Natarajan Raghunand

Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases

The external pH of solid tumors is acidic as a consequence of increased metabolism of glucose and poor perfusion. Acid pH has been shown to stimulate tumor cell invasion and metastasis in vitro and in cells before tail vein injection in vivo. The present study investigates whether inhibition of this tumor acidity will reduce the incidence of in vivo metastases. Here, we show that oral NaHCO(3) selectively increased the pH of tumors and reduced the formation of spontaneous metastases in mouse models of metastatic breast cancer. This treatment regimen was shown to significantly increase the extracellular pH, but not the intracellular pH, of tumors by (31)P magnetic resonance spectroscopy and the export of acid from growing tumors by fluorescence microscopy of tumors grown in window chambers. NaHCO(3) therapy also reduced the rate of lymph node involvement, yet did not affect the levels of circulating tumor cells, suggesting that reduced organ metastases were not due to increased intravasation. In contrast, NaHCO(3) therapy significantly reduced the formation of hepatic metastases following intrasplenic injection, suggesting that it did inhibit extravasation and colonization. In tail vein injections of alternative cancer models, bicarbonate had mixed results, inhibiting the formation of metastases from PC3M prostate cancer cells, but not those of B16 melanoma. Although the mechanism of this therapy is not known with certainty, low pH was shown to increase the release of active cathepsin B, an important matrix remodeling protease.

In vivo imaging of extracellular pH using 1H MRSI.

Tumor pH is physiologically important since it influences a number of processes relevant to tumorigenesis and therapy. Hence, knowledge of localized pH within tumors would contribute to understanding these processes. The destructiveness, poor spatial resolution, and poor signal‐to‐noise ratio (SNR) of current technologies (e.g., microelectrodes, 31P magnetic resonance spectroscopy) have limited such studies. An extrinsic chemical extracellular pH (pHe) probe is described that is used in combination with 1H magnetic resonance spectroscopic imaging to yield pHe maps with a spatial resolution of 1 × 1 × 4 mm3. The principle of the technique is demonstrated on a phantom. Further data are shown to demonstrate its application in vivo, and results agree with previously reported pH values. The accuracy of the reported pH measurements is <0.1 pH units, as derived from a detailed analysis of the errors associated with the technique, the description of which is included. Magn Reson Med 41:743–750, 1999.

Enhancement of chemotherapy by manipulation of tumour pH.

SummaryThe extracellular (interstitial) pH (pHe) of solid tumours is significantly more acidic compared to normal tissues. In-vitro, low pH reduces the uptake of weakly basic chemotherapeutic drugs and, hence, reduces their cytotoxicity. This phenomenon has been postulated to contribute to a ‘physiological’ resistance to weakly basic drugs in vivo. Doxorubicin is a weak base chemotherapeutic agent that is commonly used in combination chemotherapy to clinically treat breast cancers. This report demonstrates that MCF-7 human breast cancer cells in vitro are more susceptible to doxorubicin toxicity at pH 7.4, compared to pH 6.8. Furthermore 31P-magnetic resonance spectroscopy (MRS) has shown that the pHe of MCF-7 human breast cancer xenografts can be effectively and significantly raised with sodium bicarbonate in drinking water. The bicarbonate-induced extracellular alkalinization leads to significant improvements in the therapeutic effectiveness of doxorubicin against MCF-7 xenografts in vivo. Although physiological resistance to weakly basic chemotherapeutics is well-documented in vitro and in theory, these data represent the first in vivo demonstration of this important phenomenon.

Tumor acidity, ion trapping and chemotherapeutics: I. Acid pH affects the distribution of chemotherapeutic agents in vitro

Resistance to anti-cancer chemotherapies often leads to regional failure, and can be caused by biochemical and/or physiological mechanisms. Biochemical mechanisms include the overexpression of resistance-conferring proteins. In contrast, physiological resistance involves the tumor microenvironment, and can be caused by poor perfusion, hypoxia and/or acidity. This communication investigates the role of tumor acidity in resistance to a panel of chemotherapeutic agents commonly used against breast cancer, such as anthracyclines, taxanes, anti-metabolites and alkylating agents. The effects of pH on the cytotoxicity of these agents were determined, and ion trapping was confirmed by monitoring the effect of pH on the cellular uptake of radiolabeled anthracyclines. Furthermore, pH-dependent cytotoxicity and uptake were compared between parental drug sensitive MCF-7 cells and variants overexpressing p-glycoprotein (MDR-1) and Breast Cancer Resistance Protein. These data indicate that the magnitude of physiological resistance from pH-dependent ion trapping is comparable to biochemical resistance caused by overexpression of drug efflux pumps. Hence, microenvironment-based ion trapping is a significant barrier to anthracycline-based chemotherapy and can itself be a therapeutic target to enhance the efficacy of existing chemotherapies.

