Development and In Vitro Evaluation of a Nimodipine Injectable Formulation: Preformulation, Optimization and Preliminary Biological Characterization

Nimodipine is a 1,4-dihydropyridine calcium-channel blocker characterized by high lipophilicity and a preferential cerebrovascular effect. Current product information identifies nimodipine as a therapy for neurological deficits associated with aneurysmal subarachnoid haemorrhage (aSAH). Oral products are widely used, while sterile infusion formulations are also authorised in several jurisdictions for the same clinical setting. The established clinical indication should be distinguished from experimental investigations of nimodipine in other neurological or ophthalmic conditions.

The formulation problem is driven primarily by the drug’s very low aqueous solubility. An injectable solution must maintain the drug in solution during manufacture, storage and administration while meeting requirements for sterility, particulate control, chemical stability, container compatibility and safe administration. Published formulation research has therefore explored cosolvents, micellar systems, nanoemulsions and other delivery platforms.

Interest in ophthalmic delivery has also generated preclinical evidence for topical nimodipine formulations. Such work is relevant to formulation science but does not establish nimodipine as an approved glaucoma treatment. Similarly, experimental studies of calcium-channel modulation in epilepsy do not establish nimodipine as an approved antiseizure medicine. The present work therefore treats glaucoma- and epilepsy-related findings as research context rather than therapeutic indications.

1.1 Rationale for formulation development

For a poorly water-soluble molecule, cosolvency is a straightforward approach in which a water-miscible organic solvent is combined with the aqueous vehicle to increase apparent solubility. PEG 400 is widely used as a pharmaceutical cosolvent, while ethanol can substantially increase the solubility of lipophilic compounds. Polysorbate 80 may further support wetting and solubilization. Chelation and antioxidant protection can be incorporated when justified by formulation-development studies.

The formulation strategy evaluated here used PEG 400 and ethanol as the principal variables, with Polysorbate 80, disodium EDTA and sodium metabisulfite maintained at controlled levels. The purpose was not to reproduce a marketed product exactly, but to identify a composition that provided acceptable solution characteristics at laboratory scale.

1.2 Objectives

To characterize the physical and physicochemical properties of nimodipine relevant to injectable formulation development.

To improve apparent aqueous solubility using a PEG 400–ethanol cosolvent system.

To prepare and compare ten trial formulations with systematically varied cosolvent and surfactant levels.

To evaluate appearance, clarity, pH, drug content, viscosity, osmolality, particulate matter and microbiological quality.

To investigate short-term stability under refrigerated, intermediate and accelerated conditions.

To perform exploratory in vitro biological and ex vivo corneal-permeation assessments of the selected formulation.

Figure 1. Chemical structure of nimodipine.

2. BACKGROUND AND LITERATURE REVIEW

2.1 Glaucoma and neuroprotection

Glaucoma is a group of progressive optic neuropathies characterized by retinal ganglion-cell loss, optic-nerve damage and corresponding visual-field impairment. Elevated intraocular pressure is the principal modifiable risk factor, but disease progression can occur despite pressure control. This has stimulated research into neuroprotective approaches directed at mechanisms such as excitotoxicity, oxidative stress, mitochondrial dysfunction and impaired ocular perfusion.

Nimodipine has been investigated in this context because L-type calcium-channel blockade can alter calcium influx and vascular tone. A 2017 study evaluated topical nimodipine formulations containing cyclodextrin complexes and reported improved ocular delivery characteristics and an intraocular-pressure-lowering response in experimental models. These findings support continued formulation research but remain preclinical evidence.

2.2 Epilepsy and calcium signalling

Epilepsy is a neurological disorder characterized by a persistent predisposition to epileptic seizures. Abnormal neuronal excitability and altered ion-channel activity contribute to seizure generation and propagation. Calcium signalling is one component of neuronal excitability, providing a mechanistic basis for experimental investigation of calcium-channel modulators. However, clinical management of epilepsy relies on established antiseizure therapies, and nimodipine is not an approved first-line antiseizure drug.

