[CANCER RESEARCH 56. 1194- 1198. March IS. 1996)
`
`Advances in Brief
`
`Cellular pH Gradient in Tumor versus Normal Tissue: Potential Exploitation for
`the Treatment of Cancer1
`
`Leo E. Gerweck1 and Kala Seetharaman
`Edwin L Steele Laboratory. DtparrmenJ of Radiation Oncology, Massachusms General Hospital, Harvard Medical S<-hool. Boston. Massachuse11s OZ J9Z
`
`Abstract
`
`Allllougb limited data exist, electrode-measured pH values or human
`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 tmue extracellular pH) Is
`substantlally and consistently lower in tumor than in normal tissue. In
`contrast, the "P-magnetic resonance spectroscopy estimated that intra•
`cellular pH Is -ntially identical or sllg)ltly more basic in tumor com•
`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 or cancer. The extent to
`which drugs exhibiting weakly add or basic properties are Ionized ls
`strongly dependent on the pH of their milieu. Weakly acidic drugs which
`are relatively lipid soluble in their nonionlzed state may diffuse freely
`across the cell membrane and, upon entering a relatively basic lotracel•
`lular compartment, become trapped and accumulate within a cell, leading
`to substantial differences in the intraceUular/extraceUular drug distribu(cid:173)
`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(cid:173)
`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(cid:173)
`tion using 3 1P-MRS3 procedures that tissue pH is broadly resolvable
`into two comparunents: pH evaluated by electrodes primarily meas(cid:173)
`ures interstitial or extracellular tissue pH, whereas pH evaluated by
`3 1P-MRS primarily reflects the aggregate pHi of tissue. The MRS
`analyses show that the pHi of tumor and normal tissue are similar.
`i.e., :!: approximately 0.1-0.2 pH units.
`Most studies designed to exploit the relative acidity of tumor versus
`normal tissue have been based on the electrode pH data showing that
`
`Received I 0/20/95: accepted 1/25/96.
`The costs of publication of Chis anicle were defrayed in pan by Che payment of page
`charges. This anicle muse therefore be hereby marked advertisement in accordance wilh
`18 U.S.C. Section 1734 solely 10 indicate chis face.
`' Supponcd by National Cancer lns1i1ute Gran1 CA22860.
`2 To whom requests for reprints should be addressed. a1 Depanmenc of Radiation
`Oncology. Edwin l. S~le Laboratory, Massachusetts General Hospital. Harvard Medical
`School, 100 Blossom Street. COX 302, Boston, MA 02114-2617. Phone: (617) 726-8145;
`Pax: (617) 72~8145.
`3 The abbreviations used are: MRS. magne1ic resonance spectroscopy; pHi. intracel(cid:173)
`lular pH: pHe, extracellular pH.
`
`tumors are acidic, with no distinction between the intracellular and
`extracellular compartment. More recent attempts 10 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
`
`pHi. Patient-matched measurements of the extracellular and pHi in both
`human tumor and nonnal tissue by the same invesligator 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 31 P-MRS. Measurement of pH by MRS is largely Stan·
`dardized. provides accuracy of :t0. I pH units. and is noninvasive (5). Al(cid:173)
`though both the intracellular and extracellular companments 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
`3' P•MRS primarily reflects the aggregate pHi of tissue.
`A summary of the pHi values in tumors of various histology and three
`nonnal tissues is illustrated in Fig. I . 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 compil:11ion of tissue pHi
`values compiled by Vaupel e1 al. ( 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 nonnal tissues. i .e .. 7.0- 7.2. Values obtained in
`tumors of the same histology by the same investigator exhibit somewhat more
`variability (:t0.l pH units) than is obtained in similar studies of the same
`normal tissue ( :t0.05 pH units: Refs. 7 and 12). Limited studies indicate that
`the pHi of tumor tissue is slightly more basic than lhal obtained in nonnal
`tissue (7, 12). In summary, these data indicate that the pHi of tumors and
`nonnal tissues is similar and well regulated within :t0.1- 0.2 pH units or less.
`pHe of Tumor and Normal Tissue and the Cellular pH GradlenL As
`shown in a comprehensive review of the literature by Wike-Hooley el al. ( I ).
`the electrode-measured pH values in human tumors are on average approxi(cid:173)
`mately 0.4 units lower than those observed in nonnal subcutaneous and muscle
`tissues. However, substantial heterogeneity and overlap in the reported pH
`values of these tissues is apparent (I . 17. I&). Of special relevance to the
`present topic is the range of electrode pH values reported for the same nonnal
`tissue. Table I shows the mean and SD of measured electrode pH values of
`subcutaneous tissue by four different investigators. For the same nonnal 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 nonnal 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 nonnal tissue. Few studies meet these
`criteria.
`The electrode pH values obtained in 20 patients with glioblastoma is
`
`1194
`
`FRESENIUS EXHIBIT 1048
`Page 1 of 5
`
`

