(CANCER RESEARCH 56, 1194-1 198. March 15. 19%|
`
`Advances
`
`in Brief
`
`Cellular pH Gradient in Tumor versus Normal Tissue: Potential Exploitation for
`the Treatment of Cancer1
`
`Leo E. Gerweck2 and Kala Seetharaman
`
`Edwin L. Sleele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital. Harvard Medical School. Boston. Massachusetts
`
`02192
`
`and
`tumors are acidic, with no distinction between the intracellular
`extracellular
`compartment. More recent attempts
`to exploit
`tumor
`acidity involve an enhancement of intracellular acidity (by disruption
`of the cellular pH-regulating mechanisms), which leads to cell death
`at sufficiently low pH (2-4). As discussed in this article,
`the relative
`acidity of the extracellular/interstitial milieu of tumors compared to
`normal
`tissue, along with their
`invariant pHi. gives
`rise to a pH
`gradient difference between these tissues. This gradient difference
`provides a basis for the selective treatment of cancer.
`
`Materials and Methods
`
`pH of Normal and Tumor Tissue
`
`in both
`pHi. Patient-matched measurements of the extracellular and pHi
`human tumor
`and normal
`tissue hy the same
`investigator
`have not been
`reported in the literature. As discussed below,
`this complicates
`an evaluation of
`the cellular pH gradient of tissues due to the variability in pH values obtained
`with pH electrodes. For the measurement
`of pHi.
`the majority of values have
`been obtained using " P-MRS. Measurement
`of pH by MRS is largely stan
`
`(5). Al
`accuracy of ±0.1 pH units, and is noninvasive
`dardized, provides
`though both the intracellular
`and extracellular
`compartments
`of tissue contain
`phosphate, because of the relative size of the intracellular compartment
`and the
`relative concentration
`of phosphate
`in this compartment.
`pH measured using
`"P-MRS
`primarily reflects the aggregate pHi of tissue.
`
`in tumors of various histology and three
`A summary of the pHi values
`normal
`tissues
`is illustrated
`in Fig. 1. Each of the indicated
`values
`is the
`average for several
`tumors obtained by one or more investigators
`(6-15). The
`results are similar
`to those obtained in the extensive compilation of tissue pHi
`values compiled by Vaupel el id. ( 16). The pHi of tissues is relatively constant,
`ranging from approximately
`7.1 to 7.3 for the various tumor
`types and largely
`overlap those obtained in three normal
`tissues,
`i.e., 7.0-7.2. Values obtained in
`tumors of the same histology by the same investigator exhibit somewhat more
`variability
`(±0.1 pH units)
`than is obtained
`in similar
`studies of the same
`normal
`tissue (±0.05 pH units: Refs. 7 and 12). Limited studies indicate that
`the pHi of tumor
`tissue is slightly more basic than that obtained in normal
`tissue (7, 12). In summary,
`these data indicate
`that
`the pHi of tumors and
`normal
`tissues is similar and well regulated within ±0.1-0.2 pH units or less.
`pHe of Tumor and Normal Tissue and the Cellular pH Gradient. As
`shown in a comprehensive
`review of the literature by Wike-Hooley
`et al. (1),
`the electrode-measured
`pH values in human tumors are on average approxi
`mately 0.4 units lower than those observed in normal subcutaneous
`and muscle
`tissues. However,
`substantial
`heterogeneity
`and overlap in the reported pH
`values of these tissues
`is apparent
`(1. 17, 18). Of special
`relevance
`to the
`present
`topic is the range of electrode pH values reported for the same normal
`tissue. Table
`I shows the mean and SD of measured electrode pH values of
`subcutaneous
`tissue by four different
`investigators. For the same normal
`tissue,
`the pH variation between investigators
`is greater than the pH variation between
`patients analyzed by the same investigator
`(19-22). Differences
`in electrode
`calibration,
`electrode
`stability,
`local
`tissue damage
`at
`the site of electrode
`insertion,
`and the physiological
`and metabolic
`status of the patients may all
`contribute
`to the observed differences. By considering
`pH values obtained in
`both normal and tumor
`tissue, with the same electrode,
`at the same time,
`the
`interexperimental
`variation
`can be eliminated
`from the calculation
`of
`the
`difference
`in the pH of tumor and normal
`tissue. Few studies meet
`these
`criteria.
