 F508-Homozygous Cystic
Fibrosis Patients
Partial Restoration of Nasal Epithelial CFTR Function |
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ABSTRACT |
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INTRODUCTION
METHODS
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Sodium 4-phenylbutyrate (Buphenyl, 4PBA) is a new FDA approved drug for management of urea
cycle disorders. We have previously presented data suggesting that 4PBA, at clinically achievable concentrations, induces CFTR channel function on the plasma membrane of 
F508-expressing cystic fibrosis (CF) airway epithelial cells in vitro (Rubenstein, R. C., and P. L. Zeitlin, 1997. J. Clin. Invest. 100:2457-
2463). We hypothesized that 4PBA would induce epithelial CFTR function in vivo in individuals
homozygous for F508-CFTR. A randomized, double-blind, placebo-controlled trial in 18 F508-
homozygous patients with CF was performed with the maximum approved adult dose of 4PBA, 19 grams p.o. divided t.i.d., given for 1 wk. Nasal potential difference (NPD) response patterns and
sweat chloride concentrations were determined before and after study drug treatment, and 4PBA
and metabolites were assayed in plasma and urine at the end of study drug treatment. Subjects in
the 4PBA group demonstrated small, but statistically significant improvements of the NPD response
to perfusion of an isoproterenol/amiloride/chloride-free solution; this measure reflects epithelial
CFTR function and is highly discriminatory between patients with and without CF. Subjects who had
received 4PBA did not demonstrate significantly reduced sweat chloride concentrations or alterations
in the amiloride-sensitive NPD. Side effects due to drug therapy were minimal and comparable in the
two groups. These data are consistent with 4PBA therapy inducing CFTR function in the nasal epithelia of F508-homozygous CF patients.
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INTRODUCTION |
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Cystic fibrosis (CF) results from the functional absence of a
single membrane glycoprotein, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Seventy percent of
CF patients carry at least one copy of the most common mutation, the deletion of a phenylalanine residue at position 508 (F508-CFTR). The F508-CFTR protein is retained in the
endoplasmic reticulum (1) and degraded (2, 3) rather than
trafficked to the cell surface. F508-CFTR forms a functional
chloride channel in reconstituted bilayers (4) but with decreased mean open times, and therefore decreased conductance, compared with wild type CFTR (5). Thus, treatments that would promote F508-CFTR trafficking beyond the endoplasmic reticulum might restore partial CFTR chloride channel function at the cell surface.
The F508 mutation is temperature sensitive, in that the
intracellular processing block of F508-CFTR can be reversed
in vitro by incubation of cells at reduced temperatures (6), or
with protein stabilizing agents (chemical chaperones) such as
glycerol (7, 8). These data suggest that F508-CFTR is less
thermodynamically stable than the wild type CFTR, but is still
capable of assuming a functional conformation under certain
conditions. An alternative strategy for increasing F508-CFTR expression on the epithelial surface might be by increasing expression of the protein and overcoming the trafficking block by mass action. Butyric acid has been used in
vitro and in vivo to upregulate mRNA expression of genes
such as 
-globin (9, 10). Cheng and colleagues observed upregulation of F508-CFTR by butyrate in cultured cells (11);
however, circulating butyrate has a very short half-life and
must be given by continuous intravenous infusion (10, 12).
Sodium 4-phenylbutyrate (Buphenyl, 4PBA) is an oral butyrate analog that was recently approved for use in patients with urea cycle enzyme deficiencies where it functions as an ammonia scavenger. Because 4PBA, like butyrate, increases fetal hemoglobin, it is also currently in Phase I clinical trials in sickle cell disease and thalassemia (13). 4PBA also has profound effects on cell differentiation and is in Phase I cancer chemotherapy trials as a tumor differentiating agent (14, 15).
We initially tested 4PBA in vitro because it was an oral analog of butyrate. We have previously presented data suggesting that 4PBA, at clinically achievable concentrations, induces
CFTR channel function on the plasma membrane of F508-
expressing CF airway epithelial cells in vitro (16). Based on
these in vitro data, we examined the hypothesis that 4PBA will
induce epithelial CFTR function in vivo in individuals homozygous for F508-CFTR. Our data are consistent with induction of partial CFTR function in the nasal epithelia of
F508-homozygous CF patients by 4PBA therapy.
