From EP impurities to nitrosamines-Tadalafil-Tadalafil-Comprehensive analysis of impurity control and standard requirements

Tadalafil is a PDE5 inhibitor originally developed by Eli Lilly. It is mainly used to treat male erectile dysfunction (ED). The official domestic trade name is "Cialis". Because its effect can last for a long time after a single dose, up to 36 hours, it is also known as the "weekend drug".

However, for pharmaceutical quality, registration and analytical R&D practitioners, the difficulty of impurity control of this "old drug" that has been on the market for more than 20 years is not "old" at all. From conventional process impurities to nitrosamine impurities that have attracted regulatory attention in recent years, Tadalafil almost covers many typical issues in API impurity research and control, and can be called a condensed version of the "API impurity control textbook."

From a technical perspective, this article sorts out Tadalafil’s pharmacopoeia impurity framework, nitrosamine compliance points, and the actual needs for supporting standards.

1. What is Tadalafil?

Tadalafil is a selective phosphodiesterase type 5 (PDE5) inhibitor that promotes the relaxation of cavernous and pulmonary vascular smooth muscle by inhibiting the breakdown of cGMP. According to information included in the drug database Pharmacodia, tadalafil was originally developed by Eli Lilly and was first approved for marketing in the European Union in 2002. The U.S. FDA approved it under the trade name Cialis for the treatment of erectile dysfunction in 2003. In 2009, it approved 20 mg tablets (recommended daily dose of 40 mg, that is, 2 tablets) for the treatment of pulmonary arterial hypertension (trade name Adcirca). In 2011, it was further approved to extend the indications of Cialis to symptomatic benign prostatic hyperplasia. in the country, its approved indications are erectile dysfunction and benign prostatic hyperplasia.

Tadalafil (Tadalafil,171596-29-5)

The core skeleton of Tadalafil is a pyrazinopyridoindoledione fused ring structure, which contains a piperazinedione ring with a benzodioxole substituent. CAS number 171596-29-5, molecular formula C22H19N3O4, molecular weight approximately 389.4.

According to in vitro research data disclosed by Hepatox, Tadalafil's inhibitory effect on PDE5 is more than 10,000 times stronger than that of PDE1, PDE2, PDE3, PDE4 and PDE7-10, and nearly 700 times stronger than that of PDE6, which is involved in retinal light signal transduction. This subtype selectivity is also one of the structural basis for its clinical side effect spectrum to be different from similar drugs.

2. Impurity control: the logic behind pharmacopoeia provisions

The core of API quality standards is never just "content compliance", but the classification, naming and limit control of all impurities, which are usually divided into process-related impurities (residues of starting raw materials, intermediates, side reaction products), degradation products, isomers, and genotoxic impurities that need to be evaluated separately. The general chapter of the European Pharmacopoeia also aligns the control of genotoxic impurities with ICH M7 and the control of elemental impurities with ICH Q3D.

Tadalafil happens to be a molecule with a relatively complex impurity spectrum: it has two chiral centers on its skeleton. During synthesis, it is easy to produce stereoisomers with the same molecular formula as Tadalafil but different spatial configurations - this is the case for EP impurities A, B, and C in the catalog. The molecular formulas of all three are C. 22H19N3O4, which is consistent with Tadalafil itself; in addition, the synthesis route involves multi-step condensation and ring-forming reactions, and there will also be residual oxidation/structure-changing impurities with extra oxygen atoms in the molecular formula, as well as demethylene analogues (such as "Demethylene Tadalafil" named separately in the catalog). This is why the industry has named and characterized hundreds of known impurities around this molecule.

3. "Required questions" in European Pharmacopoeia monographs

Tadalafil is listed in monograph 2606 (Tadalafil) in the European Pharmacopoeia. According to an academic paper published in ScienceDirect, which conducted a "fingerprint" study on 46 batches of tadalafil API samples from 13 different manufacturers, the monograph required the use of infrared spectroscopy for identification and content determination (97.5%–102.0 %), loss on drying (not exceeding 0.5%) and other regular items, there are also inspection items for impurities A, B, C and related substances: the single limit of impurity A does not exceed 0.15%, the limits of other individual known or unknown impurities do not exceed 0.10%, and the total amount of impurities does not exceed 0.3%.

It is worth noting that this is only the portion of the monograph of the edition on which this paper is based that is explicitly quantitatively controlled. After cross-referencing the public product information of multiple international standard databases, it was found that the recognized EP impurities under Tadalafil are actually far more than A, B, and C - D, E, F, G, H, I and other numbers have consistent CAS from different sources. No. and molecular formula records (for example, EP impurity A has been confirmed to be the (6R, 12aS) stereoisomer of Tadalafil itself, and the molecular formulas of EP impurities D, E, F, and H are all C22H19N3O6, which has two more oxygen atoms than Tadalafil). This suggests that the three impurity reference standards in the early monograph are no longer enough to cover the current needs for more sophisticated impurity spectrum comparison, method development and registration application. The specific quantitative limits are still based on the latest monograph text of the current version of the Pharmacopoeia.

4. The unavoidable red line: nitrosamine impurities

If pharmacopoeia impurities are "must-test questions", nitrosamine impurities have been an "additional question" hanging over the heads of all API manufacturers in recent years.