2.3 Nimodipine delivery systems

The formulation literature demonstrates several strategies for overcoming nimodipine’s low aqueous solubility. Song and colleagues developed nimodipine-loaded mixed micelles and reported markedly increased apparent solubility, compatibility with infusion media and reduced vascular irritation compared with conventional nimodipine solution. Other investigators have explored nanoemulsions, polymeric micelles, liposomes and ophthalmic formulations. Collectively, these studies show that delivery-system design can substantially influence solubility, release, tissue exposure and tolerability.

2.4 Clinical formulation context

Authorised Nimotop infusion products provide an important benchmark for parenteral formulation science. Current product information describes a sterile 0.02% nimodipine solution containing 10 mg in 50 mL and an ethanol-containing vehicle, with administration through a controlled continuous-infusion system. Product information also identifies compatibility restrictions, light sensitivity and clinically important hypotension risks. These characteristics highlight why an experimental formulation should not be considered interchangeable with an authorised product.

The present laboratory formulation contains 10 mg nimodipine in a final volume of 100 mL and therefore differs in concentration and composition from authorised infusion products. It should be considered an investigational laboratory formulation requiring comprehensive quality and safety evaluation.

3. MATERIALS

3.1 Active pharmaceutical ingredient and excipients

Figure 2. Schematic workflow for preparation and evaluation of the nimodipine injectable formulation

Figure 3. FTIR spectra of nimodipine and the drug–excipient physical mixture.

Figure 4. pH of F1–F10 formulations (mean ± SD, n=3).

Figure 5. Drug content of F1–F10 formulations (mean ± SD, n=3).

Figure 6. Viscosity of F1–F10 formulations (mean ± SD, n=3).

Figure 7. Osmolality of F1–F10 formulations (mean ± SD, n=3).

Condition	Time	Appearance	pH	Visual finding
2–8 °C	Initial	Clear, colourless	6.85 ± 0.02	No visible particles
2–8 °C	1 month	Clear, colourless	6.84 ± 0.02	No visible particles
25 ± 2 °C/60 ± 5% RH	Initial	Clear, colourless	6.85 ± 0.02	No visible particles
25 ± 2 °C/60 ± 5% RH	1 month	Clear, colourless	7.80 ± 0.03	No visible particles
40 ± 2 °C/75 ± 5% RH	Initial	Clear, colourless	6.85 ± 0.02	No visible particles
40 ± 2 °C/75 ± 5% RH	1 month	Turbid	7.99 ± 0.03	Small particles observed

Figure 8. Change in pH of F6 during one-month storage.

Nimodipine concentration (µg/mL)	Cell viability (%)	Cytotoxicity (%)
10	97.8 ± 1.2	2.2
25	96.4 ± 1.4	3.6
50	94.7 ± 1.6	5.3
100	92.1 ± 1.8	7.9
250	88.6 ± 2.1	11.4

Figure 9. MTT cell viability across the tested nimodipine concentrations.

Concentration (µg/mL)	Scavenging activity (%)
100	9.8
250	23.5
500	46.1
750	68.2
1000	90.7

Figure 10. DPPH radical-scavenging activity of the tested sample.

Figure 11. Concentration-dependent inhibition of calcium influx by the tested sample.

Time (min)	Amount permeated (mg)	Permeation (%)	Papp (×10⁻⁶ cm/s)
30	0.38 ± 0.02	7.60 ± 0.40	—
60	0.82 ± 0.03	16.40 ± 0.60	—
90	1.24 ± 0.04	24.80 ± 0.80	—
120	1.68 ± 0.05	33.60 ± 1.00	7.84 ± 0.23

Figure 12. Ex vivo cumulative corneal permeation of the tested F6 preparation.

Permeation increased progressively over 120 min, reaching 33.60 ± 1.00% with a reported apparent permeability coefficient of 7.84 ± 0.23 × 10⁻⁶ cm/s. This experiment provides preliminary evidence of corneal transport under ex vivo conditions. It should not be interpreted as proof that an injectable formulation is suitable for ophthalmic administration.