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METHODS |
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INTRODUCTION
METHODS
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Experimental Subjects
Subjects were eligible for entry into this study if they were 14 yr of age
or older and were homozygous for F508-CFTR. Potentially eligible
subjects were identified and recruited during routine CF clinic visits at
the Johns Hopkins Hospital, or by referral from outside sources. The
study protocol was approved by the Pediatric Clinical Research Unit
and the Joint Committee on Clinical Investigation of the Johns Hopkins Medical Institutions. All subjects gave written informed consent
prior to the commencement of the study protocol.
Age was calculated to the nearest tenth of a year at the time of study entry. Pancreatic sufficiency and baseline pulmonary function were ascertained by chart review. All baseline pulmonary function tests were performed within the 3 mo prior to study entry. The study period was February to November, 1996.
Study Drug
Sodium 4-phenylbutyrate (Buphenyl) and placebo (500 mg tablets) were purchased from Ucyclid Pharma, Inc., (Hunt Valley, MD). Randomization and blinding were performed by the Johns Hopkins Hospital Investigational Drug Pharmacy.
Assays
Sweat chloride concentration was determined by the pilocarpine iontophoresis method according to published standards (17). Sweat collections were obtained from equivalent sites on each forearm and the results were averaged. All collections contained at least 1 mg sweat/m2 body surface area/minute of sweat collection. Sweat collection was performed by one of the study investigators (R.C.R.) and chloride measurements were performed by the Johns Hopkins Hospital Special Chemistry Laboratory.
Nasal potential difference (NPD) was recorded using a high impedance voltmeter, an exploring bridge (PE50 tubing) perfused with Ringer's solution, and a subcutaneous electrode (22 gauge needle filled with 4% agar in Ringer's solution) as previously described by Knowles and coworkers (18). Baseline NPD was obtained during perfusion under the inferior nasal turbinate with sterile Ringer's solution at a perfusion rate of 2 ml/min. The double-barrelled PE50 exploring catheters were advanced by 0.5 cm intervals up to a total of 3 cm to map the point of maximal negative NPD. The point of maximal NPD was relocated and maintained throughout the duration of the protocol. After a stable baseline was reached at the point of maximal NPD, the value was recorded and the perfusing solution was changed to 0.1 mM amiloride in Ringer's solution. This solution was administered for 2 min at a rate of 5 ml/min. After a stable reading was obtained and the value recorded, the perfusate was changed to 0.1 mM amiloride in a low chloride Ringer's solution; gluconate was substituted for chloride as the counterion in the low chloride Ringer's solution. The perfusion of this solution continued for 2 min at 5 ml/min, and the NPD value was recorded after a stable reading was attained. Finally, 0.1 mM isoproterenol (Isuprel, Iso) was added to the amiloride/low chloride Ringer's perfusate and was administered at 5 ml/min for 3 min. The final and sustained value of NPD was recorded. Transient (< 1 min) repolarizations were sometimes observed with either the low chloride or Isuprel perfusions; these transient changes were not considered responses unless sustained. Each data point is the average of measurements made in the right and left nares.
Quantitation of 4PBA and the metabolites, phenylacetate (PAA) and phenacetyl glutamine (PAG), in plasma and urine was performed by high performance liquid chromatography (19).
Study Protocol
Sweat chloride concentration (18/18 subjects) and NPD response pattern (10/18 subjects) were determined on the first day of the study prior to the dispensing of study drug. Subjects then received their randomized, double blind study agent (placebo or 4PBA) and were instructed to take 19 g/d of the study agent in three divided oral doses of 6, 6, and 7 g for 1 wk. On the final day of the 1 wk study period, the subjects returned, and plasma and urine samples were obtained prior to the last dose of study agent. Subjects then ingested the final dose, and 1-2 h later, sweat chloride concentration (18/18 subjects) and NPD response pattern (17/18 subjects) were determined.
Subjects signed separate consent for NPD measurements. Subjects were not excluded from study, nor was the order of dispensing the randomized, double blind study drug altered if they did not consent to either the pre-study or both NPD measurements. One patient (randomized to the placebo group) did not consent to NPD measurement, and seven patients (four randomized to the placebo group and three randomized to the 4PBA group) only consented to NPD measurements at the end of the study.
Compliance was determined by pill count; no subject reported missing more than two doses during the study period, and most subjects reported no missed doses. Side effects were recorded as reported by the subjects after extensive questioning.
