A Comprehensive Monograph on Thymalfasin (Thymosin Alpha 1): From Molecular Mechanisms to Clinical and Regulatory Perspectives

Executive Summary

Thymalfasin is a chemically synthesized, 28-amino acid immunomodulatory peptide identical to the endogenous human polypeptide Thymosin Alpha 1 (Tα1). Classified as a biologic drug, its primary therapeutic function is the restoration and enhancement of cell-mediated immunity. The core mechanism of action involves a multifaceted modulation of the immune system, centered on the promotion of T-cell differentiation and maturation. At a molecular level, Thymalfasin acts as an agonist for Toll-like receptors (TLRs), particularly TLR2 and TLR9, on antigen-presenting cells, which initiates a signaling cascade that drives a T-helper 1 (Th1) polarized immune response, characterized by the production of key cytokines such as interferon-gamma (IFN-γ) and interleukin-2 (IL-2).

Clinically, Thymalfasin has achieved widespread use, with regulatory approval in over 35 countries for the treatment of chronic hepatitis B (HBV) and hepatitis C (HCV) infections.[1] Beyond its antiviral indications, it has been extensively investigated and is used in some regions as an adjuvant to chemotherapy in various cancers and as an enhancer of vaccine responses in immunocompromised populations.[4] Its investigational scope continues to expand into areas of acute immune dysregulation, including sepsis and severe COVID-19.

A central paradox defines Thymalfasin's global status: despite its extensive clinical use, a robust safety profile demonstrated in over 11,000 patients, and a significant body of post-marketing data, it remains unapproved by major Western regulatory bodies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA).[6] Both agencies have, however, granted it orphan drug designation for the treatment of hepatocellular carcinoma, acknowledging its potential in a high-unmet-need indication.[8] This regulatory divergence highlights a potential disconnect between the drug's modulatory benefits and the stringent primary endpoints of pivotal clinical trials submitted for review. Consequently, the therapeutic identity of Thymalfasin is evolving from its original role as an antiviral agent to that of a broad-spectrum, well-tolerated immune modulator with potential applications in a new era of complex immunological challenges, including immuno-oncology and the management of immunosenescence.

1.0 Introduction to Thymalfasin: An Immunomodulatory Peptide

1.1 Historical Context and Discovery

Thymalfasin is the chemically synthesized, international non-proprietary name for the peptide Thymosin Alpha 1 (Tα1). Its origins are rooted in early immunological research focused on the function of the thymus gland. Tα1 was first identified as a biologically active component of "thymosin fraction 5," a crude extract derived from bovine thymus tissue that contained a mixture of immunologically active peptides.[10] The isolation and characterization of Tα1 as a distinct, 28-amino acid polypeptide allowed for its precise chemical synthesis, leading to the development of Thymalfasin, a product identical in sequence to the native human peptide.[1] This transition from a biological extract to a pure, synthetic compound was a critical step in its development as a consistent and well-characterized therapeutic agent.

1.2 Classification and Therapeutic Rationale

Thymalfasin is classified as a biotech or biologic drug, specifically falling into the categories of immunomodulatory agents, immunologic adjuvants, and peptide hormones.[1] The fundamental therapeutic rationale for its use is the correction or enhancement of immune function, particularly in conditions characterized by a compromised or dysfunctional T-cell-mediated immune response. This includes chronic viral infections where the immune system fails to clear the pathogen, malignancies where the immune system is suppressed by the tumor or by cytotoxic therapies, and states of immunosenescence associated with aging, where vaccine responses are often suboptimal.[2] By restoring T-cell maturation and function, Thymalfasin is intended to re-establish the body's natural capacity to combat disease.

1.3 Key Synonyms and Brand Names

For clarity in scientific and clinical literature, it is essential to recognize the various names used to identify this peptide.

• Generic Name: Thymalfasin.[10]
• Scientific Names/Synonyms: Thymosin alpha 1, Tα1, Thymosin α1, Thymosin A1.[1]
• Primary Brand Name: Zadaxin®.[1]

These terms are often used interchangeably in clinical trial reports, regulatory documents, and scientific publications.

2.0 Physicochemical Properties and Formulation

2.1 Molecular Structure and Amino Acid Sequence

Thymalfasin is a single, non-glycosylated polypeptide chain composed of 28 amino acids. A defining structural feature is the acetylation of the N-terminal serine residue, which is critical for its biological activity.[1] The complete amino acid sequence is as follows:

• IUPAC Condensed: Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH.[1]
• Single-Letter Code: SDAAVDTSSEITTKDLKEKKEVVEEAEN.[1]

2.2 Chemical and Physical Characteristics

The key chemical and physical properties of Thymalfasin are well-defined, facilitating its consistent manufacturing and formulation.