`

`caLULAR pH GRADIENT I N TUMOR AND NORMAL TISSUE
`
`vascular wall does not substantially impede the extravasation of biomolecules
`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 transpon occurs by both mechanisms.
`Both inward and outward diffusion may occur simultaneously and independ(cid:173)
`ently of carrier-mediated transpon. and under certain circumstances, become
`the predominant mechanism of transpon (28).
`For noncarrier-mediated molecules (commonly those which are not ana(cid:173)
`logues of naturally occurring biomolecules). diffusion is the sole mechanism of
`transpon. Diffusion across a non-polar lipid barrier is dependent on the lipid
`solubility or polarity of the diffusing molecule. Ionization substantially de(cid:173)
`creases lipid solubility and diffusivity. A wide variety of naturally occurring
`biomolecules (amino acids, proteins. nucleic acids. ATP. etc.) as well as
`therapeutics arc weakly acidic or basic and arc therefore charged or uncharged
`depending on the pH of their microenvironment.
`Following drug extravasation across the vessel wall into a relatively acidic
`extracellular tumor environment. the fraction of a weak acid which is charged
`decrea.~. resulting in an increased ability to diffuse across the cell membrane.
`If the pHi is relatively basic, ionization of the weak acid increases. leading to
`a decreased membrane permeability and trapping in the relatively basic com(cid:173)
`partment. Assuming that an undissociated wealc 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 fonn is:
`
`(A)
`where C; and c. arc the intracellular and extracellular drug concentrations.
`respectively. and pKa is the negative logarithm of the drug dissociation
`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(cid:173)
`ences in any of the parameters may markedly effect the drug concentration
`ratio.
`
`Results
`
`pH Gradient, pHe, and Drug Uptake. As indicated in Table I
`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 nonnal tissues is substantial and relatively invari(cid:173)
`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 nonnal 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 nonnal tissue.
`Fig. 3A illustrates the relationship between the calculated intracel(cid:173)
`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
`
`-
`
`Cl)
`
`ca a, e=
`.. 0
`O.!
`Zt-
`
`Liver
`
`Brain
`
`Sk. muscle
`
`0
`
`t-<)-t
`
`K)-j
`
`.. 0
`E = t-
`
`Misc. tumors
`
`i--0--l
`
`Breast tumors
`
`l-0--l
`
`Brain tumors
`
`f--0---i
`
`Sq. cell carcinoma
`
`>----0----i
`
`Sarcomas
`
`1---0-1
`
`Non-Hodgkins lymph.
`
`l---0--I
`
`6
`
`6.5
`
`7
`
`7.5
`
`8
`
`Mean pH1 (MRS)
`Fig. I. The ·" P-MRS estima1cd pHi of various human normal tissues and tumors.
`Confidence intervals are I SD. T1le data for li,·er are from Oberhacnsli et al. (7); for brain
`from Oberhacnsli et al. (7) and Hubesch et al. (13): for resling skclml muscle from
`Sostman tt al. (13). Semmler et al. (9). and Nideckcre, al. ( 10); for miscellaneous tumors
`from Oberhaensli et al. (7) and Ng et al. (8); for brcas1 tumors from Sijens e1 al. (6) and
`Oberh~nsli er al. (7); for brain tumors from Oberhaensli e1 al. (7) and Hubesch er al. (14);
`for squamous cell carcinomas from Ng et al. (8); for sarcomas from Sostman et al. ( 12.
`13). Dewhirs1 et al. (11). Nid«ker et al. (10). and Semmler ti al. (9); and for non•
`Hodgkin's lymphoma from Ng et al. (8) and Smilh et al. (IS).
`
`matched with the pH values obtained in the adjacent normal brain of the same
`patients ( Fig. 2A; Ref. 23 ). In 18 of 20 cases. the electrode-measured pH values
`of glioblastomas are equal to or less than those obtained in adjacent normal
`brain. In addition to these studies, Pampus (23) also masurcd the pH in 11
`patients with astrocytomas and adjacent normal brain (Fig.28). In all 11
`patients. the tumor pH was equal to or lower than the pH of the normal tissue.
`Similar results were obtained by Nacslund 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 nonnal tissue in 40 of 42 cases; the mean pH difference
`being 0.41 :!: 0.27 as is observed in comprehensive 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 nonnal tissue and
`subs1antially reduced pHe of tumor compared to normal tissue gives rise to a
`subs1antially 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. I, 17. 18. and 25 ), the average difference
`between the extracellular and pHi is approximately +0.2 pH units in normal
`tissue and -0.2 to -0.6 in tumor tissue.
`All drugs exhibit neutral. acidic. or basic propenies. For drugs which are
`weak acids ( or bases). the extent to which they are ionized is exponentially
`related to the pH of 1heir milieu. As 1he 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 the
`intracellular/extracellular equilibrium distribution of the drug.
`
`Drug Charge and Drug TrtJnsport
`
`Table I £1,ctrode estimattd pH of subcutoneous tinut'
`Measured pH values of human subcutaneous tissue obtained by various investigators.
`Differences in patients· age. physiology. electrode characteristics. and measuremen1
`procedures likely accoun1 for lhe observed differences. From Wike-Hooley et al. (I).
`lnvestiga1or
`Sample size
`Mean pH:!: SO
`van den Berg rt al. ( 19)
`7.63 :!: 0. 17
`26
`Harrison and Walker
`7.54 :!: 0.09
`40
`( 22)
`Stamm,., al. (20)
`Vidyasagar t'I al. (21)
`
`10
`II
`
`7.42 :!: 0.05
`7.33 :!: 0.03
`
`The principal barrier to the entry of a drug into an intracellular site of action
`is the cell membrane. With the exception of the blood-brain barrier. the
`1195
`
`FRESENIUS EXHIBIT 1048
`Page 2 of 5
`
`