Statistical Analysis
Data are expressed as the mean change in sweat chloride concentration and as the mean change in NPD with each perfusate. Comparison of means between treatment groups was performed using the non-parametric Mann-Whitney U test. Comparison of pre- and post-therapy values of NPD response for groups of subjects was performed using a pair-wise t test. All statistical analysis was performed using SPSS for Windows software (version 7.0).
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RESULTS |
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Subject Demographics
Each treatment group, 4PBA and placebo, contained nine subjects. As shown in Table 1, the groups were similar with respect to age, gender, pancreatic sufficiency, and baseline pulmonary function.
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Effect of 4PBA on Nasal Potential Difference Response Pattern
Measurement of baseline nasal epithelial potential difference (NPD) and the change in NPD during perfusion of a series of defined solutions is a sensitive method for in vivo evaluation of epithelial CFTR function. The protocol for these measurements has been defined by Knowles and coworkers (18) and used extensively to define the efficacy of CFTR gene transfer to the nasal mucosa by adenoviral-CFTR (20) and liposomal-CFTR gene therapy vectors (21). NPD can be affected by intercurrent viral upper respiratory infections and nasal epithelial inflammation (18). None of our subjects reported any nasal symptoms during the study period.
NPD measurements were performed on 10/18 subjects (6/9 in the 4PBA group and 4/9 in the placebo group) prior to study drug therapy, and on 17/18 subjects (9/9 in the 4PBA group and 8/9 in the placebo group) after study drug therapy (see METHODS).
The resting potential difference of the nasal mucosa primarily reflects epithelial sodium transport. There was no difference between the pre-study, post-placebo, or post-4PBA determinations (mean ± SD 
36.3 ± 5.2, 36.3 ± 9.8, and
33.3 ± 4.7, respectively). Similarly, in the subjects studied
prior to and after study drug therapy, there was no significant
change in the basal NPD after 4PBA therapy compared to the
placebo group (data not shown).
The response to amiloride, the inhibition of epithelial sodium transport, was assessed next and results in a depolarization, or positive change in the NPD. In the second phase, after
blockade of the sodium transport by amiloride, chloride was
removed from the perfusate and replaced with gluconate. This
creates a transepithelial chloride gradient and can lead to a
small repolarization, or negative change, in the NPD of normal subjects. Subsequent perfusion of the nasal epithelium
with the 
2-adrenergic agonist isoproterenol (Isuprel, Iso) in
the low chloride/amiloride perfusate activates CFTR and
leads to further polarization, or negative change, in the NPD
of normal subjects. The overall change in NPD after change to
low chloride perfusate and addition of Isuprel (in the continued presence of amiloride), the "Isuprel/Low Cl
" response,
is highly discriminatory among individuals with and without
CF, and separates subjects with and without CF better than
the individual response to either low chloride perfusion or Isuprel perfusion (18). Individuals without CF show a repolarization, or further negative NPD change, after Isuprel/Low Cl
perfusion. The repolarization in subjects without CF can result in hyperpolarization of the nasal epithelia, or a final NPD
more negative than the basal NPD. In contrast, subjects with
CF typically demonstrate little change in potential or a further
depolarization, or positive NPD change, after Isuprel/Low Cl
perfusion. Representative NPD response profiles for a subject without CF, a study subject who received placebo, and a study subject who received 4PBA are shown in Figure 1. Basal NPD,
the amiloride-sensitive potential, and the Isuprel/Low Cl-sensitive potential are marked in Figure 1A and were compared between groups. Table 2 shows a summary of NPD measurements performed prior to the administration of study drug.
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Figure 2 summarizes the NPD data obtained after administration of study drug for 1 wk (n = 8 in the placebo group, n = 9 in the 4PBA group). We did not observe a significant difference between the two groups with respect to depolarization of
the NPD in response to amiloride. The absolute magnitude of
the NPD after amiloride perfusion was (mean ± SD) 16.8 ± 3.7 mV before study drug therapy, 16.9 ± 4.3 mV in the placebo group, and 15.0 ± 4.5 mV in the 4PBA group. We also
did not observe a significant change in the basal NPD between
the two groups (see above). These data are consistent with little alteration of the basal or amiloride-sensitive NPD after
4PBA therapy, and lead to the conclusion that epithelial sodium transport was not affected by 4PBA therapy under the
conditions of this study.