• Molecular Formula: C129​H215​N33​O55​.[1]
• Molecular Weight: The computed molecular weight is approximately 3108.3 g/mol (or 3108.3 Da).[1]
• Appearance: Thymalfasin is supplied as a sterile, white to off-white lyophilized (freeze-dried) powder.[15]
• Solubility: It is soluble in water. Reported solubility values vary slightly, with figures up to 2 mg/mL noted in regulatory documents and 1 mg/mL in technical data sheets.[21] Reconstitution is typically performed with sterile water for injection.[15]

2.3 Formulation, Excipients, and Stability

The commercial formulation of Thymalfasin, marketed as Zadaxin®, is designed for stability and subcutaneous administration.

• Standard Formulation: A single-use vial typically contains 1.6 mg of Thymalfasin (as thymosin alpha 1), 50 mg of mannitol, and a sodium phosphate buffer system used to adjust the pH to approximately 6.8.[17] Mannitol serves as a bulking agent, providing structure to the lyophilized cake and ensuring rapid dissolution upon reconstitution.
• Storage and Stability: The lyophilized powder is stable when stored under recommended conditions, which include desiccation at temperatures below -18°C.[15] After reconstitution with sterile water, the resulting solution is stable for 2 to 7 days when stored at 4°C. For extended storage of the solution, the addition of a carrier protein such as human serum albumin (HSA) or bovine serum albumin (BSA) is recommended to prevent adsorption to container surfaces. Freeze-thaw cycles of the reconstituted solution should be avoided to maintain peptide integrity.[15]

The table below consolidates the primary identifiers and physicochemical properties of Thymalfasin, providing a quick-reference summary.

Table 1: Key Identifiers and Physicochemical Properties of Thymalfasin

Parameter	Value	Source(s)
DrugBank ID	DB04900	1
Type	Biotech	10
CAS Number	62304-98-7	1
Other CAS	69521-94-4	1
Molecular Formula	C129​H215​N33​O55​	1
Molecular Weight	3108.3 g/mol	1
Amino Acid Sequence	Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN	1
Appearance	White to off-white lyophilized powder	15
Solubility	Soluble in water	15

3.0 Pharmacodynamics and Cellular Mechanism of Action

The biological activity of Thymalfasin is rooted in its ability to modulate multiple facets of the innate and adaptive immune systems. Its mechanism is not that of a simple, linear immunostimulant but rather that of a complex immune modulator, capable of both enhancing and regulating immune responses.

3.1 Primary Role in T-Cell-Mediated Immunity

The cornerstone of Thymalfasin's pharmacodynamic effect is the augmentation of T-lymphocyte function.[10] In various in vitro and in vivo models, it has been shown to orchestrate the development and activation of T-cells. This includes promoting the differentiation of pluripotent stem cells and immature thymocytes into mature T-cells, leading to measurable increases in the populations of key T-cell subsets, including CD3+ (total T-cells), CD4+ (T-helper cells), and CD8+ (cytotoxic T-lymphocytes).[23] Furthermore, Thymalfasin exerts a protective effect on T-cell precursors by antagonizing glucocorticoid-induced apoptosis of thymocytes, a mechanism that may help preserve the T-cell pool during periods of stress or immunosuppressive therapy.[10]

3.2 Interaction with Toll-Like Receptors (TLRs) and Downstream Signaling

A critical molecular insight into Thymalfasin's mechanism is its function as an agonist for pattern recognition receptors, specifically Toll-like receptors (TLRs). Evidence indicates that it interacts directly with TLR9 on plasmacytoid dendritic cells and TLR2 on myeloid dendritic cells.[10] This interaction serves as a bridge between the peptide and the innate immune system, initiating a well-defined intracellular signaling cascade. Activation of TLRs leads to the recruitment of adaptor proteins like MyD88 and subsequent activation of key transcription factors, including nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1) via the JNK/p38 pathway.[10] The activation of these pathways is fundamental to the expression of a wide array of genes involved in immune activation, including those for pro-inflammatory cytokines, chemokines, and co-stimulatory molecules. This TLR-to-T-cell activation cascade provides a comprehensive molecular-to-cellular explanation for the observed clinical effects in viral infections and cancer, where robust T-cell responses are paramount.

3.3 Modulation of Cytokine and Chemokine Profiles

A major consequence of Thymalfasin-induced immune cell activation is a profound shift in the cytokine environment. It preferentially promotes a T-helper 1 (Th1)-biased immune response, which is essential for effective cell-mediated immunity against intracellular pathogens and tumors. This is achieved by stimulating the production of signature Th1 cytokines, most notably interferon-gamma (IFN-γ) and interleukin-2 (IL-2).[2] The increased availability of IL-2, coupled with Thymalfasin's ability to upregulate the expression of the high-affinity IL-2 receptor on T-cells, creates a positive feedback loop that amplifies T-cell proliferation and effector function.[10]

The drug's activity is not limited to simple stimulation. It also appears to function as an "immune rheostat," capable of both upregulating and downregulating immune responses to maintain homeostasis. For instance, while promoting pro-inflammatory Th1 cytokines, it has also been shown to increase the production of the anti-inflammatory cytokine IL-10 and stimulate the activity of indoleamine-2,3-dioxygenase (IDO), which can lead to an increase in regulatory T-cells (Tregs).[15] This dual functionality is crucial, as it allows Thymalfasin to enhance necessary immune attacks against pathogens or cancer cells while simultaneously mitigating the risk of excessive inflammation or a runaway cytokine storm, a common and dangerous side effect of more potent immunotherapies like high-dose IL-2. This balanced mechanism likely underlies its remarkable safety profile.