Renal and Systemic pH Imaging by Contrast-Enhanced MRI

Perturbations of renal and systemic pH accompany diseases of the kidney, such as renal tubular acidosis, and the ability to image tissue pH would be helpful to assess the extent and severity of such conditions. A dual‐contrast‐agent strategy using two gadolinium agents, the pH‐insensitive GdDOTP5− and the pH‐sensitive GdDOTA‐4AmP5−, has been developed to generate pH maps by MRI. The renal pharmacokinetics of the structurally dissimilar pH‐insensitive contrast agents GdDTPA2− and GdDOTP5− were found to be similar. On that basis, and on the basis of similarity of structure and charge, the renal pharmacokinetics of GdDOTP5− and GdDOTA‐4AmP5− were assumed to be identical. Dynamic T1‐weighted images of mice were acquired for 1 hr each following boluses of GdDOTP5− and GdDOTA‐4AmP5−. The time‐varying apparent concentration of GdDOTP5− and the time‐varying enhancement in longitudinal relaxation rate following GdDOTA‐4AmP5− were calculated for each pixel and used to compute pH images of the kidneys and surrounding tissues. MRI pH maps of control mice show acidic regions corresponding to the renal papilla, calyx, and ureter. Pretreatment of mice with the carbonic anhydrase inhibitor acetazolamide resulted in systemic metabolic acidosis and accompanying urine alkalinization that was readily detected by this dual‐contrast‐agent approach. Magn Reson Med 49:249–257, 2003.

High resolution pHe imaging of rat glioma using pH-dependent relaxivity

Previous studies using MR spectroscopy have shown that the extracellular pH (pHe) of tumors is acidic compared to normal tissues. This has a number of important sequelae that favor the emergence of more aggressive and therapy‐resistant tumors. New MRI methods based on pH‐sensitive T1 relaxivity are an attractive alternative to previous spectroscopic methods, as they allow improvements in spatial and temporal resolution. Recently, pH‐dependent GdDOTA‐4AmP5‐ and a pH‐independent analog, GdDOTP5‐, were used to image renal pH in mice. The current study has used a similar approach to image pHe in rat gliomas. Significant differences were observed compared to the renal study. First, the relaxivity of GdDOTP5‐ was found to be affected by the higher extracellular protein content of tumors. Second, the pixel‐by‐pixel analysis of the GdDOTP5‐ and GdDOTA‐4AmP5‐ pharmacokinetics showed significant dispersion, likely due to the temporal fluctuations in tumor perfusion. However, there was a robust correlation between the maximal enhancements produced by the two boluses. Therefore, to account for the local time‐courses differences, pHe maps were calculated at the time of maximal enhancement in each pixel. Finally, the comparison of the pHe and the time to maximal intensity maps revealed an inverse relationship between pHe and tumor perfusion. Magn Reson Med, 2006.

pH and drug resistance. I. Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines.

A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells. Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump. However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities. Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells. Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX). The resting steady-state pHi was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity. These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines. The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated. In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did. Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors. Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane. Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells. Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not. Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover. The potential impact of this behavior on drug resistance is examined in a companion manuscript.

Tumor acidity, ion trapping and chemotherapeutics: II. pH-dependent partition coefficients predict importance of ion trapping on pharmacokinetics of weakly basic chemotherapeutic agents