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The Isuprel/Low Cl response changed significantly after
1 wk of drug therapy. The Placebo group (n = 8) demonstrated modest depolarizations, or positive NPD responses,
while the PBA group (n = 9) had significantly less depolarization, and in some cases, repolarization, or negative NPD responses, in response to Isuprel/Low Cl perfusion (see also
Figure 1B and C). These data represent a significant change
towards a non-CF NPD response pattern in subjects who had
received 4PBA, and are consistent with increased chloride current due to epithelial CFTR activity in these 4PBA-treated F508-homozygotes. The absolute magnitude of the NPD after Isuprel/Low Cl perfusion was (mean ± SD) 11.6 ± 3.6 mV before study drug treatment, 11.1 ± 4.1 mV for the placebo group, and 13.7 ± 4.2 mV for the 4PBA group. These
data further suggest that the NPD after Iso/Low Cl perfusion
was more polarized in the 4PBA group. The magnitude of Isuprel/Low Cl-induced repolarization seen in these subjects is
less than that observed in subjects without CF (16 mV on average) (18), but is similar to that observed in CF patients who
had received nasal administration of an Adenovirus2/CFTR-2
gene therapy vector (20), or nasal administration of a plasmid
encoding CFTR cDNA either alone or complexed with cationic lipid liposome (21).
The improvement in Isuprel/Low Cl response was predominantly due to a statistically significant improvement in
the response to Low Cl perfusion (change in NPD from "b"
to "c" in Figure 1A). There was also a trend toward NPD improvement with the addition of Isuprel to the amiloride/low
chloride perfusate (change in NPD from "c" to "d" in Figure
1A). These data are summarized in Table 3.
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Figure 3 shows the NPD response to Isuprel/Low Cl perfusion in the 10 patients (n = 4 Placebo, n = 6 4PBA) who had
these measurements performed both pre- and post-study. The
four subjects who had received placebo demonstrated no significant change in their NPD response to Isuprel/Low Cl perfusion. This contrasts with the data obtained from the subjects who had received 4PBA; all of these subjects had less depolarization or frank repolarization of their NPD in response to
Isuprel/Low Cl perfusion after 4PBA therapy.
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4PBA Metabolites in Plasma and Urine
Human subjects completely metabolize 4PBA to phenylacetate (PAA) by -oxidation (19). The PAA is eliminated by
conjugation with glutamine to form phenacetyl glutamine
(PAG) which is excreted in the urine; this metabolic pathway
is the mechanism by which 4PBA acts as an ammonia scavenger in patients with urea cycle disorders and hyperammonemia (19). Plasma PAA and urinary PAG are also endpoints of
normal tyrosine metabolism; low level detection of these species in control subjects is not unexpected.
Four of nine subjects in the 4PBA group had detectable 4PBA in their plasma (concentration range 0.044-2.02 mM), while 7/9 had detectable PAA in their plasma (concentration range 0.46-1.56 mM). All nine 4PBA subjects had elevated urinary concentrations [PAG]urine compared with the placebo group. None of the placebo subjects had 4PBA or PAA detected in their plasma. Formal pharmacokinetic analysis, which was not performed in this pilot study, is required to confirm whether higher drug levels lead to greater improvement.
Effect of 4PBA Therapy on Sweat Chloride Concentration
In CF, the sweat duct epithelium is impermeable to chloride due to the absence of functional CFTR (22). This leads to inadequate chloride reabsorption and elevated chloride concentrations in the sweat, and is the basis for the most common diagnostic test for CF. We tested whether 4PBA would decrease sweat chloride concentration in these subjects.
All subjects had sweat chloride tests in duplicate before and after study drug therapy. All sweat chloride concentrations, both before and after study drug therapy, were in excess of 80 mEq/L (range 90-140 mEq/L), and therefore consistent with the diagnosis of CF in an adult subject.
Figure 4 summarizes the change in sweat chloride concentration after study drug therapy, i.e., the post-treatment concentration minus the pre-treatment value. A negative value
represents a decrease in sweat chloride. The changes were averaged within each treatment group and the means were compared. There was no significant change in sweat chloride concentration due to 4PBA therapy when compared to the placebo
group. These data suggest that 4PBA, at a dose of 19 g/d for
1 wk, did not induce sufficient CFTR function in the sweat
duct epithelia of F508-homozygous subjects to significantly decrease their sweat chloride concentration.