3.4 Effects on Other Immune Cells and Pathways

Beyond its central role in T-cell biology, Thymalfasin influences a broad range of other immune components:

• Natural Killer (NK) Cells: It directly enhances the cytotoxic activity of NK cells, a critical component of the innate immune system responsible for the early recognition and elimination of virally infected and malignant cells.[1]
• Antigen Presentation: Thymalfasin improves the efficiency of antigen presentation by macrophages and dendritic cells. It achieves this in part by upregulating the expression of Major Histocompatibility Complex (MHC) Class I molecules on the surface of these cells, which makes them more effective at presenting foreign or tumor antigens to cytotoxic T-lymphocytes.[24]
• Humoral Immunity: By enhancing T-helper cell function, Thymalfasin indirectly boosts humoral immunity, increasing the production of antibodies in response to T-cell dependent antigens. This provides the mechanistic basis for its use as a vaccine adjuvant, particularly in individuals with weakened immune systems.[2]
• Antiviral Activity: The drug exhibits a dual mode of action against viruses, both directly inhibiting viral replication and restoring host immune function to facilitate viral clearance.[28]

4.0 Pharmacokinetic Profile (ADME)

The pharmacokinetic (PK) profile of Thymalfasin is characterized by rapid absorption and elimination, with no evidence of accumulation, which contributes significantly to its favorable safety and tolerability.

4.1 Absorption

Following subcutaneous (SC) injection, the recommended route of administration, Thymalfasin is rapidly and effectively absorbed into the systemic circulation.[10] Pharmacokinetic studies in healthy volunteers and patient populations consistently show that peak serum concentrations (Cmax) are achieved approximately 1 to 2 hours after administration.[22]

4.2 Distribution

Once absorbed, Thymalfasin appears to distribute primarily within the extracellular fluid. This is supported by a calculated apparent volume of distribution (Vz/f) in the range of 30 to 40 liters, a value consistent with distribution in the body's extracellular water compartments rather than extensive tissue sequestration.[31]

4.3 Metabolism

Specific metabolic pathways for Thymalfasin have not been fully elucidated, and this information is noted as "Not Available" in major drug databases.[10] However, as a 28-amino acid polypeptide, it is presumed to be catabolized by proteases and peptidases throughout the body into smaller, inactive peptide fragments and constituent amino acids, which are then re-utilized in endogenous metabolic pools. This proteolytic degradation is a common metabolic fate for therapeutic peptides.

4.4 Excretion

A significant portion of the administered dose is cleared via the kidneys. Studies have reported that urinary excretion accounts for between 31% and 60% of a given dose following both single and multiple administrations.[23]

4.5 Half-Life and Dosing Implications

Thymalfasin has a short elimination half-life of approximately 2 hours.[13] This rapid clearance profile means that the drug does not accumulate in the body even with repeated dosing, such as the twice-weekly regimen commonly used for chronic hepatitis B.[23] This lack of accumulation is a key pharmacokinetic feature that minimizes the risk of dose-related toxicities and simplifies dosing schedules.

The table below summarizes the key pharmacokinetic parameters for subcutaneously administered Thymalfasin.

Table 2: Summary of Key Pharmacokinetic Parameters

Parameter	Value / Description	Source(s)
Route of Administration	Subcutaneous	23
Time to Peak Concentration (Tmax​)	Approximately 1–2 hours	22
Elimination Half-life (T1/2​)	Approximately 2 hours	23
Volume of Distribution (Vz​/f)	30–40 L (suggests extracellular distribution)	31
Accumulation	No evidence of accumulation with multiple doses	23
Route of Excretion	Primarily renal; 31% to 60% of dose excreted in urine	23

5.0 Clinical Evidence and Therapeutic Applications

The clinical development of Thymalfasin has spanned several decades and a wide range of diseases, reflecting its broad immunomodulatory activity. Its therapeutic role has evolved over time, shifting from a primary antiviral agent to a versatile adjuvant and immune restorative therapy.

