Introduction

Cytotoxic chemotherapy drugs have transformed cancer treatment over the past sixty years and remain the first-line systemic therapy for many cancers, even as newer therapeutics such as tyrosine kinase inhibitors and immune checkpoint inhibitors have been introduced into clinical practice. The mechanisms of chemotherapy drugs rely on “killing” cancer cells, and their efficacy is closely linked to the dosage administered1,2. To maximize therapeutic outcomes, treatment typically involves the repeated administration of chemotherapy drugs at their maximum-tolerated dose (MTD), the highest dose that can be given without causing unacceptable toxicity. However, because chemotherapy drugs inhibit cell proliferation, they also affect normal cells, resulting in toxic side effects that can negatively impact the quality of life and physical functioning of cancer patients3,4,5. Consequently, effective cancer treatment should not only extend patient survival but also maintain or improve their quality of life3,4,6. Understanding the underlying causes of chemotherapy-induced toxicity is critical for optimizing treatment, refining dose and dosing schedules, and ultimately achieving the dual goals of efficacy and patient well-being.

Docetaxel (DTX) is an antimicrotubular agent that disrupts the assembly of microtubules by interfering with their polymerization and depolymerization. Due to its low solubility (< 20 ng/mL) and permeability, DTX is formulated with 50% Tween 80 and 50% ethanol and administered at its MTD, typically 75 to 100 mg/m², as a 1-hour intravenous (IV) infusion every three weeks7. The MTD-based regimen of DTX is associated with significant toxic effects, including hypersensitivity reactions related to Tween 80, myelosuppression, and peripheral neuropathy, which often necessitate dose reductions due to intolerability in clinical practice5,8. To address these challenges, various clinical trials have explored alternative dosing schedules. Weekly or biweekly DTX regimens at 30–40 mg/m2 have demonstrated improved cancer treatment outcomes7,9,10. However, weekly DTX administration increases the frequency of hospital visits and raises treatment costs.

To enable flexible and convenient administration while reducing hospital visits in metronomic chemotherapy, numerous oral DTX formulations have been developed11,12,13. Our laboratory also developed oral DTX granules and demonstrated their efficacy in inhibiting prostate tumor growth in a lung metastatic prostate tumor mouse model11. In this study, mice received either oral DTX granules or intraperitoneal (IP) commercial DTX injection at a dose of 5 mg/kg every 3 days for 24 days, with no observed toxicity. However, a recent study reported that IP administration of DTX at 1 mg/kg per day caused intestinal toxicity and resulted in mouse death after just 7 days of dosing14. This finding raised concerns about the safety of using low, frequent dose regimens, such as oral DTX for metronomic chemotherapy. To test the safety of oral DTX administration, we conducted a comprehensive toxicity study of oral DTX granules. The results showed that overall oral DTX granules were safe for once daily over 27 days and the MTD of oral DTX granules was 50 mg/kg for female mice and 25 mg/kg for male mice for once daily dosing15. Although tissue distribution of DTX has been previously studied, comprehensive comparisons among different formulations and administration routes remain limited. Therefore, the objective of this study was to evaluate the impact of formulations and administration routes on DTX tissue distribution. We measured the tissue distribution of DTX following IP DTX injection, oral DTX solution (prepared using commercial DTX injection), oral DTX granules at a 5 mg/kg dose over 7 h, and IV DTX injection at 2 h. Pharmacokinetic (PK) analysis was also conducted to provide further insights.

Materials and methods

Materials

DTX was purchased from LC Laboratories (Woburn, MA). DTX Injection USP (20 mg/mL), equivalent to Taxotere, was purchased from Accord Healthcare, Inc. (Durham, NC). TPGS was obtained as gifts from BASF (Florham Park, NJ). Miglyol 812 is a mixed caprylic (C8:0) and capric (C10:0) fatty acid triglyceride and was obtained as gifts from Cremer (Eatontown, NJ). Aeroperl 300 was a gift from Evonik (Parsippany, NJ). Methanol and acetonitrile (HPLC grade) were purchased from Fisher Scientific (Pittsburgh, PA). Phosphoric acid was purchased from Sigma–Aldrich (St. Louis, MO).

