Tubulin/microtubule
Microtubules/Tubulin. Paclitaxel, the active pharmaceutical ingredient, binds to the beta-tubulin subunit of microtubules, promoting their assembly from tubulin dimers and stabilizing them against depolymerization. This stabilization disrupts the dynamic equilibrium of microtubules, leading to mitotic arrest at the G2/M phase of the cell cycle and ultimately inducing apoptosis in rapidly dividing cancer cells.

Taxanes are a key chemotherapy component for several malignancies, including metastatic breast cancer (MBC), ovarian cancer, and advanced non-small cell lung cancer (NSCLC). Despite the clinical benefit achieved with solvent-based (sb) taxanes, these agents can be associated with significant and severe toxicities. Albumin-bound paclitaxel (Abraxane; nab®-Paclitaxel), a novel solvent-free taxane, has demonstrated higher response rates and improved tolerability when compared with solvent-based formulations in patients with advanced MBC and NSCLC. The technology used to create nab-paclitaxel utilizes albumin to deliver paclitaxel, resulting in an advantageous pharmacokinetic (PK) profile. This review discusses the proposed mechanism of delivery of nab-paclitaxel, including an examination into a hypothesized greater ability to leverage albumin-based transport relative to sb-paclitaxel. An advantageous PK profile and the more efficient use of albumin-based transport may contribute to the preclinical finding that nab-paclitaxel achieves a 33% higher tumor uptake relative to sb-paclitaxel. Another possible contributing factor to the tumor accumulation of nab-paclitaxel is the binding of albumin to secreted protein acidic and rich in cysteine (SPARC), although the data supporting this relationship between SPARC and nab-paclitaxel remain largely correlative at this point. Recent data also suggest that nab-paclitaxel may enhance tumor accumulation of gemcitabine in pancreatic cancer treated with both agents. Additionally, a possible mechanistic synergy between nab-paclitaxel and capecitabine has been cited as the rationale to combine the two agents for MBC treatment. Thus, nab-paclitaxel appears to interact with tumors in a number of interesting, but not fully understood, ways. Continued preclinical and clinical research across a range of tumor types is warranted to answer the questions that remain on the mechanisms of delivery and antitumor activity of nab-paclitaxel[1].

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In vitro, Nab-Paclitaxel exhibits potent cytotoxicity against a wide range of human cancer cell lines, including those of the breast (MCF-7), lung (A549), and ovary (SKOV-3). Its IC50 values are comparable to, or slightly lower than, those of solvent-based paclitaxel (Taxol), indicating that the albumin nanoparticle formulation does not diminish the anti-tumor activity of the paclitaxel payload.

Classic thought on drug distribution holds that the free, or unbound, fraction of drug is the active fraction because drug bound to proteins or other macromolecules might be unable to cross cell membranes. In clinical studies, sb-paclitaxel has been shown to be highly protein bound in plasma, with Cremophor EL further decreasing the free/unbound fraction of drug. To examine the effect that formulation has on the pharmacokinetics (PK) of paclitaxel, a randomized crossover PK study was carried out in patients with cancer receiving either sb-paclitaxel at 175 mg/m2 q3w over a 3-hour infusion or nab-paclitaxel at 260 mg/m2 q3w over a 30-minute infusion. In this study, mass spectrometry was used to identify whether circulating paclitaxel was free/unbound or not. In this report, paclitaxel associated with Cremophor EL would not have been identified as unbound drug. The key finding of the study was that the formulation of nab-paclitaxel allowed a much higher fraction of unbound paclitaxel for nab-paclitaxel vs. sb-paclitaxel (6.3% vs. 2.4%, p < 0.001). Furthermore, the maximal concentration of unbound paclitaxel was about 10-fold higher for nab-paclitaxel (1284 ng/mL vs. 122 ng/mL, p < 0.000001), and the systemic exposure (AUCINF) of unbound paclitaxel was about 3-fold higher for nab-paclitaxel (1159 h*ng/mL vs. 410 h*ng/mL, p < 0.000005). A likely explanation for these differences lies in the entrapment of paclitaxel in Cremophor EL-based micelles in the solvent-based formulation of paclitaxel. Consistent with the effect of micellar entrapment on paclitaxel distribution, Sparreboom et al. found that nab-paclitaxel achieves a higher plasma clearance and a larger volume of distribution vs. sb-paclitaxel in preclinical studies. It has been suggested that micellar entrapment could affect the PK linearity of sb-paclitaxel. Table 3 lists selected PK studies on sb-paclitaxel and nab-paclitaxel and gives the authors' characterizations of PK linearity for the two agents[1].