5.1 Foundational Indication: Chronic Hepatitis B Virus (HBV) Infection

The treatment of chronic HBV was the first major clinical application for Thymalfasin, leading to its approval in over 35 countries.[1]

• Monotherapy Efficacy: Evidence from randomized controlled trials (RCTs) has been mixed but generally supportive. A key RCT demonstrated that a 6-month course of Thymalfasin (1.6 mg twice weekly) resulted in a complete virological response (clearance of both HBV DNA and HBeAg) in 40.6% of patients at 18 months, a statistically significant improvement over the 9.4% response rate in untreated controls.[32] A large study in Japanese patients confirmed its long-term efficacy, showing ALT normalization in 36.4% and HBeAg clearance in 22.8% of patients 12 months after completing therapy.[33] However, another multicenter, placebo-controlled trial did not find a statistically significant difference in its primary endpoint, though a positive trend was observed (14% vs. 4% complete response).[34]
• Combination Therapy: Pilot studies combining Thymalfasin with interferon or nucleoside analogues have shown encouraging results, with some reporting complete sustained response rates as high as 70%.[29]
• Distinct Clinical Characteristics: A notable feature of Thymalfasin therapy in HBV is a frequently observed delayed therapeutic response, where virological clearance occurs months after the treatment course has ended.[17] Additionally, transient elevations in alanine aminotransferase (ALT), known as "ALT flares," can occur during treatment. These are often interpreted as a positive sign of a restored immune response targeting infected hepatocytes and are generally not a reason for treatment discontinuation.[23]

5.2 Combination Therapy in Chronic Hepatitis C Virus (HCV) Infection

Thymalfasin is also approved in many countries for the treatment of chronic HCV, almost exclusively as part of a combination regimen.[1]

• Efficacy Profile: As a monotherapy, Thymalfasin is not considered effective against HCV.[2] Its clinical value lies in its ability to augment the efficacy of interferon-based therapies. Studies have consistently suggested that adding Thymalfasin to standard or pegylated interferon, with or without ribavirin, can improve virological response rates in both treatment-naïve patients and those who have previously failed interferon therapy.[18]
• Pivotal Phase III Trial Results: A large, randomized Phase III trial in European patients who were non-responders to previous Peg-IFN/ribavirin therapy (NCT01178996) provided critical insights. The study found that the addition of Thymalfasin did not significantly increase the rate of the primary endpoint, sustained virologic response (SVR). However, a key secondary finding was that among patients who completed the full course of therapy, Thymalfasin significantly reduced the rate of virological relapse.[29]

The evolution of highly effective direct-acting antivirals (DAAs) for both HBV and HCV has largely rendered interferon-based regimens, and by extension Thymalfasin's role in this specific context, obsolete in many developed nations.[2] This shift has catalyzed the exploration of Thymalfasin in other therapeutic areas where immunomodulation remains a primary goal.

5.3 Adjuvant Therapy in Oncology

A major focus of ongoing research is the use of Thymalfasin as an adjuvant to conventional cancer treatments, aiming to counteract therapy-induced immunosuppression and enhance anti-tumor immunity.

• Hepatocellular Carcinoma (HCC): Thymalfasin holds orphan drug designation from both the FDA and EMA for HCC.[1] A Phase II RCT evaluated its use in combination with transarterial chemoembolization (TACE) for unresectable HCC. While the results did not reach statistical significance due to the small sample size, the combination arm showed a strong positive trend with a nearly doubled median overall survival (110.3 weeks vs. 57.0 weeks) and a higher rate of tumor response compared to TACE alone. Notably, the Thymalfasin group experienced no bacterial infections, versus four in the control group, highlighting its potential to protect against chemotherapy-related complications.[38]
• Malignant Melanoma: A large, multicenter Phase II trial in patients with stage IV melanoma produced compelling results. The combination of Thymalfasin with dacarbazine (DTIC) chemotherapy tripled the overall response rate and extended median overall survival by nearly three months compared to the standard-of-care arm (DTIC plus low-dose interferon alpha).[39] These positive data led to an agreement with the FDA on a Special Protocol Assessment (SPA) for a pivotal Phase III registration trial.
• Non-Small Cell Lung Cancer (NSCLC): Preclinical studies have shown that Thymalfasin can inhibit the growth and migration of NSCLC cells, particularly those expressing high levels of the immune checkpoint ligand PD-L1.[15] Clinical data suggest that adjuvant Thymalfasin can significantly reduce the incidence and severity of chemoradiation-induced lymphopenia and may improve overall survival in patients with resectable or locally advanced NSCLC.[40]

5.4 Application as a Vaccine Adjuvant

Thymalfasin's ability to enhance T-cell dependent antibody responses provides a strong rationale for its use as a vaccine adjuvant, especially in populations with compromised immune function.

• Approved Use: It is indicated in some countries as an adjuvant for both influenza and hepatitis B vaccines in patients undergoing chronic hemodialysis, a group known for poor vaccine responses.[10]
• Clinical Evidence: A pilot study in hemodialyzed patients demonstrated that co-administration of Thymalfasin with an adjuvanted pandemic H1N1 influenza vaccine (Focetria) significantly enhanced immunogenicity. The Thymalfasin groups were able to meet the stringent Committee for Medicinal Products for Human Use (CHMP) criteria for seroprotection and seroconversion, which the vaccine-only group did not.[41]
• Ongoing Research: A Phase I clinical trial (NCT05541293) is currently evaluating the safety and efficacy of Thymalfasin as an enhancer of the immune response to COVID-19 vaccine boosters in adults aged 65 and older, a key population affected by immunosenescence.[42]

5.5 Investigational Frontiers: Sepsis, COVID-19, and Cystic Fibrosis

The most recent research has focused on conditions involving acute immune dysregulation, where Thymalfasin's modulatory properties may be particularly beneficial.