Mice

Male CD-1 mice (20–30 g) were purchased from Charles River (Wilmington, Massachusetts). The experiments were conducted according to an approved protocol by the by the Institutional Animal Care and Use Committee (IACUC) at the University of North Texas Health Science Center. All animal experiments were conducted with IACUC guidelines and regulations. The animal studies were designed and reported according to the ARRIVE guideline. Animals that required euthanasia were euthanized by CO2 followed by cervical dislocation as recommended by the American Veterinary Medical Association Panel of Euthanasia.

Preparation of DTX granules

DTX granules (10% drug loading) were prepared as previously published11. Briefly, DTX (15 mg) was mixed with Miglyol 812 (45 mg) and TPGS (45 mg) at 50 °C for 20 min. Aeroperl 300 (54 mg) was then added and mixed. After cooling to room temperature, DTX granules were prepared.

Characterization of the physical state of DTX in DTX granules

The physical state of DTX in DTX granules was evaluated by both fourier transform infrared spectroscopy (FTIR) using a Nicolet iS5 FTIR spectrometer (Thermo Scientific, USA) and powder X-ray diffraction (XRD) using a Bruker D8 Advance (Bruker, Karlsruhe, Germany). For the FTIS measurements, pure DTX powder was used to identify the crystal peaks of DTX. An unprocessed physical mixture and a processed physical mixture that was prepared as described above without Aeroperl 300 were used as controls. Samples were scanned over a wave number range of 4000 –400 cm− 1. For XRD measurement, DTX powder prepared by mixing pure DTX powder and Aeroperl 300 at 10% drug loading was used to identify DTX peaks in XRD. Samples were measured over a 2θ range of 8–25° with a scan rate of 1.0 s per step.

Tissue sample collection

Male CD-1 mice (20–30 g) were randomly divided to four groups (four mice per group). The treatment groups received either an IP commercial DTX injection prepared according to the manufacturer instruction, an oral DTX solution prepared by diluting commercial DTX injection with tap water, or oral DTX granules suspended in tap water. All treatments were administered at an equivalent dose of 5 mg/kg DTX. Mice were sacrificed at 0.5, 1, 2, 4 and 7 h post-doing. EDTA-treated blood samples were centrifuged at 4000 rpm for 10 min at 4 °C to separate plasma. Tissues, including liver, lung, kidney, spleen, heart, brain, mesenteric lymph node, were directly collected without perfusion. Stomach, and small intestine were washed with water to remove contents before collection. All tissues were stored at − 80 °C until analyzed within three months. In addition, commercial DTX injection was given to mice (n = 4) by IV injection and with tissues collected at 2 h post-dosing.

Docetaxel sample analysis by LC-MS

DTX concentrations in tissue samples were measured by a validated LC-MS method with modification11. Briefly, DTX plasma samples were mixed with paclitaxel (internal standard) and then were extracted by using acetonitrile containing 0.1% formic acid at room temperature for 10 min with shaking. For other tissue samples, tissue was weighted and homogenized with a methanol-water solution (50:50, v/v). Paclitaxel was mixed into the tissue homogenates and acetonitrile containing 0.1% formic acid was added to extract DTX as described above. Following centrifugation at 15,000 rpm for 10 min, the supernatants were mixed with water containing 0.1% formic acid and then 5 µl of samples were injected into an Agilent 1260 Infinity HPLC coupled with an Agilent 6460 triple-quadrupole mass spectrometry for measurements. Positive electrospray (+ ESI) and multiple reaction monitoring modes (MRM) were optimized with 500 V nozzle voltage, 4000 V capillary, 380 °C sheath gas temperature, 325 °C gas temperature, 12 L/min sheath gas flow, 10 L/min gas flow, and 60 Psi nebulizer. The m/z transitions were optimized and selected as 830.3 + >549.2 + for DTX and 876.3 + >308.3 + for paclitaxel. The HPLC mobile phases consisted of A (water containing 0.1% formic acid) and B (acetonitrile containing 0.1% formic acid). The gradient elution was used as shown in Table 1.

Table 1 The gradient elution program for docetaxel LC-MS analysis.
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The LC-MS method was validated in terms of linearity, precision, accuracy, carryover, and specificity as previously reported16. The retention time of DTX and paclitaxel was 3.21 min and 3.33 min, respectively. Linearity range of DTX in all tissues was from 2.5 to 2500 ng/mL. The correlation coefficient was higher than 0.998 for all tissues.