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Side Effects
Side effects due to study drug therapy were minimal and comparable in the two groups (Table 4). The most common side effect when 4PBA is used in patients with urea cycle disorders, nausea and upset stomach (23), was not reported by any study patient. One patient who had received 4PBA had a diarrheal illness from Day 3 to Day 5 of study drug therapy. The patient continued to receive study drug (the diarrhea was not reported to the investigators until Day 5), and the diarrhea had resolved by Day 6 of the trial when her post-therapy evaluation was completed. This subject did not have detectable plasma PAA, but had a low level of 4PBA in plasma and elevated [PAG]urine (see above). Her NPD showed improvement and is included in the data of Figure 3.
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We conclude that the observed diarrhea was an intercurrent illness rather than an adverse reaction to 4PBA because the diarrhea resolved without discontinuation of 4PBA therapy. Also, diarrhea has not been reported as a complication of 4PBA therapy when used for the approved indications (23).
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DISCUSSION |
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INTRODUCTION
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DISCUSSION
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Summary
Therapy with 4PBA in F508-homozygous patients with CF
at a dose of 19 g/d for 1 wk was well tolerated. 4PBA therapy
was associated with the partial restoration of chloride transport in the nasal epithelium, but was not associated with a
change in the 4PBA recipients' sweat chloride concentration.
These data are consistent with 4PBA inducing CFTR function
in the nasal epithelia of F508-homozygous patients with CF.
Partial Correction of NPD Response
Our data demonstrated a partial restoration of the NPD response to Isuprel/Low Cl perfusion after 4PBA therapy, but
not a complete correction. A dose escalation and safety trial of
4PBA with formal pharmacokinetics is currently in progress
to address whether higher doses lead to further improvement.
It is possible that chemical modification of the 4PBA molecule might lead to increased efficacy with respect to NPD and sweat chloride. Proof of the concept that a butyrate agonist will induce some CFTR function would support a systematic inquiry for more active compounds. However, as the mechanism of 4PBA action is, as yet, unclear, it is similarly unclear as to what modifications of 4PBA might improve efficacy. Also, modifications of 4PBA might result in a compound that would not be an approved pharmaceutical agent, and therefore more difficult to test in vivo in patients with CF.
Alternatively, the observed level of correction at a dose of
19 g/d might be the maximal response that can be achieved by 4PBA therapy. Although F508-CFTR has typical CFTR-like
activity in vitro in both a reconstituted system (4) and in the
nuclear/endoplasmic reticular membrane (24), the single channel conductance on the cell surface of transfected cells has a
reduced open probability (5). This decreased open probability
may limit recovery of the Isuprel/Low Cl response in vivo.
Other mutations that affect channel conductance, but not
CFTR trafficking, (25) such as R117H (substitution of histidine residue at position 117 for an arginine), are often present
in CF patients with clinically mild lung disease and pancreatic
sufficiency. We have examined the NPD of one such patient
(genotype F508-R117H) who is pancreatic sufficient and has
an elevated sweat chloride concentration. This subject had a
basal NPD of 37.5 mV, an amiloride-sensitive potential of
18.5 mV (depolarization), and a 5 mV response (repolarization) to Isuprel/Low CL perfusion (P. L. Zeitlin and R. C. Rubenstein, unpublished observations). Thus, the NPD responses to Isuprel/Low Cl perfusion in 4PBA-treated F508-homozygous CF patients are approaching that of a CF patient
with pancreatic sufficiency and milder lung disease.
Measurement of the NPD in five CF patients with another
partially functional CFTR mutation, A455E (substitution of
glutamate for alanine at position 455) have recently been published (26). While all study subjects had mild lung disease by
pulmonary function testing, only one had a repolarization of
NPD in response to Iso/Low Cl perfusion. This is in apparent
contrast to the case above. It is possible, even though R117H
and A455E are both partially functional CFTR mutations,
that their interactions with other (as yet undetermined) cellular constituents might differ, and thereby alter NPD responses. These data suggest that further rigorous studies will be required to determine if there exists a correlation of NPD responses with genotype, and if intermediate NPD responses are
predictive of milder clinical manifestations of CF.