This regulatory storm began in mid-2018: N-nitrosodimethylamine (NDMA) and other nitrosamine impurities were detected in valsartan raw materials. The European Medicines Agency (EMA) immediately initiated an assessment and expanded it to a systematic review of all human drugs. Nitrosamine compounds were classified as "probable human carcinogens" based on animal experiments. The U.S. FDA has joined forces with international regulatory agencies to establish the "Carcinogenic Potency Classification Method" (CPCA), which classifies carcinogenic potency levels based on structural characteristics such as the number and distribution of α-hydrogen atoms in molecules, and calculates the daily acceptable intake limit (AI limit).

Why is Tadalafil on this risk list? The piperazinedione structure retains a secondary amine (-NH-) group, which is one of the potential sources of risk for the formation of nitrosamine impurities: Once the secondary amine comes into contact with trace residues of nitrosating reagents such as nitrite during production or storage, the corresponding N-nitroso derivatives may be generated. This is also the reason why structures such as "N-nitroso-tadalafil" and "tadalafil di-nitroso impurities" are named and characterized separately.

The acceptable intake limit of nitrosamine impurities is usually at the nanogram level per day. When converted to the concentration of the raw material drug, it is mostly on the ppm level, and in some cases even approaches ppb. Conventional HPLC ultraviolet detection can no longer meet the sensitivity requirements, and LC-MS is generally required. /MS cooperates with isotope internal standards (deuterated, 13C labeled) for accurate quantification - this is the reason why isotopic labels such as "Tadalafil-d3", "Tadalafil-13C2, d3" and "N-nitroso-tadalafil-D3" exist.

CATO provides tadalafil series of reference substances

By superimposing the above requirements, a laboratory must fully handle the quality control and registration declaration of tadalafil. The actual standards required are far more than just "API + three EP impurities". They must at least cover:

• The API itself and its chromatographic system suitability/standards for content determination;

• Impurities that have been named in the Pharmacopoeia (A, B, C and subsequent supplementary numbers under EP/USP);

• Process-related impurities and degradation products for method development and impurity spectrum assignment;

• Nitrosamine impurities and their isotope-labeled internal standards are used for trace quantitative analysis;

• The isotope-labeled API itself is used for metabolism, pharmacokinetics or mass spectrometry internal standard studies.

(The above are some representative products, the complete product list can be obtained from CATO)

As a dual-system ISO that has passed CNAS (RM0030) and ANAB (AR-2832) at the same time A 17034-approved reference material/standard sample producer, CATO provides HPLC, NMR, and MS triple structure confirmation for the Tadalafil series standard products. The COA discloses a fixed value uncertainty of ±2.0% (95% CI). Spot specifications are shipped on the same day, and non-spot specifications support customized synthesis, minimizing the waiting period from "discovering an unknown peak" to "getting the reference substance" in the laboratory.

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References

1. Pharmacodia drug database, Tadalafil target/indication/first batch year information, https://data.pharmacodia.com/drugs/details/12002835551391696551

2. FierceBiotech, FDA Approves ADCIRCA (Tadalafil) Tablets for the Treatment of Pulmonary Arterial Hypertension (approved in May 2009, recommended daily dose 40mg), https://www.fiercebiotech.com/biotech/fda-approves-adcirca-tadalafil-tablets-for-treatment-of-pulmonary-arterial-hypertension

3. FiercePharma, FDA approves Cialis to treat benign prostatic hyperplasia (approved for BPH indication in October 2011), https://www.fiercepharma.com/pharma/fda-approves-cialis-to-treat-benign-prostatic-hyperplasia

4. Hepatox Drug-Induced Liver Injury Professional Network, Tadalafil PDE subtype selectivity data, https://www.hepatox.org/drug/show/455

5. Pharmaceutical Data Center, Tadalafil tablets product registration information (Chinese trade name "Cialis", English trade name Cialis), https://pharm.ncmi.cn/zysjk/ypjkyp/202312/t20231205_398315.html

6. Clustering of Tadalafil API Samples According to their Manufacturer in the Context of API Falsification Detection, ScienceDirect (Tadalafil European Pharmacopoeia Monograph 2606 Impurity A/B/C Limit Data), https://www.sciencedirect.com/science/article/abs/pii/S0022354923002022

7. Clearsynth, Tadalafil EP Impurity A product page ((6R,12aS) stereoisomer structure confirmation), https://clearsynth.com/product/tadalafil-ep-impurity-a

8. European Medicines Agency (EMA), Nitrosamine impurities (Regulatory Background and Guidance on Nitrosamine Impurities), https://www.ema.europa.eu/en/human-regulatory-overview/post-authorisation/referral-procedures-human-medicines/nitrosamine-impurities

9. US FDA, Determining Recommended Acceptable Intake Limits for N-nitrosamine Impurities in Pharmaceuticals (CPCA Carcinogenic Potency Classification Method), https://www.fda.gov/drugs/spotlight-cder-science/determining-recommended-acceptable-intake-limits-n-nitrosamine-impurities-pharmaceuticals

10. Veeprho, Tadalafil Nitroso Impurity 5 product description (Tadalafil secondary amine structure and nitrosylation site), https://veeprho.com/impurities/tadalafil-nitroso-impurity-5/

11. European Pharmacopoeia 10th ed. (General chapter on the alignment of ICH M7 genotoxic impurities and ICH Q3D elemental impurities), https://dokumen.pub/european-pharmacopoeia-10nbsped-9789287189127.html