Pharmacokinetic modeling

The PK analysis was performed with a non-compartmental approach using Phoenix WinNonlin software (version 8.4) (Princeton, NJ) to calculate PK parameters. The following PK parameters were estimated: peak plasma concentration (Cmax), time to peak plasma concentration (Tmax), area under the plasma concentration-time curve from time 0 to the last measurable concentration (AUClast), area under the plasma concentration-time curve from time 0 to infinity (AUCinf), terminal elimination half-life (t1/2), apparent clearance (CL/F), apparent volume of distribution (Vd/F). The tissue-to-plasma ratio (T/P ratio) and bioavailability (F) were calculated based on AUClast. Assuming the bioavailability of the IP formulation as 100%, the relative F values of the other two formulations were determined.

Statistical analysis

All parameters are represented as mean ± standard deviation (SD). The unpaired Student’s t-test was performed to compare between two groups and the p value < 0.05 was considered statistically significant.

Results and discussion

DTX crystals in DTX granules

Differential scanning calorimetry (DSC), FTIR and XRD are common methods used to identify the crystalline state of drugs. In our previous DSC analysis, the melting point peak observed in DTX powder was absent in the DTX granules11. A similar observation was made for sorafenib granules in the DSC analysis; however, sorafenib granules exhibited crystal peaks in the FTIR analysis17. These finding suggest that both DTX and sorafenib likely melted with the lipid and surfactant during the heating process in the DSC measurement. Therefore, we measured DTX granules by FTIR and XRD. As shown in Fig. 1A, the characteristic DTX crystal peaks at 708.93 cm⁻¹ and 1712.26 cm⁻¹ were present in the DTX granules, indicating that DTX remained in its crystalline form within the granules. In the XRD analysis (Fig. 1B), DTX remained in crystal forms in DTX granules even after one year of storage despite the shifts of peaks compared with DTX powder. These results confirm that the formulation strategy did not solubilize DTX in the granules. Previously, we reported binary lipid systems (BLS), consisting of a single lipid and a single water-soluble surfactant18. We demonstrated that coating a BLS onto pro-drug crystals in solid granules enhanced the oral absorption of water-insoluble drugs18,19. Among the BLS combinations in our previous report, the system composed of Miglyol 812 and TPGS forms stable microemulsions upon contact with water. The FTIR and XRD data presented here confirm that DTX crystals in the granules were coated with the BLS of Miglyol 812 and TPGS. Despite DTX not being dissolved within the granules, these DTX granules significantly increased tissue uptake of DTX and inhibited tumor growth in a prostate cancer mouse model, as we previously published11.

Fig. 1
figure 1

The physical state of DTX in DTX granules. (A) The FTIR measurements of DTX granules. Black: pure DTX powder; Purple: blank granule; Blue: physical mixture; Red: DTX granules. (B) The XRD measurements of DTX in DTX powder (control) and DTX granules. Black: freshly prepared DTX granules; Red: DTX powder; Blue: DTX granules stored at room temperature for one year. Both methods confirmed that DTX was present in its crystalline form in DTX granules.

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Tissue distribution of DTX formulations over 7 h

The MTD of DTX for IV administration in mice is 40 mg/kg1. Based on this, we selected a dose of 5 mg/kg for our animal studies, which is well-tolerated in mice. IV DTX injection was used as a reference, and its tissue distribution was measured at 2 h post-dosing (Fig. 2). To compare with oral DTX granules, commercial DTX injection was administered orally as a solution formulation containing fully dissolved DTX. The tissue distribution for each group is presented in Fig. 3.

Interestingly, DTX exhibited high lung uptake because DTX concentrations in the liver and lungs were comparable regardless of formulation types and administration routes (Fig. 3). For example, at 0.5 h post-dosing, concentrations in the liver vs. lung were 2167 ng/g vs. 2495 ng/g for IP DTX injection, 612 ng/g vs. 369 ng/g for oral DTX injection, and 135 ng/g vs. 213 ng/g for oral DTX granules. These findings suggest that the lung selectivity of DTX may be related to its molecular properties.

IV DTX injection demonstrated pronounced lung selectivity, with the highest DTX concentrations observed in the lungs among all tested tissues (Fig. 2). The formulation of DTX injection, a micelle composed of Tween 80 and alcohol (50%/50%, v/v), may play a role in this selectivity. Upon IV injection, the lungs are the first organ reached by the drug, and the small capillary vessels in the lungs may trap DTX micelles, leading to high concentrations in the lungs20. This could explain the efficacy of DTX in non-small cell lung cancer, where it serves as a first-line treatment. Our results suggest that the lung selectivity of IV DTX injection arises from a combination of molecular properties, formulation characteristics, and administration route.