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Although investigations into the distribution of unbound drug are important, it is also critical to consider that in human blood, paclitaxel is highly bound to proteins and other biomolecules. As stated previously, Kumar et al. demonstrated that when sb-paclitaxel is administered to humans, approximately 95% of paclitaxel binds to other molecules. Albumin and alpha-1-acid glycoprotein contributed equally to this binding, with a minor fraction of paclitaxel bound to lipoproteins. Furthermore, albumin is known to be a ubiquitous carrier of biomolecules in the blood, which prompts the consideration of how albumin may influence the transport of nab-paclitaxel to tumors. Circulating albumin must cross endothelial cells to reach tumors, and albumin has been reported to accomplish this in at least 2 ways (Fig. 1): receptor-mediated transcytosis and the enhanced permeation and retention (EPR) effect. It has been hypothesized that nab-paclitaxel may take advantage of each of these mechanisms to reach the tumor microenvironment[1].

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In vivo, Nab-Paclitaxel has demonstrated superior anti-tumor efficacy compared to solvent-based paclitaxel (Taxol) in multiple murine xenograft models of human cancer, including breast and non-small cell lung cancer (NSCLC). It achieves higher intratumoral concentrations of paclitaxel, leading to a greater reduction in tumor volume. Its improved tolerability profile, due to the absence of Cremophor EL, allows for higher tolerated doses and a higher maximum tolerated dose (MTD).

Standard non-cellular assays are not applicable for the Nab-Paclitaxel nanoparticle formulation, as it requires cellular uptake to release the active drug. However, for the active moiety (paclitaxel), the mechanism can be studied in a tubulin polymerization assay. Purified bovine brain tubulin is incubated with GTP and paclitaxel. The rate and extent of tubulin polymerization are measured by the increase in optical density at 340 nm (OD340), providing a direct measure of the drug's ability to stabilize microtubules.

In vitro cellular assays are performed using standard 2D monolayer cultures or 3D tumor spheroids of cancer cells (e.g., MCF-7, MDA-MB-468). Cells are seeded in 96-well plates and treated with serial dilutions of Nab-Paclitaxel for 48-72 hours. Cell viability is measured using an MTT, MTS, or CellTiter-Glo assay to determine the GI50 (50% growth inhibition concentration). For cell cycle analysis, cells are fixed, stained with propidium iodide, and analyzed by flow cytometry to quantify the accumulation of cells in the G2/M phase.

Studies have shown that a large fraction of injected albumin-conjugated molecules accumulate in proximity to tumors. As discussed above, albumin may reach tumors by receptor-mediated transport mechanisms or by the EPR effect. It has been hypothesized that cancer cells may consume albumin from the tumor microenvironment and then metabolize it, possibly enhancing tumor growth. Desai et al. conducted experiments in mice bearing xenograft tumors from injected human breast cancer cells to determine whether formulation played a role in the tumor uptake of paclitaxel. In these experiments, paclitaxel in both formulations was radioactively labeled and the amount of labeled paclitaxel that eventually reached tumors was quantified. When equal amounts were injected, the researchers found that a third more paclitaxel from the nab-paclitaxel formulation was taken up by tumors. The authors suggested that nab-paclitaxel reached a higher tumor accumulation vs. sb-paclitaxel due to both the lack of drug-sequestering solvent micelles and enhanced albumin-mediated transcytosis. A subsequent report of similar experiments suggested that nab-paclitaxel may achieve some degree of tumor selectivity relative to sb-paclitaxel, although the mechanisms responsible for this possibility were not characterized. [1]

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Another molecular mechanism proposed to play a potential role in the tumor accumulation of nab-paclitaxel is the prevalence of albumin-binding proteins such as secreted protein acidic and rich in cysteine (SPARC) in proximity to tumors. According to this theory, proteins such as SPARC may exist at higher-than-normal levels in the tumor interstitium. Thus, these proteins could sequester paclitaxel bound to albumin in tumors at levels higher than those in healthy tissues. High SPARC expression correlates with disease progression across a range of tumor types; however, some clinical data have suggested a correlation between SPARC expression in the tumor and/or tumor microenvironment and positive clinical outcomes in patients receiving nab-paclitaxel. Other studies have failed to show such a correlation. Thus, further studies delineating the molecular relationship between SPARC and nab-paclitaxel are warranted.[1]