Others have examined the possibility of enhancing the
channel activity of F508-CFTR in vitro with agents such genistein (27), 8-cyclopentyl-1,3-dipropylxanthine (CPX) (28), or
milrinone (29). Genistein activates F508-CFTR in vitro by
increasing the channel's open probability (27). CPX directly
binds to and stimulates F508-CFTR-mediated chloride conductance (28). Milrinone, a phosphodiesterase inhibitor, decreases dephosphorylation of F508-CFTR, and prolongs the
lifetime of the activated channel (29). Milrinone also causes improvement of the NPD measured in the F508-homozygous
transgenic mouse model (30). Should further activation of
F508-CFTR at the cell surface be clinically necessary in humans, combination therapy with 4PBA and one of these agents
might prove more efficacious than 4PBA alone.
Improvement of NPD but not Sweat Chloride
Our data demonstrated improvement of the NPD response
that was consistent with CFTR activity on the nasal epithelial
surface of F508-homozygous individuals after 4PBA treatment, but showed no significant change in sweat chloride concentration. While this appears contradictory, it is likely that
this should be the anticipated result. Patients with mutations
that decrease, but do not eliminate, CFTR activity such as
R117H, have elevated sweat chloride concentrations characteristic of CF, but are typically pancreatic sufficient and have
relatively mild lung disease (25). In the Johns Hopkins Hospital Cystic Fibrosis Center, there are two patients with genotype R117H/F508. Both of these patients are pancreatic sufficient and have relatively mild lung disease for age by
pulmonary function testing and were diagnosed with CF on
the basis of an abnormal sweat test. The NPD response of one
of these patients is detailed above and is intermediate to that
of non-CF patients and F508-homozygous CF patients at
baseline. These observations are consistent with NPD measurements being more sensitive to partial CFTR function than
sweat chloride determination.
It is also possible that our trial did not control all of the variables that can affect sweat chloride concentration. The amount of dietary sodium will modify sweat chloride concentration and was not controlled in this pilot trial. Higher NaCl ingestion causes modest increases in sweat chloride concentration (31) that would blunt a positive drug effect. At a dose of 19 g/d, 4PBA therapy leads to ~ 2.5 g/d of additional sodium intake. Further studies will address this issue.
Optimal Dosage and Dosing Regimen for 4PBA
Our study did not address an optimal dose or obtain formal
pharmacokinetics for 4PBA. Considerable variation in plasma
PAA concentrations and urinary PAG concentrations were
observed. 4PBA is a fatty acid and its absorption may vary
among CF subjects. It is unlikely that inter-subject variation in
4PBA metabolism occurs since humans demonstrate a highly
efficient -oxidization system and complete conversion of
4PBA to PAG (19).
A formal dose escalation protocol will be necessary to find the best schedule for administration of 4PBA to CF patients. One could speculate that intermittent 4PBA dosage might be as efficacious as daily therapy if the beneficial effect of 4PBA on NPD decays over days to weeks.
Conclusions
We have demonstrated improved NPD responses consistent
with partial CFTR activity in the nasal epithelia of F508-
homozygous CF patients after 1 wk of therapy with 4PBA, an
approved pharmaceutical agent, in a randomized, double-blind, placebo-controlled trial. Sweat chloride concentration
did not change after this therapy and may require higher drug
doses. These data indicate that 4PBA is a promising potential
pharmacologic therapy for CF patients who have the F508-CFTR mutation.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Ronald C. Rubenstein, M.D., Ph.D., Pediatric Pulmonary, The Johns Hopkins Hospital
Park 316, 600 N. Wolfe St., Baltimore, MD 21287. E-mail: rrubenst{at}welchlink.welch.jhu.edu
(Received in original form June 24, 1997 and in revised form September 22, 1997).
Acknowledgments: The authors thank Ms. Lois Brass-Ernst, R.N., for assistance with patient recruitment and the NPD measurements, Dr. Saul Brusilow for helpful discussions, Dr. Brusilow and Ms. Ellen Gordes for measurements of 4PBA and its metabolites, and Ms. Dawn Martin, and Mr. Matthew Harley for expert technical assistance.
This work was supported by NIH P01 HL51811 to P.L.Z., The John Hopkins Hospital Pediatric Clinical Research Unit (NIH RR00052), and a Leroy Matthews Physician Scientist Award from the Cystic Fibrosis Foundation to R.C.R.
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References |
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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
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