`

`CELLULAR pH GRAOIENr IN TUMOR AND NORMAL TISSUE
`
`(A)
`
`20
`18
`16
`
`GlloblHtoma
`
`•
`•
`•
`•
`•
`•
`
`~
`
`6
`4
`
`2
`
`14
`•
`• 12
`-
`•
`•
`C 10
`.!
`iii •
`•
`•
`•
`• • o~--..... ---,,--.-------,---1
`
`Aetrocytoma
`
`•
`•
`•
`•
`•
`•
`•
`
`(8)
`
`10
`
`•
`
`I
`
`4
`
`2
`
`Fig. 2. pH electrode measllltd pHe values con(cid:173)
`currently oblained in rumors and nonnal 1issue in
`the same pati<nl. For glioblastoma (A) and aslJ'O(cid:173)
`cy1oma (8), the data an: from Pampus (23). for
`u1erine cancer ( C) from Naeslund and Swenson
`(24). and for melanoma (D) from Ashby and
`Cantab (25).
`
`1 . 4 1 . 1 I.I 7 . 0 7.2 7.4 7.1
`Extracellular pH
`
`0 ~---.--"T'"--,.---""T""---,.---1
`s., 1.2
`I.I 7.0
`7 .4
`7.8
`Extracellular pH
`
`s (C)
`
`4
`
`Uterine
`cancer
`
`C
`
`~
`
`•
`- 3
`.! -• 2
`1 •
`0
`s.o 1.4 •.• 7 . 2 7 . 1 1.0 1.4
`Extracellular pH
`
`•
`•
`
`Sullcutaneoue
`Tiu~
`
`• (D)
`• llelano•• •
`•
`•
`•
`•
`•
`
`7
`
`5
`
`4
`
`3
`
`2
`
`1
`
`0
`1.4
`
`7 . 3
`7.0
`1.7
`Extracellular pH
`
`7.6
`
`basic pHe conditions. the fraction of the drug that becomes ionized
`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. 3B. 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 curve and 7.4 in the lower curve. Very
`weak acids (pKa > 9) are essen1ially 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(cid:173)
`tion. For a pKa of 5 (Fig. 38, 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 ponion of the weak
`acid is ionized and confined to the extracellular compartment.
`A number of studies have investigated drug partitioning into arti(cid:173)
`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 intravesicular.extravesicular (or Disc~lon
`cellular) concentration ratios. pKa is influenced by factors such as the
`solvent in which it is dissolved, ionic strength, and temperature (29,
`Although the cellular pH gradient differs in tumor and normal
`32). Additionally, the numerical value of the predicted distribution
`tissue, the pHe and, therefore, the magnitude of the gradient within a
`1196
`
`ratio does not precisely match the observed distribution if the ionized
`drug is not completely membrane impermeable or is rapidly metab(cid:173)
`olized or sequestered in the intracellular companment. Neverthe(cid:173)
`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. (31) measured the intracellu(cid:173)
`lar:extracellular 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(cid:173)
`lar concentration was uninfluenced by pH. Also in accordance with
`theory. the intracell ular 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(cid:173)
`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 of these analogues is readily apparent and
`substantial.
`
`FRESENIUS EXHIBIT 1048
`Page 3 of 5
`
`