`The electrode
`
`pH values obtained
`
`in 20 patients with glioblastoma
`
`is
`
`Abstract
`
`pH values of human
`Although limited data exist, electrode-measured
`tumors and adjacent normal
`tissues, which are concurrently
`obtained by
`the same
`investigator
`in the same patient,
`consistently
`show that
`the
`electrode pH (believed to primarily represent
`tissue extracellular
`pH) is
`substantially
`and consistently
`lower in tumor than in normal
`tissue.
`In
`contrast,
`the '"P-magnetic
`resonance
`spectroscopy
`estimated that intra-
`
`identical or slightly more basic in tumor com
`cellular pH is essentially
`pared to normal
`tissue. As a consequence,
`the cellular pH gradient
`is
`substantially
`reduced or reversed in tumor compared to normal
`tissue:
`in
`normal
`tissue the extracellular pH is relatively basic, and in tumor tissue
`the magnitude of the pH gradient
`is reduced or reversed. This difference
`provides an exploitable avenue for the treatment of cancer. The extent
`to
`which drugs exhibiting weakly acid or basic properties
`are ionized is
`strongly dependent on the pH of their milieu. Weakly acidic drugs which
`are relatively
`lipid soluble
`in their nonionized state may diffuse freely
`across the cell membrane
`and, upon entering a relatively basic intracel-
`lular compartment, become trapped and accumulate within a cell, leading
`to substantial differences
`in the intracellular/extracellular
`drug distribu
`tion between tumor and normal
`tissue for drugs exhibiting
`appropriate
`pKas.
`
`Introduction
`
`Evidence accumulated over the past 50 years and more has shown
`that electrode-evaluated
`human tumor pH is on average,
`lower than
`the pH of normal
`tissues
`( 1). Few strategies, however, have been
`successfully developed to exploit
`this pH difference for the treatment
`of cancer. Two factors have hampered the exploitation of this differ
`ence. One factor
`is the overlap of electrode-measured
`tumor and
`normal
`tissue pH values that
`is observed when values obtained by
`various investigators
`are pooled and compared. This overlap appears
`to be due to largely undefined technical
`factors associated with the
`electrode measurement
`of tissue pH, as well as differences
`in the
`physiological
`and metabolic status of the patients at the time of the
`analyses. A second fundamental
`factor is the more recent demonstra
`tion using " P-MRS ' procedures
`that tissue pH is broadly resolvable
`
`pH evaluated by electrodes primarily meas
`into two compartments:
`ures interstitial or extracellular
`tissue pH. whereas pH evaluated by
`"P-MRS
`primarily reflects
`the aggregate pHi of tissue. The MRS
`
`the pHi of tumor and normal
`show that
`analyses
`0.1-0.2
`pH units.
`i.e., ±approximately
`Most studies designed to exploit
`the relative acidity of tumor versus
`normal
`tissue have been based on the electrode pH data showing that
`
`tissue are similar.
`
`Received 10/20/95; accepted 1/25/96.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must
`therefore be hereby marked advertisement
`in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`1Supported hy National Cancer
`Institute Grant CA22860.
`of Radiation
`" To whom requests
`for reprints
`should be addressed,
`at Department
`Oncology, Edwin L. Steele Laboratory. Massachusetts General Hospital. Harvard Medical
`School. 100 Blossom Street. COX 302. Boston, MA 02114-2617. Phone: (617) 726-8145;
`Fax: (617) 726-8145.
`used are: MRS, magnetic resonance spectroscopy;
`1The abbreviations
`lular pH: pHe. extracellular pH.
`
`intracel-
`
`pHi,
`
`1194
`
`MYLAN INST. EXHIBIT 1048 PAGE 1
`
`MYLAN INST. EXHIBIT 1048 PAGE 1
`
`

`

`CELLULAR pH GRADIENT IN TUMOR AND NORMAL TISSUE
`
`_ w.0
`
`- w
`o.«2
`
`Liver
`
`Brain
`
`Sk. muscle
`
`Misc.
`
`tumors
`
`Breast
`
`tumors
`
`O
`
`Brain tumors
`
`Sq. cell
`
`carcinoma
`
`Sarcomas
`
`hOH
`
`t-OH
`
`I—O-
`
`!—O—!
`
`I—
`
`I—C
`
`I—C
`
`Non-Hodgkins
`
`lymph.
`
`I—O-
`
`'
`
`'
`
`I
`6.5
`
`'
`
`'
`
`'
`
`I
`7
`
`'
`
`'
`
`'
`
`'
`
`'
`
`I
`7.5
`
`8
`
`Mean pH. (MRS)
`i
`tissues and tumors.