Ion-trapping theory predicts that alkalinization of tumor extracellular pH will enhance the anti-tumor activity of weak-base chemotherapeutics. We have previously demonstrated that chronic and acute treatment of tumor-bearing mice with sodium bicarbonate results in tumor-specific alkalinization of extracellular pH. Furthermore, bicarbonate pretreatment enhances the anti-tumor activity of doxorubicin and mitoxantrone in two different mouse tumor models. Previous work has indicated subtle, yet significant differences between the pH sensitivities of the biodistribution and anti-tumor efficacies of doxorubicin and mitoxantrone in vitro. The present study demonstrates that systemic alkalinization selectively enhances tumor uptake of radiolabeled mitoxantrone, but not doxorubicin. Results using these two drugs are quantitatively and qualitatively very different, and can be explained on the basis of differences in the octanol-water partition coefficients of their charged forms. These results suggest that inducing metabolic alkalosis in patients would have a positive effect on response to mitoxantrone therapy. However, the therapeutic index would not increase if sodium bicarbonate also caused increased retention of mitoxantrone in susceptible normal tissues in the host. The major dose-limiting organ systems for mitoxantrone are heart, liver, bone marrow, spleen and blood cells. Bicarbonate was found to have no significant effect on the distribution of mitoxantrone to any of these tissues except for spleen. However, neither spleen weights nor lymphocyte counts were adversely affected by NaHCO(3) pretreatment, indicating that this co-therapy does not enhance myelosuppression due to mitoxantrone therapy. These findings suggest that metabolic alkalosis would produce a net gain in mitoxantrone therapeutic index.

pH and drug resistance. II. Turnover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs.

Resistance to chemotherapeutic agents is a major cause of treatment failure in patients with cancer. The primary mechanism leading to a multidrug-resistant phenotype is assumed to be plasma-membrane localized overexpression of drug efflux transporters, such as P-glycoprotein (P-gp). However, acidic intracellular organelles can also participate in resistance to chemotherapeutic drugs. In this study, we investigated, both experimentally and theoretically, the effect of acidic vesicle turnover on drug resistance. We have developed a general model to account for multiple mechanisms of resistance to weakly basic organic cations, e.g. anthracyclines and Vinca alkaloids. The model predicts that lower cytosolic concentrations of drugs can be achieved through a combination of high endosomal turnover rates, a low endosomal pH, and an alkaline-inside pH gradient between cytosol and the extracellular fluid. Measured values for these parameters have been inserted into the model. Computations using conservative values of all parameters indicate that turnover of acidic vesicles can be an important contributor to the drug-resistant phenotype, especially if vesicles contain an active uptake system, such as H+/cation exchange. Even conservative estimates of organic cation-proton antiport activity would be sufficient to make endosomal drug extrusion a potent mechanism of resistance to weakly basic drugs. The effectiveness of such a drug export mechanism would be comparable to drug extrusion via drug pumps such as P-gp. Thus, turnover of acidic vesicles can be an important factor in chemoresistance, especially in cells that do not overexpress plasma membrane-bound drug pumps like P-glycoprotein.

Phase Ib Study of PEGylated Recombinant Human Hyaluronidase and Gemcitabine in Patients with Advanced Pancreatic Cancer

Purpose: This phase Ib study evaluated the safety and tolerability of PEGylated human recombinant hyaluronidase (PEGPH20) in combination with gemcitabine (Gem), and established a phase II dose for patients with untreated stage IV metastatic pancreatic ductal adenocarcinoma (PDA). Objective response rate and treatment efficacy using biomarker and imaging measurements were also evaluated. Experimental Design: Patients received escalating intravenous doses of PEGPH20 in combination with Gem using a standard 3+3 dose-escalation design. In cycle 1 (8 weeks), PEGPH20 was administrated twice weekly for 4 weeks, then once weekly for 3 weeks; Gem was administrated once weekly for 7 weeks, followed by 1 week off treatment. In each subsequent 4-week cycle, PEGPH20 and Gem were administered once weekly for 3 weeks, followed by 1 week off. Dexamethasone (8 mg) was given pre- and post-PEGPH20 administration. Several safety parameters were evaluated. Results: Twenty-eight patients were enrolled and received PEGPH20 at 1.0 (n = 4), 1.6 (n = 4), or 3.0 μg/kg (n = 20), respectively. The most common PEGPH20-related adverse events were musculoskeletal and extremity pain, peripheral edema, and fatigue. The incidence of thromboembolic events was 29%. Median progression-free survival (PFS) and overall survival (OS) rates were 5.0 and 6.6 months, respectively. In 17 patients evaluated for pretreatment tissue hyaluronan (HA) levels, median PFS and OS rates were 7.2 and 13.0 months for “high”-HA patients (n = 6), and 3.5 and 5.7 months for “low”-HA patients (n = 11), respectively. Conclusions: PEGPH20 in combination with Gem was well tolerated and may have therapeutic benefit in patients with advanced PDA, especially in those with high HA tumors. Clin Cancer Res; 22(12); 2848–54. ©2016 AACR.