`

`CELLIJLAR pH GRADIENT IN TUMOR AND NORMAL TISSUE
`
`3.0 - r - - - - - - - - - - - - - - - - - - , 3.0 ...... - - - - - - - - - - - - - - - - - - - - - ,
`(A)
`(B)
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`5
`
`pHI = 7.2, pKa = 7.0
`
`6
`
`7
`
`8
`
`9
`
`1.5
`
`1.0
`
`0.5
`
`o.o
`
`5
`
`6
`
`7
`
`8
`
`9
`
`C
`
`0 i .. -C ...
`(.) . g) CJ
`.. -C ,E -
`
`CJ CII c-o.:
`
`::, ~
`
`pKa
`Fig. 3. A. calculated effect of variable pHe on the ratio of the intracellular:extracellular distribution of a weak add. For the example shown. the pHi is 7 .2. and the drug pKa is 7.0.
`B. calculated effect of variable pKa on the ratio of the intracellular:extracellular concentr:11ion of a weak acid. For the example shown, the pHi is 7.2, and the pHe is 6.8 or 7.4.
`
`Extracellular pH
`
`possibilities for exploiting the pH gradient exist. Using an in vitro cell
`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.
`The pH gradient difference between tumor and nonnal tissue pro(cid:173)
`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
`chaJJenging but appropriate aspect of this evaluation is the develop(cid:173)
`ment and utilization of experimental tumor models and procedures for
`the evaluation of tumor and nonnal tissue toxicity as a function of the
`tissues' pH gradient and drug pKa (36-38).
`
`Acknowledgments
`
`We thank Ors. Bruce Chabner, Yves Boucher, Claus Kristensen, and Fan
`Yuan for their helpful suggestions during the preparation of the manuscript.
`
`particular tumor are not unifonn. Studies with miniature pH electrodes
`show that the pH within a tumor may vary from values which are
`similar to those in nonnal tissue to substantially more acidic values ( I,
`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
`µm 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 nonnally exposed to the lowest drug con(cid:173)
`centration, and especially relevant to radiation therapy, to low
`concentrations of oxygen.
`Although several chemotherapeutics exhibit acidic or basic prop(cid:173)
`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
`
`Table 2 Measured and calculated intracellular concentration ratios of misonidawle
`and acidic or basic analogues
`Measured and calculated intracellular concentration ratios of misonidazole and acidic
`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 ).
`
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`K. H. Human neoplasm pH and response to radiation therapy. P-31 MR specuoscopy
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`9. Semmler, W., Gademann, G .. Bachen-Bawnann. P .. Zabel, H.J., Lorenz. W. J .. and
`van Kaick, G. Monitoring human tumor response 10 therapy by means of P-31 MR
`spectroscopy. Radiology. 166: 533- 539. 1988.
`10. Nidecker, A. C., Muller. S .. Aue. W. P .. Seelig. J .. Fridrich, R .• Remagcn, W.,
`1197
`
`Analogue
`
`pKa
`Neutral
`7.2
`8.9
`
`Intracellular concentration ratio
`
`(pHe = 6.6/pHe = 7 .6)
`Observed"
`Calculated&
`
`1.0
`2.2
`0.22
`
`1.5
`0.36
`
`Misonidazole
`Azomycin (acid)
`Ro 03899 (base)
`" From Dennis et al. (31 ).
`b Calculated concentration ratios based on the measured pHi of approximately 6.87 at
`pHe = 6.6. and pHi of 7.45 at pHe = 7.6.
`
`FRESENIUS EXHIBIT 1048
`Page 4 of 5
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`

`

`CELLULAR pH GRADIENT IN TUMOR AND NORMAL TISSUE
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