`estimated pHi of various human normal
`Fig. I. The "P-MRS
`Confidence intervals are I SD. The data Corliver are from Oberhaensli et ill. (7): for brain
`from Oberhaensli
`ft
`tit. (7) and Hubesch el ai
`(13):
`for resting skeletal muscle from
`Soslman el al. (13). Semmler et til. (9). and Nidecker et al. (10): for miscellaneous
`tumors
`from Oberhaensli
`et al. (7) and Ng et al. (8): for breast
`tumors from Sijens el al. (n) and
`Oberhaensli et al. (7); for brain tumors from Oberhaensli et al. (7) and Hubesch et al. ( 14);
`for squamous cell carcinomas
`from Ng el al. (8): for sarcomas
`from Sostman el al. ( 12.
`13). Dewhirst
`et al.
`(II). Nidecker
`et al.
`(10). and Semmler et al.
`(9): and for non-
`Hodgkin's
`Ivmphoma from Ng et al. (8) and Smith et al. (15).
`
`of biomolecules
`impede the extravasation
`vascular wall does not substantially
`whose molecular weight
`is a few thousand
`or
`less (26). Similarly,
`in the
`absence of binding, drugs of molecular weight of approximately M, 10.000 or
`less freely diffuse (similar
`to water)
`in the interstitium (27). Entry of a drug
`into the cell may occur via either carrier or noncarrier-mediated
`processes
`(diffusion),
`and. commonly, membrane transport occurs by both mechanisms.
`Both inward and outward diffusion may occur simultaneously
`and independ
`ently of carrier-mediated
`transport, and under certain circumstances,
`become
`the predominant mechanism of transport
`(28).
`those which are not ana
`For noncarrier-mediated molecules
`(commonly
`logues of naturally occurring biomolecules).
`diffusion is the sole mechanism of
`transport. Diffusion across a non-polar
`lipid barrier
`is dependent on the lipid
`solubility or polarity of the diffusing molecule.
`lonization
`substantially
`de
`creases
`lipid solubility and diffusivity. A wide variety of naturally occurring
`biomolecules
`(amino acids, proteins,
`nucleic
`acids. ATP. etc.) as well as
`therapeutics
`are weakly acidic or basic and are therefore charged or uncharged
`depending on the pH of their microenvironment.
`into a relatively acidic
`Following drug extravasation
`across the vessel wall
`extracellular
`tumor environment,
`the fraction of a weak acid which is charged
`decreases,
`resulting in an increased ability to diffuse across the cell membrane.
`If the pHi is relatively basic, ¡onizationof the weak acid increases,
`leading to
`a decreased membrane permeability
`and trapping in the relatively basic com
`partment. Assuming that an undissociated weak acid freely passes between the
`intracellular
`and extracellular
`compartment,
`and the charged molecule does
`not,
`then as shown by Roos and Boron (29).
`the ratio of the intracellular:
`extracellular
`drug concentration
`of both the charged and uncharged form is:
`+ 10pH°-pKa)
`
`C/Q. = (I
`
`(A)
`
`drug concentrations,
`and extracellular
`where C¡and Ct. are the intracellular
`the drug dissociation
`logarithm of
`respectively,
`and pKa is the negative
`constant
`(the pH at which 50% of the drug is dissociated). A similar expression
`describes
`the behavior of weak bases. Because of the exponential
`relationship
`between the cellular drug concentration
`and pHe. pHi. and pKa. small differ
`ences in any of the parameters may markedly effect
`the drug concentration
`ratio.
`
`matched with the pH values obtained in the adjacent normal brain of the same
`patients (Fig. 14; Ret'. 23). In 18 of 20 cases,
`the electrode-measured
`pH values
`
`Results
`
`pH Gradient, pHe, and Drug Uptake. As indicated in Table 1
`and Fig. 2. literature reported electrode pH values of human s.c. tissue
`vary significantly.
`In spite of
`this variability,
`the pHe difference
`between tumor and normal
`tissues is substantial and relatively invari
`ant when concurrently measured by the same investigator
`in the same
`patient
`(Fig. 2). Over a relevant pH range,
`the magnitude of the pH
`gradient across the cell membrane
`and not
`the absolute pH values
`provides the driving force for the selective distribution of weak acids
`and bases. For example, assuming the pHe of normal
`tissue ranges
`from 7.6 to 7.2, with the tumor pHe being 0.4 units lower, the ratio of
`the intracellular
`drug concentration
`in tumor versus normal
`tissue
`ranges
`from 2.4 to 2.3 (based on a pHi of 7.2 in both tissues,
`pKa = 6.0, Equation A). Substantial
`variation
`in pHe does not
`significantly impact
`the expected preferential uptake of weak acids
`(pKa < 6) in tumor compared to normal
`tissue.
