Preparation of Fludarabine Phosphate_Industrial Additives

Overview^[1]

Fludarabine phosphate, chemical name 9-β-D-arabinofuranosyl-2-fluoroadenine-5′-phosphate, is used to treat chronic lymphocytic leukemia. The synthesis of fludarabine phosphate usually uses guanosine as the starting material, and synthesizes fludarabine through a multi-step reaction, and then performs an esterification reaction with phosphorus oxychloride. Fludarabine phosphate is an antimetabolite fluorinated purine nucleoside analogue that is highly selective for lymphocytes. It can inhibit the repair of DNA in cells in the resting phase and the synthesis of DNA in cells in the dividing phase, and It has the effect of promoting the apoptosis of these cells and has shown good curative effect. It is currently widely used to treat various blood system diseases, especially chronic lymphocytic leukemia. Fludarabine phosphate improves disease remission rate by promoting tumor cell apoptosis. Its pro-apoptotic method is mainly achieved by regulating the following pathways: death receptor pathway, mitochondrial pathway, P53, ceramide, Bcl-2 family and some apoptosis promoters and inhibitors. .

Preparation^[1][3]

Freeze-dried powder injection:

Add 1530ml of water for injection into the liquid preparation tank. The temperature of the liquid preparation tank is 10°C. Then add 50g of fludarabine phosphate. After stirring thoroughly, add an appropriate amount of 5mol/L sodium hydroxide solution and stir until the fludarabine phosphate reaches the required temperature. Labine is completely dissolved; then add 40g of mannitol and stir to make the solution become a clear solution; adjust the pH value of the solution to 7.7 with 5mol/L sodium hydroxide solution, add 270ml of water for injection, and mix well.

Add 5.4g of activated carbon to the clear solution, stir and adsorb for 30 minutes, then use a 0.45 μm microporous filter membrane to filter and decarburize while circulating, a 0.2 μm filter membrane for one-time sterilization filtration, and a 0.2 μm filter membrane. Secondary terminal sterilization and filtration, the filtrate obtained is put into the medicinal solution bottle for filling.

The filtrate is divided into bottles, each bottle contains 25 mg of fludarabine phosphate, half-stoppered, and put into a freeze-drying machine for freeze-drying. Freeze-drying is divided into three stages: pre-freezing, primary drying and secondary drying;

Pre-freezing stage: lower the shelf temperature to -3°C at a rate of 0.5°C/min, keep warm for 0.5 hours, then drop to -30°C at a rate of 0.72°C/min, and stop cooling. Keep the temperature for 1 hour, slowly raise the temperature to -5℃, keep the temperature for 1 hour, then cool down to -45℃ at a speed of 0.72℃/min, continue to keep the temperature for 4 hours, and evacuate until the vacuum in the box reaches 10pa;

Primary drying stage: The shelf temperature is raised in a stepwise manner. Heating and holding are carried out alternately. Each time the temperature is raised for 10 minutes, then held for 5 minutes. The heating rate is 0.34°C/min. When the temperature rises to -3°C, continue Keep warm for 4 hours;

Secondary drying stage: Raise the shelf temperature to 18°C ​​at a rate of 0.55°C/min and keep it warm for 1 hour. The shelf will continue to rise to 40°C at a rate of 0.2°C/min. The products in the secondary drying process After the temperature reaches 35℃, continue to keep warm for 3 hours.

The entire freeze-drying process is completed, the plug is fully pressed, and it is shipped out of the box after passing the test.

Capsules:

Fludarabine phosphate solution

1. The pharmacodynamic properties are solved in a pH 6.0 aqueous solution, and the solution is coated on the microcrystalline cellulose pellets. Finally, the coated pellets are wrapped with a gastric-soluble moisture-proof film coating, with a weight increase of 0.3%, and put into capsules. Ready in the shell.

Pharmacological effects^[2]

This product is a fluorinated nucleotide analogue of the antiviral drug vidarabine, namely 9-β-D-arabinic acid-furyl adenine (ara-A), which is relatively resistant to adenosine deamination. Enzyme deamination.