6. DISCUSSION

The preformulation findings establish the principal formulation challenge: nimodipine is poorly soluble in aqueous media but dissolves readily in PEG 400 and ethanol. This behaviour is consistent with the high lipophilicity of the molecule and supports the use of cosolvency as an initial formulation strategy. The progressive changes in PEG 400, ethanol and Polysorbate 80 allowed the formulation space to be explored without changing the total cosolvent volume.

All ten formulations were visually clear and homogeneous. This is important for an injectable solution because visible precipitation or particulate contamination would be unacceptable. However, visual clarity alone cannot establish chemical stability or physical stability over the product lifetime. The stability study demonstrates this distinction: F6 remained clear under refrigeration for one month, while the formulation showed substantial pH drift at 25 °C and turbidity with small particles at 40 °C/75% RH.

The pH behaviour is particularly important. F6 changed from 6.85 to 7.80 at 25 °C and to 7.99 with turbidity at 40 °C/75% RH. These changes indicate that the formulation requires further investigation of degradation pathways, buffer capacity, packaging interactions and temperature sensitivity. A one-month study cannot support a commercial shelf-life claim.

The physicochemical profile of F6 was favourable within the tested series. Drug content was 99.62 ± 0.18%, viscosity was 5.28 ± 0.04 cP and osmolality was 296 ± 2 mOsm/kg. These values are useful for comparative formulation screening, but injectable-product suitability also depends on sterility assurance, endotoxin control, visible and sub-visible particles, container closure integrity, compatibility with administration sets, extractables/leachables, pH, degradation products and systemic tolerability.

The formulation composition differs from authorised Nimotop infusion products. Current product information describes a 10 mg/50 mL sterile infusion solution with ethanol and macrogol 400, together with specific administration and compatibility requirements. The experimental formulation contains 10 mg/100 mL and adds Polysorbate 80, disodium EDTA and sodium metabisulfite. Therefore, direct equivalence with a marketed product should not be claimed.

The biological experiments provide supportive mechanistic observations but should be interpreted cautiously. The MTT results indicate preserved cell viability over the tested range. DPPH activity increased with concentration, and calcium-influx inhibition showed a concentration-dependent response. These assays can support early formulation research but cannot establish efficacy in glaucoma or epilepsy. The corneal-permeation experiment is similarly exploratory and does not convert an injectable preparation into an ophthalmic dosage form.

7. QUALITY AND REGULATORY CONSIDERATIONS

7.1 Sterile product development

A parenteral formulation requires a higher level of control than a conventional oral dosage form. Development should address bioburden, endotoxins, sterility assurance, aseptic processing or validated terminal sterilization where feasible, particulate matter, container-closure integrity, compatibility with the intended administration system, and stability of the active and excipients. The current results represent an early laboratory evaluation and do not replace formal sterile-product qualification.

7.2 Container and administration compatibility

Authorised nimodipine infusion information identifies compatibility restrictions with certain plastics and requires controlled administration equipment. Accordingly, future work should include systematic compatibility testing with the intended vial, stopper, syringe, tubing and infusion set. Adsorption, leachables, precipitation during dilution and light exposure should be investigated before clinical translation.

7.3 Stability programme

The observed pH drift and turbidity under elevated-temperature storage demonstrate why a formal stability programme is essential. Long-term and accelerated studies should be designed according to the intended storage condition, with appropriate time points and validated analytical methods. Chemical assay, related substances/degradation products, pH, appearance, particulate matter and container-closure integrity should be monitored.

7.4 Biological evidence

The current biological studies should be classified as preliminary. A future programme should specify the cell line, passage number, test concentration basis, vehicle controls, positive controls, exposure time, replicate structure and statistical analysis. For ocular research, corneal integrity, tissue viability and drug recovery should be documented. For neurological applications, pharmacokinetic exposure and validated disease-relevant models would be needed.

7.5 Statistical considerations

The reported results are primarily descriptive and are based on triplicate measurements. Future optimization should use a predefined experimental design with statistical modelling rather than selecting a formulation from individual highest values. A quality-by-design approach can identify critical material attributes and critical process parameters and establish a design space supported by experimental evidence.