`Fig. 3A illustrates the relationship between the calculated intracel
`lular and extracellular drug concentration at variable pHe. assuming
`the drug is a weak acid with a pKa of 7.0, and the pHi is 7.2. Under
`
`tissue
`Table I Electrode estimated ¡)Hof subcutaneous
`Measured pH values of human subcutaneous
`tissue obtained by various investigators.
`Differences
`in patients'
`age. physiology,
`electrode
`characteristics,
`and measurement
`procedures
`likely account
`for the observed differences. From Wike-Hooley el al. ( 1).
`
`to or less than those obtained in adjacent normal
`are equal
`of glioblastomas
`brain.
`In addition to these studies, Pampus (23) also measured the pH in 11
`patients with astrocytomas
`and adjacent
`normal brain (Fig.2B).
`In all
`II
`patients,
`the tumor pH was equal
`to or lower than the pH of the normal
`tissue.
`Similar
`results were obtained by Naeslund and Swenson (24). who measured
`the tissue pH in uterine cancer and normal
`tissue (Fig.2C).
`and Ashby and
`Cantab (25)
`in patients with melanoma
`(Fig.2D). For the four sets of data
`shown in Fig. 2. the matched pH values in the tumor were equal
`to or lower
`than those obtained in normal
`tissue in 40 of 42 cases;
`the mean pH difference
`being 0.41 ±0.27 as is observed ¡ncomprehensive
`reviews of unmatched data
`(I. 17. 19). However,
`in contrast
`to these pooled data compilations, matching
`of the measured pH values for investigator
`and patient, and time of analysis,
`markedly reduces the overlap of pHe values between tumor and normal
`tissue.
`The relatively
`invariant
`and similar pHi of
`tumor
`and normal
`tissue and
`substantially
`reduced pHe of tumor compared to normal
`tissue gives rise to a
`substantially different cellular pH gradient
`in these tissues. For an average pHi
`of 7.2 for both tumor and normal
`tissue and an pHe of 7.4 in normal
`tissue and
`6.8-7.2
`in tumor tissue (Fig.
`I; Refs. 1. 17. 18. and 25). the average difference
`between the extracellular
`and pHi
`is approximately
`+0.2 pH units in normal
`tissue and —¿(cid:3)0.2to —¿(cid:3)0.6in tumor
`tissue.
`
`All drugs exhibit neutral, acidic, or basic properties. For drugs which are
`weak acids (or bases),
`the extent
`to which they are ionized is exponentially
`related to the pH of their milieu. As the presence or absence of charge on a
`molecule will influence its lipophilicity.
`slight differences
`in pH may markedly
`influence
`the ability of these drugs
`to traverse
`the cell membrane
`and (he
`intracellular/extracellular
`equilibrium distribution of the drug.
`
`Drug Charge and Drug Transport
`
`The principal barrier to the entry of a drug into an intracellular
`is the cell membrane. With the exception
`of
`the blood-brain
`
`site of action
`barrier,
`the
`
`el al.(20)Vidyasagar
`
`el al. (21)Sample
`
`1195
`
`Investigatorvan
`
`den Berg et al.(19)Harrison
`
`andWalker(22)Stamm
`
`
`size2640III11Mean
`
`
`
`pH ±SD7.63
`
`±0.177.54
`
`±0.097.42
`
`±0.057.33
`
`±0.03
`
`MYLAN INST. EXHIBIT 1048 PAGE 2
`
`MYLAN INST. EXHIBIT 1048 PAGE 2
`
`

`

`CELLULAR pH GRADIENT IN TUMOR AND NORMAL TISSUE
`
`Attrocytoma
`
`Normal
`Braln
`
`(B)
`
`io-
`
`B e
`
`'
`
`4-
`
`2-»
`
`Normal
`Braln
`
`(A)
`
`Glioblastoma
`
`20
`
`18
`16-
`
`14-
`
`12-
`10-
`
`8 6
`
`-
`4-
`
`2-
`
`C0
`
`)
`
`«B
`O.