• Sepsis: A completed Phase IV trial (NCT02883595) demonstrated that Thymalfasin was effective in improving monocyte function in patients with sepsis.[43] Multiple reviews and preclinical studies support its potential to curb mortality in sepsis by restoring T-cell function and balancing the inflammatory response.[2]
• COVID-19: A prospective, randomized pilot trial (NCT04487444) in hospitalized COVID-19 patients with hypoxemia and lymphocytopenia found that Thymalfasin treatment led to a significantly faster recovery of CD4+ T-cell counts compared to standard of care.[44] A comprehensive review of over 30 clinical trials concluded that Thymalfasin has demonstrated significant effectiveness and safety in treating COVID-19.[6]
• Cystic Fibrosis (CF): Preclinical research in a mouse model of CF has yielded promising results, showing that Thymalfasin can correct multiple tissue defects by reducing inflammation and, importantly, increasing the maturation, stability, and functional activity of the defective CFTR protein.[15] It is currently in Phase II clinical development for this indication.[45]

The following table provides a consolidated overview of significant clinical trials that have defined the therapeutic profile of Thymalfasin across its major indications.

Table 3: Overview of Significant Clinical Trials by Indication

Indication	Trial ID / Reference	Phase	Design	Key Findings	Source(s)
Chronic Hepatitis B	Chien et al., 1998	III	Randomized, Controlled	6-month course led to 40.6% complete virological response at 18 months vs. 9.4% in controls (p=0.004). Delayed response noted.	32
Chronic Hepatitis B	Mutchnick et al., 1999	III	Randomized, DB, Placebo-Controlled	No statistically significant difference in complete response (14% vs. 4%), but a positive trend was observed.	34
Chronic Hepatitis C	Rasi et al., 2004 (Review)	III	Randomized, Controlled	Adding Thymalfasin to Peg-IFN/ribavirin did not improve SVR but significantly reduced relapse rates in non-responders.	29
Hepatocellular Carcinoma	Patt et al., 2009	II	Randomized, Active-Controlled	Combination with TACE showed numerically higher survival (110.3 vs. 57.0 weeks) and fewer bacterial infections vs. TACE alone.	38
Malignant Melanoma	SciClone Press Release, 2007	II	Randomized, Open-Label	Combination with dacarbazine tripled the overall response rate and extended overall survival vs. dacarbazine + interferon.	39
Sepsis	NCT02883595	IV	N/A	Found to be effective in improving monocyte function for sepsis.	43
COVID-19	NCT04487444	N/A	Prospective, Randomized, Open-Label	Increased CD4+ T-cell counts faster than standard of care in hospitalized patients with hypoxemia and lymphocytopenia.	44
Vaccine Adjuvant (Influenza)	Carraro et al., 2012	Pilot	Randomized, Controlled	Enhanced immunogenicity of H1N1v vaccine in hemodialyzed patients, meeting CHMP criteria for seroprotection.	41

6.0 Safety, Tolerability, and Drug Interaction Profile

A defining characteristic of Thymalfasin, consistently demonstrated across decades of clinical use in diverse patient populations, is its exceptional safety and tolerability profile.

6.1 Comprehensive Adverse Event Profile

Thymalfasin is remarkably well-tolerated. In clinical experience involving over 3,000 individuals and post-marketing data from over 600,000 patients, clinically significant adverse reactions attributable to the drug are rare, with an overall incidence of drug-related adverse events reported at less than 1%.[7] This favorable profile stands in sharp contrast to other potent immunomodulators like interferon and IL-2, which are often associated with severe systemic side effects.[26]

• Common Adverse Events: The most frequently reported adverse experiences are mild and localized to the injection site, consisting primarily of discomfort, redness, and swelling.[11]
• Rare Adverse Events: Infrequent and mild systemic effects have been reported, including erythema (skin redness), transient muscle atrophy, polyarthralgia (joint pain) combined with hand edema, and rash.[17]
• Laboratory Abnormalities: A transient increase in serum alanine aminotransferase (ALT) to more than twice the baseline value can occur, particularly during the treatment of chronic hepatitis B. This "ALT flare" is generally not considered a sign of drug toxicity but rather an indication of a beneficial immunologic response against infected liver cells. Therapy is typically continued unless signs or symptoms of liver failure are observed.[17]

6.2 Contraindications and Populations of Concern

The contraindications and precautions for Thymalfasin are primarily related to its immune-enhancing mechanism of action.