7.6 Clinical and regulatory positioning

Nimodipine is clinically established for aneurysmal subarachnoid haemorrhage, whereas use in glaucoma and epilepsy remains investigational. The present formulation should therefore be positioned as a preclinical pharmaceutical-development study. No conclusion regarding clinical efficacy, clinical safety or suitability for human administration should be drawn from the current data.

8. CONCLUSION

A laboratory-scale nimodipine injectable formulation was developed using PEG 400 and ethanol as the principal cosolvents, with Polysorbate 80, disodium EDTA and sodium metabisulfite as supporting excipients. Preformulation studies demonstrated poor aqueous solubility and improved solubilization in PEG 400 and ethanol. FTIR evaluation showed retention of characteristic nimodipine bands in the tested physical mixture.

Among the ten trial formulations, F6 contained 25 mL PEG 400, 15 mL ethanol and 0.45% v/v Polysorbate 80 per 100 mL and was selected for further evaluation based on its overall physicochemical profile. F6 showed pH 6.85 ± 0.02, drug content 99.62 ± 0.18%, viscosity 5.28 ± 0.04 cP and osmolality 296 ± 2 mOsm/kg. No microbial growth was observed in the SCDM test performed, and no visible particulate matter was detected.

The preliminary stability findings were informative: F6 remained clear with minimal pH change at 2–8 °C over one month, whereas marked pH changes occurred at 25 °C and 40 °C/75% RH, with turbidity and small particles observed at the highest temperature. Accordingly, the formulation cannot be described as stable for long-term storage on the basis of the current dataset.

The exploratory MTT, DPPH, calcium-influx and ex vivo corneal-permeation studies generated concentration- or time-dependent responses. These observations support further investigation but do not demonstrate clinical efficacy or establish a therapeutic role for injectable nimodipine in glaucoma or epilepsy.

9. LIMITATIONS

• Only one month of stability data were available; long-term stability and formal shelf-life determination were not established.
• Complete compendial sterility testing, bacterial endotoxin testing and sterility-assurance validation were not demonstrated in the available dataset.
• Container-closure integrity, extractables/ leachables and administration-set compatibility require dedicated studies.
• Chemical stability and degradation products were not comprehensively characterized by a stability-indicating analytical method.
• The biological assays were preliminary and require fuller methodological and statistical documentation.
• The ex vivo corneal study used post-mortem tissue and does not establish suitability for ophthalmic administration of an injectable product.
• No in vivo pharmacokinetic, toxicological or efficacy study was conducted.
• Clinical efficacy for glaucoma or epilepsy was not established.

10. FUTURE WORK

Future development should prioritize validated assay and related-substance methods, complete sterile-product quality assessment, container and administration compatibility, formal stability studies, endotoxin testing, validated aseptic processing, pharmacokinetic characterization and appropriately designed preclinical safety studies. If glaucoma remains a target, a dedicated ophthalmic formulation should be developed rather than assuming that an injectable composition is suitable for ocular administration.

DECLARATIONS

Ethics Statement

No live-animal experiment was conducted in the present study. The ex vivo corneal permeation experiment was performed using post-mortem goat corneal tissue. The tissue was obtained and used in accordance with applicable institutional and local requirements governing the collection and use of post-mortem animal tissues.

Consent for Publication

Not applicable.

Funding

The authors received no external funding or financial support for the present study.

Conflict of Interest

The authors declare that there are no financial or non-financial conflicts of interest related to the present work.

Data Availability

All numerical data generated and analyzed in this study are presented in the tables and figures of the manuscript. Additional data may be made available by the corresponding author upon reasonable request, subject to applicable institutional requirements.

Author Contributions

All authors contributed to the conception and design of the study, formulation development, experimental work, data analysis, interpretation of results, manuscript preparation, critical revision of the manuscript, and approval of the final version.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the support and technical assistance provided by the respective laboratory and institutional facilities during the conduct of this study.

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