`
`6.4
`
`6.6
`6.8
`7.0
`Extracellular
`
`7.2
`pH
`
`7.4
`
`7.6
`
`5.8
`
`6.2
`
`6.6
`
`7.0
`
`7.4
`
`7.8
`
`Extracellular
`
`pH
`
`(D)
`
`Malanoma
`
`Subcutaneous
`Tissue
`
`8 7 6
`
`-
`
`5-
`
`4-
`
`3-
`
`2-
`
`1-
`
`(C)
`
`Uterin«
`Canear
`
`4-
`
`1-
`
`S.
`
`Normal
`Tissue
`
`6.0
`
`6.4
`
`7.6
`7.2
`6.8
`Extracellular
`
`8.0
`pH
`
`8.4
`
`6.4
`
`7.0
`6.7
`Extracellular
`
`7.3
`pH
`
`7.6
`
`Fig. 2. pH electrode measured pHe values con
`currently obtained in tumors and normal
`tissue in
`the same patient. For glioblastoma
`(A ) and astro-
`cytoma (fi).
`Ihe data are from Panipus (23),
`for
`uterine cancer
`(C)
`from Naeslund and Swenson
`(24), and for melanoma
`(¿>)from Ashby and
`Cantab (25).
`
`ionized
`the fraction of the drug that becomes
`basic pHe conditions,
`and confined to the extracellular
`compartment
`predominates. As the
`pHe approaches
`the pKa of the drug, an increasing fraction of the drug
`loses its charge, rendering it free to diffuse across the cell membrane.
`Upon entering the relatively basic intracellular compartment,
`the drug
`becomes
`ionized and trapped,
`leading to an increased intracellular
`concentration.
`pKa and Drug Uptake. The influence of pKa on the calculated
`cellular drug distribution ratios is shown in Fig. 3ß.Two examples are
`illustrated.
`In both cases the pHi
`is assumed to be 7.2:
`the pHe is
`assumed to be 6.8 in the upper cun'e and 7.4 in the lower curve. Very
`weak acids (pKa > 9) are essentially nonionized under physiological
`pH conditions. However,
`for pKas which are similar to the pH of their
`milieu, small differences
`in pH markedly effect
`the extent of ioniza-
`tion. For a pKa of 5 (Fig. 3ß,upper curve) ionization is greater at 7.2
`than 6.8, and the drug becomes trapped in the compartment
`in which
`it is ionized. Similarly, at an pHe of 7.4, a greater portion of the weak
`acid is ionized and confined to the extracellular
`compartment.
`A number of studies have investigated drug partitioning into arti
`ficial
`lipid vesicles
`and cells as a function of pH and drug pKa
`(30-32).
`In addition to pH and pKa. several additional
`factors have
`been shown to affect
`the predicted intravesicularextravesicular
`(or
`cellular) concentration ratios. pKa is influenced by factors such as the
`solvent
`in which it is dissolved,
`ionic strength, and temperature
`(29,
`32). Additionally,
`the numerical value of the predicted distribution
`
`ratio does not precisely match the observed distribution if the ionized
`drug is not completely membrane
`impermeable or is rapidly metab
`olized or sequestered
`in the intracellular
`compartment. Neverthe
`less,
`systematic
`in vitro evaluation
`of cellular
`drug uptake
`as a
`function of drug pKa and pH yields results which are substantially
`consistent with theory. Dennis et al. (3l ) measured the intracellu-
`lanextracellular
`distribution
`of misonidazole
`and weak acid and
`base analogues
`of misonidazole
`in V79 cells. Results
`from their
`studies are shown in Table 2. In accordance with theory,
`for the
`neutral drug misonidazole
`at equilibrium,
`the measured
`intracellu
`lar concentration was uninfluenced
`by pH. Also in accordance with
`theory,
`the intracellular
`concentration
`of the weak acid azomycin
`was higher at an pHe of 6.6 than at 7.6 (identical
`extracellular
`drug
`concentration).
`Similarly,
`as predicted for the weak base Ro 03899.
`the intracellular
`concentration
`was higher
`at pH 7.6 than 6.6.
`Although
`the observed
`distribution
`ratios do not match the pre
`dicted (calculated)
`ratios calculated
`on the basis of the drug pKa
`and the experimentally
`estimated pHi.
`the impact of the pKa on the
`cellular
`distribution
`these
`analogues
`is readily
`apparent
`and
`substantial.