• Contraindications: The drug is contraindicated in patients with a known history of hypersensitivity to thymosin alpha 1 or any component of the formulation. Because its therapeutic effect relies on augmenting the immune system, it is also considered contraindicated in patients who are being deliberately immunosuppressed, such as organ transplant recipients, unless the potential benefits of therapy are judged to clearly outweigh the potential risks.[23]
• Precautions:
• Autoimmune Disease: Caution should be exercised when administering Thymalfasin to patients with pre-existing autoimmune disorders, as the theoretical risk exists that enhancing the immune system could exacerbate their condition.[11]
• Pregnancy and Lactation: Its use in pregnant women should be reserved for cases where it is clearly needed, as adequate studies have not been conducted. It is not known whether the drug is excreted in human milk, so caution is advised when administering it to nursing women.[17]
• Pediatric Use: Safety and effectiveness have not been established in patients under the age of 18 years.[23]

6.3 Analysis of Known Drug-Drug Interactions

Formal drug-drug interaction studies with Thymalfasin are limited, and product labeling advises caution.

• Pharmacodynamic Interactions:
• Other Immunomodulators: Caution is advised when administering Thymalfasin in combination with other immunomodulating drugs (e.g., interferons, interleukins) due to the potential for additive or synergistic effects that could lead to over-stimulation of the immune system.[11]
• Immunosuppressants: Concomitant use of immunosuppressive drugs, such as corticosteroids or certain chemotherapy agents, could theoretically antagonize the immunostimulatory action of Thymalfasin, potentially rendering it less effective.[11]
• Pharmacokinetic Interactions: DrugBank has flagged potential interactions based on theoretical risk, though clinical case reports are scarce. These include:
• An increased risk of methemoglobinemia when combined with various local anesthetics (e.g., Benzocaine, Bupivacaine, Ropivacaine) and other agents like Cocaine, Diphenhydramine, and Phenol.[10]
• An increased risk of thrombosis when combined with erythropoiesis-stimulating agents such as Darbepoetin alfa, Erythropoietin, and Peginesatide.[10]
• Formulation Incompatibility: To avoid potential physicochemical incompatibilities, Thymalfasin should not be mixed with any other drug in the same syringe or infusion.[23]

The table below summarizes the known and potential drug-drug interactions for Thymalfasin.

Table 4: Known and Potential Drug-Drug Interactions

Interacting Drug/Class	Potential Effect	Mechanism / Note	Source(s)
Immunosuppressants (e.g., Corticosteroids)	Decreased efficacy of Thymalfasin	Pharmacodynamic antagonism; immunosuppressants counteract the immunostimulatory effects.	11
Other Immunomodulators (e.g., Interferons)	Potential for over-stimulation of the immune system	Additive or synergistic pharmacodynamic effects. Caution is advised.	11
Erythropoiesis-Stimulating Agents (e.g., Erythropoietin)	Increased risk of thrombosis	The mechanism is not specified but is listed as a potential interaction.	10
Local Anesthetics (e.g., Benzocaine, Ropivacaine) & certain other drugs	Increased risk of methemoglobinemia	The mechanism is not specified but is listed as a potential interaction.	10

7.0 Global Regulatory Landscape and Market Access

The regulatory history of Thymalfasin is marked by a significant divergence between its status in the United States and Europe versus its widespread acceptance and use in many other parts of the world. This contrast forms a central paradox in the drug's overall profile.

7.1 Regulatory Status in the United States (FDA)

• Not Approved: Thymalfasin (Zadaxin®) has not received marketing approval from the U.S. Food and Drug Administration (FDA) for any indication and is not commercially available in the U.S..[7]
• Orphan Drug Designation: The FDA has acknowledged its therapeutic potential by granting it orphan drug designation for the treatment of hepatocellular carcinoma on March 6, 2000.[8] It also held orphan status for other conditions, such as DiGeorge Syndrome and malignant melanoma, between 1991 and 2006, which allowed for its use in clinical trials and on a compassionate use basis.[7]
• Clinical Development and FDA Interaction: The drug has completed numerous Phase II and Phase III trials in the U.S. for indications including chronic hepatitis B and C, and malignant melanoma.[4] A notable milestone was the FDA's agreement on a Special Protocol Assessment (SPA) for a Phase III trial in stage IV melanoma, indicating that the trial design was considered adequate to support a future regulatory submission.[39]
• FDA Concerns and Recent Actions: Despite its extensive clinical trial history, the FDA has not approved the drug. Publicly available review documents have pointed to mixed efficacy results in pivotal hepatitis B trials and have noted a lack of comprehensive submitted nonclinical toxicity study data.[22] More recently, in 2023, the FDA placed restrictions on the compounding of Tα1 by 503A pharmacies, citing a need for more data. This decision has been contested by some researchers and clinicians, who argue that the drug's extensive history of safe use in over 11,000 trial participants and widespread global post-marketing experience does not support such restrictions.[6]

7.2 Regulatory Status in Europe (EMA)

• Not Approved for Marketing: Similar to the U.S., Thymalfasin does not have a standard marketing authorisation from the European Medicines Agency (EMA).
• Orphan Designation: The EMA's Committee for Orphan Medicinal Products (COMP) granted Thymalfasin orphan designation (EU/3/02/110) for the treatment of hepatocellular carcinoma on July 30, 2002.[1] The EMA's public summary of the opinion noted that at the time of designation, no other medicinal products for HCC had been authorized in the European Union, highlighting the unmet medical need.[9] This designation provides incentives for development but is not a marketing authorization.