Fig. 2
figure 2

Tissue distribution of IV DTX injection at a 5 mg/kg dose in mice after 2 h dosing (n = 4). Tissues were collected and analyzed by a validated LC-MS method.

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DTX also showed the uptake by the lymphatic system. High DTX concentrations were detected in the mesenteric lymph nodes for orally administered DTX (both oral DTX solution and oral DTX granules) and even for IP administered DTX (IP DTX injection). It is known that lipophilic drugs could enter the circulation system by the lymphatic transport21, and DTX, with a Log P 4.6, exhibits this characteristic. The feature of enhanced lymphatic uptake could be particularly beneficial for treating metastatic cancers, where cancer cells often migrate to lymph nodes. Additionally, this feature contributes to the oral absorption of DTX formulations, as evidenced by the data for oral DTX solution (Fig. 3B) and oral DTX granules (Fig. 3C). This mechanism can be leveraged to improve the oral bioavailability of DTX. Indeed, oral DTX formulations have been shown to enhance oral absorption through increased lymphatic uptake22.

The oral DTX solution contained fully dissolved DTX, whereas the oral DTX granules contained DTX in crystalline form. As a solution formulation, the oral DTX soluiton exhibited faster uptake than the granules, resulting in a higher Cmax and peak tissue uptake at 0.5 h (Fig. 3B). In contrast, the Cmax and peak tissue uptake for oral DTX granules occurred at 2 h (Figs. 3C). Despite these differences in uptake timing, the overall tissue uptake between oral DTX solution and oral DTX granules showed no significant difference over the 7-h period. Notably, DTX concentrations in the stomach and small intestine from oral DTX granules were comparable to those from oral DTX injection at each time point (p >0.05) (Fig. 3B,C). As we recently discussed, coating a BLS onto drug crystals enhances drug dissolution18. The results here demonstrate that the BLS-based DTX granules effectively addressed the poor solubility of DTX for oral administration.

Fig. 3
figure 3

Tissue distribution of IP DTX injection (A), oral DTX solution (B) and oral DTX granules (C). Mice (n = 3–4) were given each treatment at an equivalent dose of 5 mg/kg DTX. Tissues were collected and analyzed by a validated LC-MS method.

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Pharmacokinetic analysis of DTX tissue distribution

The concentration-time profiles of DTX following IP DTX injection, oral DTX solution, and oral DTX granules at a 5 mg/kg dose in mice over a 7-h period are shown in Fig. 4, with the primary PK parameters summarized in Tables 2, 3 and 4. After 7 h, DTX concentrations in most tissue samples were below the detection limitation of the LC-MS methods, except in the stomach and small intestine. Thus, we limited the measurement period to 7 h. To assess the adequacy of this sampling window, we calculated the AUC percentage (AUC₀–₇h/AUC₀–∞) for all analyses in Tables 2, 3 and 4. Of the 30 values, 24 were greater than 70%. Overall, AUC₀–₇h provided a reasonable approximation of AUC₀–∞ in the majority of cases. In the blood, both IP DTX injection and oral DTX solution exhibited an elimination half-life of approximately 1.6 h, indicating rapid clearance of DTX from the bloodstream. This rapid elimination was further confirmed with IV DTX injection, where the plasma concentration of DTX decreased to 47 ng/mL within 2 h (Fig. 2). These findings demonstrate that regardless of the formulation or administration route, DTX is quickly cleared from the blood but accumulates in tissues, showing significant uptake in tissues (Figs. 2 and 3). As a result, conventional methods that rely on drug concentrations in the blood and PK profiles to assess therapeutic efficacy are not well-suited for evaluating DTX. Given that tumors are in tissues and side effects primarily occur in tissues, assessing tissue concentrations and distribution provides a more accurate basis for evaluating the efficacy and potential side effects of DTX.