`
`of
`
`Discussion
`
`in tumor and normal
`Although the cellular pH gradient differs
`tissue,
`the pHe and, therefore,
`the magnitude of the gradient within a
`1196
`
`MYLAN INST. EXHIBIT 1048 PAGE 3
`
`MYLAN INST. EXHIBIT 1048 PAGE 3
`
`

`

`CELLULAR pH GRADIENT IN TUMOR AND NORMAL TISSUE
`
`o "ir
`'**
`(U
`
`. s
`2 x
`
`u m
`§10-55
`
`O e
`
`0.0
`
`Extracellular
`
`pH
`
`pKa
`
`the pHi is 7.2, and the drug pKa is 7.0.
`distribution of a weak acid. For the example shown,
`Fig. 3. A, calculated effect of variable pHe on the ratio of the intracellulanextracellular
`B. calculated effect of variable pKa on the ratio of the intracellulanextracellular
`concentration of a weak acid. For the example shown,
`the pHi
`is 1.1, and the pHe is 6.8 or 7.4.
`
`tumor arc not uniform. Studies with miniature pH electrodes
`particular
`the pH within a tumor may vary from values which are
`show that
`similar to those in normal tissue to substantially more acidic values (1,
`33). Most
`likely,
`the tumor pHe decreases along the length and as a
`function of the radial distance
`from the supplying arterial vessel.
`Substantiation of this possibility is indicated by the studies of Martin
`and Jain (34), who demonstrated a decrease in pH over a range of <50
`/itm radially from supplying vessels in a rabbit ear chamber model. As
`the pH probe employed in these studies was a weak acid, the observed
`changes in tumor tissue likely underestimated the actual pHe decrease
`radially from the supplying vessel. Nevertheless,
`these observations
`are consistent with the expectations
`that the pHi:pHe gradient may be
`expected to increase in those cells most distal
`from the supplying
`blood vessel. The overall effect would be to enhance drug uptake and
`killing of cells which are normally exposed to the lowest drug con
`centration,
`and especially
`relevant
`to radiation
`therapy,
`to low
`concentrations
`of oxygen.
`exhibit acidic or basic prop
`Although several chemotherapeutics
`erties, few exhibit acidic properties with pKas in the range of 4.5-6.5,
`i.e., the range that would appreciably enhance cellular uptake of the
`drug in tumor tissue. Not only could weak acids enhance drug uptake
`in the less accessible and resistant portions of a tumor, but weak bases
`of the appropriate pKas may be used to enhance the uptake of drugs
`such as radioprotectors
`in normal
`tissues. Other conceptually similar
`
`concentration
`Table 2 Measured and calculated inlracellular
`und acidic or basic analogues
`and acidic
`Measured and calculated intracellular concentration ratios of misonidazole
`or basic analogues. Values measured in V79 cells under hypoxic conditions at a constant
`extracellular
`drug concentration
`for the various analogues. The calculated intracellular
`drug concentration is based on the experimentally estimated pHi. From Dennis et al. (31).
`
`ratios of misonidazole
`
`AnalogueMisonidazole
`
`Intracellular concentration
`
`ratio
`
`=
`Observed"1.0
`
`= 7.6)
`Calculated*1.50.36
`
`Azomycin (acid)
`Ro 03899 (base)pKaNeutral
`" From Dennis et al. (31).
`'' Calculated concentration ratios based on the measured pHi of approxim
`pHe = 6.6. and pHi of 7.45 at pHe = 7.6.
`
`2.2
`0.226.6/pHe
`
`7.2
`8.9(pHe
`
`ately 6.87 at
`
`1197
`
`for exploiting the pH gradient exist. Using an in vitro cell
`possibilities
`system, Jensen et al. (35) showed that the weak base chloroquine,
`an
`etoposide antagonist, virtually eliminated etoposide cytotoxicity at an
`pHe of 7.4, but was excluded from cells at an pHe of 6.5, resulting in
`a pronounced etoposide cytotoxicity.
`tissue pro
`The pH gradient difference between tumor and normal
`vides a strong rationale for the design and evaluation of the efficacy
`of drugs as a function of their pKas and the cellular pH gradient. A
`challenging but appropriate
`aspect of this evaluation is the develop
`ment and utilization of experimental
`tumor models and procedures for
`the evaluation of tumor and normal
`tissue toxicity as a function of the
`tissues' pH gradient and drug pKa (36-38).
`
`Acknowledgments
`
`and Fan
`We thank Drs. Bruce Chabner, Yves Boucher, Claus Kristensen,
`Yuan for their helpful suggestions during the preparation of the manuscript.
`
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