7.3 Approvals and Clinical Use in Other Regions

In stark contrast to its status in the U.S. and E.U., Thymalfasin is a widely approved and utilized therapeutic in numerous other countries.

• Widespread Approval: It is approved for sale in over 35 countries, with a significant market presence in Asia, South America, and the Middle East.[1]
• Key Approved Indications: The most common approved indications are the treatment of chronic hepatitis B and chronic hepatitis C.[4] In many of these regions, it is also approved as an adjuvant to chemotherapy for certain cancers and as a vaccine enhancer.[4]
• Examples of Approving Countries: The first approval for Thymalfasin was in China in December 1995 for chronic hepatitis B.[51] Other countries where it has been approved include Argentina, Peru, the Philippines, Singapore, Mexico, Venezuela, and South Korea.[4]

The pronounced difference between its widespread global use and its unapproved status with the FDA and EMA suggests a fundamental divergence in regulatory interpretation of the available data. This may stem from an "evidence versus endpoint" disconnect, where the drug demonstrates clear biological activity and an excellent safety profile, but the pivotal trials submitted to Western regulators may not have met the pre-specified, often very stringent, primary endpoints required for approval (e.g., achieving a statistically significant improvement in SVR in a difficult-to-treat HCV non-responder population). The drug's benefits may be more modulatory and supportive—such as reducing relapse rates, preventing infections, or improving quality of life—effects that may not be fully captured by traditional primary endpoints focused solely on viral clearance or tumor shrinkage. This regulatory paradox remains a defining feature of Thymalfasin's history.

8.0 Synthesis, Manufacturing, and Quality Control

Thymalfasin is a fully synthetic peptide, a characteristic that allows for high purity and batch-to-batch consistency, distinguishing it from earlier, less-defined biological extracts.

8.1 Chemical Synthesis: Solid-Phase Peptide Synthesis (SPPS)

The manufacturing of Thymalfasin relies on chemical synthesis rather than recombinant DNA technology.[1] The established method for this process is Solid-Phase Peptide Synthesis (SPPS), a technique pioneered by Bruce Merrifield that allows for the stepwise construction of a peptide chain on an insoluble resin support.[52]

• Fragment Condensation Strategy: While a linear, one-by-one addition of all 28 amino acids is possible, a more efficient and industrially scalable approach is the fragment condensation strategy. In this method, the full peptide sequence is broken down into smaller, more manageable fragments (e.g., residues 1-8, 9-19, and 20-28). Each fragment is synthesized separately via SPPS and then purified. Subsequently, these purified fragments are coupled together in solution or on the solid phase to construct the full-length Thymalfasin peptide.[54] This strategy offers several advantages: it significantly reduces the overall synthesis cycle time, simplifies the purification of intermediates, and generally results in a final crude product with higher purity and fewer deletion-sequence impurities compared to the linear approach.[54]
• Technical Details: The SPPS process for Thymalfasin fragments utilizes standard peptide chemistry, including the use of polymeric resins (e.g., Rink amide resin, 2-chlorotrityl chloride resin), temporary N-terminal α-amino protecting groups (most commonly the fluorenylmethyloxycarbonyl, or Fmoc, group), acid-labile side-chain protecting groups (e.g., tert-butyl (tBu), tert-butyloxycarbonyl (Boc)), and various coupling reagents (e.g., HOBt/DIC, PyBOP, HBTU) to facilitate efficient peptide bond formation.[53]

8.2 Formulation and Lyophilization Process

After the full-length peptide is synthesized, cleaved from the resin, and purified (typically by reverse-phase high-performance liquid chromatography, or RP-HPLC), it is prepared for its final dosage form.

• Formulation: The purified Thymalfasin peptide is dissolved in an aqueous solution containing excipients. As previously noted, the standard formulation includes mannitol as a bulking agent and a phosphate buffer to maintain the pH at an optimal level for stability (around 6.8).[17]
• Lyophilization: This aqueous solution is then subjected to sterile filtration to remove any microbial contamination. It is then aseptically filled into vials and undergoes a controlled vacuum freeze-drying (lyophilization) process. This involves freezing the solution, followed by sublimation of the water under vacuum, to produce a stable, sterile, lyophilized powder that is ready for reconstitution prior to injection.[56]

8.3 Impurity Profile and Quality Considerations

The quality control of a synthetic peptide therapeutic is critical to ensure its safety and efficacy.