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The in vivo efficacy of Nab-Paclitaxel is typically evaluated in a subcutaneous human xenograft model in athymic nude mice. For example, female nude mice are injected with MDA-MB-231 human breast cancer cells in the flank. Once tumors reach a size of ~150-200 mm3, mice are randomized into treatment groups and administered Nab-Paclitaxel intravenously (e.g., via tail vein injection) at doses of 10, 20, or 30 mg/kg (paclitaxel equivalent) on a q3d or q4d schedule. Tumor volumes (length × width2/2) are measured every 2-3 days with calipers for up to 30 days. Body weight is monitored as a measure of tolerability.

Nab-Paclitaxel exhibits favorable pharmacokinetic properties compared to solvent-based paclitaxel (Taxol). Following intravenous administration, it has a shorter distribution half-life, a larger volume of distribution (Vd), and a significantly higher clearance (CL). Most importantly, the albumin-bound formulation achieves a higher maximum concentration (Cmax) and a greater area under the curve (AUC) in tumors, due to the enhanced permeability and retention (EPR) effect and active albumin transport (gp60/caveolin-1 pathway).

Toxicology of Nab-Paclitaxel is well-documented from its clinical use. Its dose-limiting toxicities (DLTs) include neutropenia (low neutrophil count), sensory neuropathy (numbness, tingling), and fatigue. Importantly, because it avoids the Cremophor EL vehicle used in standard paclitaxel, Nab-Paclitaxel is associated with a significantly lower incidence of severe hypersensitivity reactions, and premedication with antihistamines and steroids is not routinely required.

[1]. Yardley DA. nab-Paclitaxel mechanisms of action and delivery. J Control Release. 2013 Sep 28;170(3):365-72.

Nab-Paclitaxel was initially developed to avoid the toxicity associated with Cremophor EL, a common surfactant in sb-paclitaxel. Unlike sb-paclitaxel and docetaxel, nab-paclitaxel does not use nonionic surfactants to dissolve paclitaxel, which are known to cause toxicity, and encapsulates paclitaxel in solvent-based micelles. The formulation of nab-paclitaxel contains human serum albumin at concentrations similar to those found in blood albumin. Nab-paclitaxel is prepared using a high-pressure homogenization process, mixing albumin and paclitaxel to form particles with an average diameter of 130 nm. This process does not covalently link albumin to paclitaxel. After injection, the nano-albumin-paclitaxel particles dissolve into a soluble albumin-paclitaxel complex, where paclitaxel may bind to or dissociate from albumin (injected or endogenous) or other biomolecules, or may exist in a free/unbound state (see the "Mechanism of Administration" section). Since nano-albumin-paclitaxel is formulated with albumin, it can be reconstituted with simple saline. Therefore, nano-albumin-paclitaxel does not require the use of steroids or antihistamines to prevent hypersensitivity reactions. Perhaps because the administration process of nano-albumin-paclitaxel is not affected by the solvent, a higher dose can be given than albumin-paclitaxel (sb-paclitaxel). In a pivotal phase III MBC trial, nab-paclitaxel and sb-paclitaxel were used as first-line treatment for MBC patients at the doses indicated on the label. Patients treated with nab-paclitaxel received a 49% higher dose of paclitaxel than those treated with sb-paclitaxel, suggesting that nab-paclitaxel can achieve a higher dose intensity [1].

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Nab-Paclitaxel (brand name Abraxane) is an FDA-approved chemotherapeutic agent. It is approved for the treatment of metastatic breast cancer, locally advanced or metastatic non-small cell lung cancer (NSCLC), and metastatic adenocarcinoma of the pancreas (in combination with gemcitabine). The albumin-bound technology significantly improves the drug's tolerability and efficacy by delivering higher doses of paclitaxel to the tumor site.

White to off-white solid powder

Nanoparticle albumin-bound Paclitaxel; Nanoparticle albumin-bound ABI-007

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)