The exposure levels of oral DTX granules and oral DTX solution were comparable in the plasma and across various organs (Tables 2 and 3). There could be different mechanisms (e.g. metabolisms) that result in the concentration-time difference of the studied formulations in different tissues. Overall, a relatively consistent rank of low to high distribution of the three tested formulations in all the tissues was observed. The results demonstrated that DTX granules effectively addressed the poor solubility issue of DTX, as the F of oral DTX granules was significantly higher than that of oral DTX solution (p < 0.05) (Tables 2 and 3). However, the F of both oral formulations remained below 10% compared to the IP injection. DTX, a BCS IV drug, is characterized by both poor solubility and poor permeability. The low absorption of oral DTX solution highlights DTX’s limited permeability in the gastrointestinal tract. Furthermore, both oral DTX solution and oral DTX granules showed significant residual DTX concentrations in the stomach and small intestine, underscoring the need for a strategy to significantly improve the permeability of DTX. Enhancing permeability, in addition to improving solubility, is crucial for the development of effective oral DTX formulations.

The Tmax for oral DTX solution was 0.625 h, compared to 2 h for oral DTX granules, indicating faster absorption with the solution formulation. The Cmax after oral granules administration was higher than that of oral solution (50 ng/mL vs. 39 ng/mL). As expected, IP DTX injection showed the highest plasma concentrations, with a plasma AUClast approximately 23-fold greater than that of oral DTX injection and 14-fold greater than that oral DTX granules. However, IP DTX injection also resulted in significantly higher uptake in the kidneys and spleen. Given that DTX is a first-line chemotherapy drug for non-small cell lung cancer, we calculated the T/P ratio of lung to plasma, which was 5.75 for IP DTX injection (Table 2), 21.4 for oral DTX solution (Table 3), and 7.21 for oral DTX granules (Table 4). These findings suggest that oral formulations exhibited greater lung selectivity than IP injection. On the other hand, the T/P ratio of kidney to plasma was 38.9 for IP DTX injection, 14.9 for oral DTX solution, and 10.2 for oral DTX granules, indicating a higher risk of kidney toxicity with IP injection. This observation aligns with our previous toxicity studies showing that high concentrations of DTX can damage kidney function15. Additionally, IP DTX injection resulted in high concentrations in the stomach and small intestine, which explains the intestinal toxicity caused by IP DTX at 1 mg/kg per day14. In clinical practice, DTX injection is administered via IV infusion at its MTD, resulting in high initial DTX concentrations in the blood and non-target tissues, which can lead to side effects. In contrast, as shown here, oral DTX formulations do not produce elevated DTX levels in non-target tissues, enabling metronomic chemotherapy with low, frequent dosing to inhibit tumor growth while minimizing side effects11.

Fig. 4
figure 4

The concentration-time profiles of DTX in tissues. Yellow: IP DTX injection; Orange: oral DTX injection; Purple: oral DTX granules. Mice (n = 3–4) were given each treatment at an equivalent dose of 5 mg/kg DTX.

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Table 2 The primary Pharmacokinetic parameters of IP DTX injection (n = 4).
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Table 3 The primary Pharmacokinetic parameters of oral DTX solution (n = 4).
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Table 4 The primary Pharmacokinetic parameters of oral DTX granules (n = 4).
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Conclusion

In this study, we conducted a comprehensive comparison of the tissue distribution of DTX formulations administered via IV injection, IP injection, oral solution, and oral granules. DTX demonstrated rapid tissue distribution regardless of the formulation type or administration route. These findings emphasize that measuring tissue concentrations is more critical than blood concentrations for understanding the efficacy and side effects of DTX treatments. Despite containing DTX in crystalline form, oral BLS-based DTX granules achieved comparable exposure to oral DTX solution in which the drug was fully dissolved. This indicates that DTX granules successfully addressed the issue of poor solubility. However, strategies to enhance permeability are necessary to further improve the oral absorption of DTX. In this study, oral DTX treatments exhibited greater lung selectivity compared to IP DTX injection, while IP injection resulted in higher drug uptake in the kidneys, raising potential concerns about renal toxicity for IV and IP injections. These findings were observed at a dose of 5 mg/kg, and tissue distribution and kinetics may vary with dose. Therefore, additional studies using multiple dosing levels would be necessary to fully understand these effects. In addition, our study does not elucidate the mechanism underlying the observed lung selectivity of DTX. However, it does confirm that the lung selectivity is an inherent property of the drug regardless of formulations and administration routes. Further mechanistic studies will be important to clarify the basis of the selective distribution. This study enhances understanding of the tissue distribution of DTX treatments and provides valuable insights for the development and evaluation of novel DTX formulations and treatments, particularly for advancing oral DTX formulations.