• Impurity Profile: Potential impurities that must be monitored and controlled include peptide-related impurities, such as deletion sequences (from incomplete coupling reactions) or modified sequences, and process-related impurities, which can include residual solvents, reagents from the synthesis (e.g., coupling agents), and catalysts.[22]
• Quality Attributes: Key quality attributes for Thymalfasin include purity, identity, peptide content, and the absence of aggregation. The FDA has noted the lack of a United States Pharmacopeia (USP) drug substance monograph for Thymalfasin, which would standardize its quality specifications. The potential for immunogenicity, which can be influenced by impurities or peptide aggregates, is also a critical consideration for any peptide therapeutic and requires careful control during manufacturing and formulation.[22]

9.0 Expert Analysis and Future Outlook

9.1 Synthesis of Evidence: Reconciling Efficacy, Safety, and Regulatory Status

Thymalfasin presents a compelling case study in drug development, defined by a significant paradox: its widespread global approval and extensive real-world use stand in stark contrast to its unapproved status in the United States and Europe. The synthesis of the available evidence reveals that this is not a simple matter of the drug being effective or ineffective. Rather, it reflects a complex interplay between the nature of the drug's biological activity, the design of its pivotal clinical trials, and the differing evidentiary standards of global regulatory bodies.

The drug's safety profile is unequivocally strong, supported by data from tens of thousands of patients showing a very low incidence of adverse events. Its mechanism as a pleiotropic immune modulator—enhancing T-cell and NK-cell function while also possessing regulatory feedback loops—is well-supported by preclinical data. The disconnect arises in the translation of this mechanism into clinical trial outcomes that satisfy the rigorous demands of Western regulators. For instance, in the treatment of HCV, while Thymalfasin demonstrated a clear biological effect by reducing relapse rates, it failed to meet the primary endpoint of improving overall SVR. This suggests that its clinical value may be more nuanced and supportive than that of a primary, disease-curing agent. The drug's true benefit may lie in its ability to optimize the patient's immune response to a primary insult (a virus, a tumor, a vaccine), an effect that may not always be captured by traditional, narrowly defined clinical endpoints.

9.2 Unmet Needs and Potential Future Therapeutic Niches

The evolution of medicine has shifted Thymalfasin's therapeutic niche. While its role in hepatitis has diminished with the advent of direct-acting antivirals, its unique properties position it favorably for several modern, high-unmet-need areas:

• Oncology Adjuvant: The most promising frontier for Thymalfasin is in immuno-oncology. Its ability to mitigate chemotherapy-induced lymphopenia and protect against infections is of clear value. More importantly, its mechanism of action suggests a strong potential for synergy with immune checkpoint inhibitors (ICIs). By enhancing T-cell function, upregulating MHC expression, and activating dendritic cells, Thymalfasin could help convert immunologically "cold" tumors (non-responsive to ICIs) into "hot" tumors, thereby increasing the efficacy of agents like PD-1/PD-L1 inhibitors. This is a critical area of unmet need in oncology.[40]
• Vaccine Adjuvant for Vulnerable Populations: As global populations age, immunosenescence poses a significant public health challenge, leading to poor vaccine responses. The clinical data showing Thymalfasin's ability to enhance vaccine immunogenicity in hemodialysis patients and its ongoing study in older adults for COVID-19 boosters highlight a vital potential application. A safe, effective adjuvant for the elderly and other immunocompromised groups is a major unmet need.[41]
• Management of Acute Immune Dysregulation: Conditions like sepsis and severe viral pneumonias (e.g., COVID-19) are characterized not just by immunosuppression but by a dysregulated immune response. Thymalfasin's dual-action profile—enhancing T-cell function while also possessing anti-inflammatory and regulatory potential—makes it an ideal candidate for restoring immune homeostasis in these critical care settings.

9.3 Recommendations for Future Clinical Development and Research

To unlock the full potential of Thymalfasin and resolve its regulatory paradox, future development efforts should be strategically re-focused:

1. Design Trials with Appropriate Endpoints: Future clinical trials, particularly in oncology, should incorporate endpoints that reflect Thymalfasin's modulatory role. Beyond traditional tumor response rates, key secondary and exploratory endpoints should include changes in the tumor microenvironment, mitigation of treatment-related adverse events (e.g., infection rates, duration of neutropenia), improvements in quality of life, and detailed immunological biomarker analyses (e.g., T-cell repertoire, cytokine profiles).
2. Prioritize Combination Studies with ICIs: Well-designed, robust clinical trials combining Thymalfasin with immune checkpoint inhibitors are urgently needed to formally test the strong preclinical and mechanistic rationale for synergy. Such trials could establish a new standard of care in tumors with low response rates to ICIs alone.
3. Address the Regulatory Data Gap: For the drug to gain consideration from the FDA and EMA, sponsors must generate and submit a complete regulatory package. This includes comprehensive nonclinical toxicology studies conducted to modern standards and a detailed chemistry, manufacturing, and controls (CMC) section that establishes a USP monograph and thoroughly characterizes the product's impurity profile and stability.

In conclusion, Thymalfasin is a well-characterized, exceptionally safe immunomodulatory peptide whose clinical journey is far from over. By moving beyond its legacy indications and embracing its identity as a versatile adjuvant and immune homeostatic agent, and by designing future clinical trials to capture these nuanced benefits, Thymalfasin may yet become a cornerstone therapy in the management of cancer, infectious diseases, and the challenges of an aging immune system.

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