinically meaningful efficacy in large, well-designed, late-stage clinical trials. Regulatory approval hinges on such evidence, and to date, C-peptide has not crossed this threshold.

The future of C-peptide as a therapeutic agent will depend on overcoming these challenges. This will likely require:

• Innovative Formulations: Continued research into novel, stable, and conveniently administered long-acting C-peptide formulations or delivery systems is essential.
• Refined Clinical Trial Designs: Future trials may benefit from more precise patient stratification (e.g., targeting patients at earlier stages of complications or those with specific biomarkers indicating potential responsiveness), carefully selected and validated clinical endpoints, and potentially longer durations to observe effects on hard clinical outcomes.
• Mechanistic Clarity: Further elucidation of C-peptide's receptor(s) and the full intricacies of its signaling pathways will be crucial for optimizing therapeutic strategies.
• Combination Approaches: Exploring C-peptide in combination with other therapies, or as part of integrated strategies like β-cell replacement that includes C-peptide secretion, may offer new avenues.

Despite the setbacks and the long road of development, the established biological activity of C-peptide and its potential to address the significant unmet needs in the management of diabetic complications suggest that it remains a molecule of considerable interest. Continued research is not only vital for potentially unlocking its therapeutic utility but also for deepening our understanding of the complex pathophysiology of diabetes and its long-term consequences. The journey of C-peptide underscores the importance of re-evaluating molecules once dismissed as inactive, as they may hold unappreciated physiological significance and therapeutic potential.

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