Fludarabine phosphate is rapidly dephosphorylated into fludarabine (2F-ara-A), which can be taken up by cells and then phosphorylated by intracellular deoxycytidine kinase to become active. Triphosphate 2F-ara-ATP. This metabolite can inhibit DNA synthesis by inhibiting ribonucleotide reductase, DNA polymerase α, δ and Σ, DNA primase and DNA ligase. In addition, it can also partially inhibit RNA polymerase II to reduce protein synthesis.

Although the mechanism of action of 2F-ara-ATP is not very clear in some aspects, it is inferred that it mainly inhibits cell growth by affecting the synthesis of DNA, RNA and protein, among which inhibiting the synthesis of DNA is its main effect. In addition, in vitro studies have shown that after lymphocytes from chronic lymphocytic leukemia (CLL) were treated with 2F-ara-A, they experienced extensive DNA fragmentation and cell death characterized by apoptosis.

Safety information

1) Systemic toxicity

In acute toxicity studies, single doses of fludarabine phosphate that were two orders of magnitude higher than the therapeutic dose could cause severe toxic symptoms or death. As predicted by cytotoxic drugs, bone marrow, lymphoid organs, gastrointestinal mucosa, kidneys, and male gonads are affected. Serious adverse reactions, including severe neurotoxicity and some with fatal consequences, have been observed in patients at doses close to recommended therapeutic doses (3-4 times) (see Overdose).

Systemic toxicity studies following multiple doses of fludarabine phosphate above critical doses also showed expected effects in rapidly proliferating tissues. Morphological changes worsen with increasing dosage and duration of administration, but the observed changes are generally believed to be reversible. In principle, existing treatment experience with fludarabine phosphate shows that it has similar toxicological characteristics in humans, although��Other adverse reactions, such as neurotoxicity, have been observed (see Adverse Reactions).

2) Embryotoxicity

The results of animal embryotoxicity studies indicate that fludarabine phosphate has the potential to cause teratogenesis. Given that, like other antimetabolites that primarily interfere with the differentiation process, there is only a small safety margin between teratogenic doses in animals and therapeutic doses in humans, the therapeutic use of fludarabine phosphate for injection is inconsistent with its teratogenic effects in humans. related to relative risk.

3) Potential genotoxicity and carcinogenicity

Research on fludarabine phosphate found that it can cause DNA damage in sister chromosome exchange experiments; it can cause chromosomal abnormalities in in vitro cytogenetic experiments; and it can increase micronucleus in mice. core rate. But experiments on genetic mutations and major lethality experiments on male mice yielded negative results. Therefore, the mutagenic potential of fludarabine phosphate is mainly expressed in somatic cells rather than germ cells.

The known effects of fludarabine phosphate at the DNA level and the results of mutagenic experiments give people reason to speculate that fludarabine phosphate may cause cancer. Since the speculation that injection of fludarabine phosphate leads to an increased risk of secondary tumors can only be confirmed with epidemiological data, no animal experiments have been conducted to directly study the tumorigenic effects of fludarabine phosphate.

4) Local tolerance

Animal experimental results of intravenous injection of fludarabine phosphate showed that there was no obvious local irritation reaction at the injection site. Even when the injection site is inappropriate, such as paravenous, intraarterial, and intramuscular injection of an aqueous solution containing 7.5 mg/mL fludarabine phosphate, there is no corresponding local irritation reaction.

In animal experiments, similar gastrointestinal damage was observed after oral or intravenous injection of fludarabine phosphate. This finding supports the hypothesis that fludarabine phosphate-induced enteritis is a systemic effect.

Pharmacokinetics^[2]

1. Fludarabine (2F-ara-A) plasma and urine pharmacokinetics

To date, rapid intravenous bolus injection, short-term infusion followed by continuous intravenous infusion and oral fludarabine phosphate (2F-ara-AMP) followed by fludarabine (2F-ara-A) have been studied. Dynamics were studied. 2F-ara-A shows similar pharmacokinetic characteristics in CLL and Lg-NHL patients. In cancer patients, no clear correlation was found between 2F-ara-A pharmacokinetics and therapeutic efficacy. The development of neutropenia and hematocrit changes suggests that the cytotoxicity of fludarabine phosphate inhibits hematopoiesis in a dose-dependent manner.

1) Distribution and metabolism

2F-ara-AMP is a water-soluble prodrug of fludarabine (2F-ara-A), which can be rapidly and quantitatively dephosphorylated into the nucleotide 2F-ara-A in the human body. Another metabolite, 2F-ara-hypoxanthine, is the major metabolite in dogs, but only trace amounts are observed in humans. A single dose of 2F-ara-AMP was infused at 25 mg/m2 for 30 minutes in CLL patients. At the end of the infusion, 2F-ara-A reached an average peak plasma concentration of 3.5-3.7 μM. After the fifth dose of infusion, there was a moderate accumulation of corresponding 2F-ara-A concentrations, and at the end of the infusion, the average peak plasma concentration reached 4.4-4.8 μM. During the 5-day treatment regimen, 2F-ara-A plasma trough concentrations increased approximately 2-fold. The accumulation of 2F-ara-A can be eliminated after several treatment cycles. The post-peak level decline is divided into three phases. The half-life of the initial phase is about 5 minutes, the half-life of the intermediate phase is 1-2 hours, and the half-life of the terminal phase is about 20 hours.

Comparison between pharmacokinetic studies of 2F-ara-A showed that the average total plasma clearance (CL) of 2F-ara-A was 79ml/min/m2 (2.2ml/min/kg), with an average The volume of distribution (Vss) is 83l/m2 (2.4l/kg), and the data varies greatly between individuals. After intravenous injection of fludarabine phosphate, 2F-ara-A plasma concentration and area under the plasma concentration-time curve (AUC) increased linearly with the drug dose, while the half-life, plasma clearance and distribution volume remained unchanged, consistent with the prompt Drugs that have a dose-linear relationship are dose-independent.

2) Clear

2F-ara-A is mainly excreted by the kidneys, and 40-60% of the intravenous dose is excreted in the urine. Total drug intake and output experiments with H-2F-ara-AMP in laboratory animals found that the radioactive label could be completely recovered from the urine.

3) Patient characteristics

Patients with renal insufficiency experience a decrease in total clearance, indicating the need to reduce the dose of the drug. In vitro studies on human plasma proteins show that 2F-ara-A has no significant protein-binding tendency.

2. Intracellular pharmacokinetics of fludarabine triphosphate

2F-ara-A can be actively transported into leukemia cells, where it is first rephosphorylated into monophosphate, followed by bisphosphate and triphosphate. Triphosphate 2F-ara-ATP is the main metabolite in cells and the only metabolite with cytotoxic effects currently known. The median time to reach peak concentration of 2F-ara-ATP in leukemia lymphocytes of CLL patients is 4 hours, and the peak concentration varies significantly, with a median value of approximately 20 μM. The level of 2F-ara-ATP in leukemia cells was much higher than the peak value of 2F-ara-A in plasma, indicating that the drug accumulates at the target site. In vitro culture experiments of leukemic lymphocytes showed that extracellular 2F-ara-A exposure (including the concentration of 2F-ara-A and culture time) was linearly related to the intracellular 2F-ara-ATP concentration. The median half-life of 2F-ara-ATP clearance from target cells is 15 and 23 hours.

Drug interactions^[2]

1. In a clinical study, a high incidence of fatal disease occurred when fludarabine phosphate was combined with pentostatin (deoxycofamycin) in the treatment of refractory chronic lymphocytic leukemia (CLL). Pulmonary toxicity. Therefore, concomitant use of pentostatin is not recommended when using this product.

2. The therapeutic effect of fludarabine phosphate will be weakened by dipyridamole and other adenosine absorption inhibitors.

��relationship. The median half-life of 2F-ara-ATP clearance from target cells is 15 and 23 hours.

Drug interactions^[2]

1. In a clinical study, a high incidence of fatal disease occurred when fludarabine phosphate was combined with pentostatin (deoxycofamycin) in the treatment of refractory chronic lymphocytic leukemia (CLL). Pulmonary toxicity. Therefore, concomitant use of pentostatin is not recommended when using this product.

2. The therapeutic effect of fludarabine phosphate will be weakened by dipyridamole and other adenosine absorption inhibitors.