Tolerance to coxibs in patients with intolerance to non-steroidal anti-inflammatory drugs -LRB- NSAIDs -RRB- : a systematic structured review of the literature Adverse events triggered by non-steroidal anti-inflammatory drugs -LRB- NSAIDs -RRB- are among the most common drug-related intolerance reactions in medicine ; they are possibly related to inhibition of cyclooxygenase-1 . Coxibs , preferentially inhibiting cyclooxygenase-2 , may therefore represent safe alternatives in patients with NSAID intolerance . We reviewed the literature in a systematic and structured manner to identify and evaluate studies on the tolerance of coxibs in patients with NSAID intolerance . We searched MEDLINE -LRB- 1966 -- 2006 -RRB- , the COCHRANE LIBRARY -LRB- 4th Issue 2006 -RRB- and EMBASE -LRB- 1966 -- 2006 -RRB- up to December 9 , 2006 , and analysed all publications included using a predefined evaluation sheet . Symptoms and severity of adverse events to coxibs were analysed based on all articles comprising such information . Subsequently , the probability for adverse events triggered by coxibs was determined on analyses of double-blind prospective trials only . Among 3,304 patients with NSAID intolerance , 119 adverse events occurred under coxib medication . All adverse events , except two , have been allergic/urticarial in nature ; none was lethal , but two were graded as life-threatening -LRB- grade 4 -RRB- . The two non-allergic adverse events were described as a grade 1 upper respiratory tract haemorrhage , and a grade 1 gastrointestinal symptom , respectively . In 13 double-blind prospective studies comprising a total of 591 patients with NSAID intolerance , only 13 adverse reactions to coxib provocations were observed . The triggering coxibs were rofecoxib -LRB- 2/286 -RRB- , celecoxib -LRB- 6/208 -RRB- , etoricoxib -LRB- 4/56 -RRB- , and valdecoxib -LRB- 1/41 -RRB- . This review documents the good tolerability of coxibs in patients with NSAID intolerance , for whom access to this class of drugs for short-term treatment of pain and inflammation is advantageous . Introduction Non-steroidal anti-inflammatory drugs -LRB- NSAIDs -RRB- are the most commonly used therapeutics in the outpatient management of pain and inflammation in a wide spectrum of diseases . Their primary mode of action is the blockade of prostaglandin synthesis by cyclooxygenases -LRB- COX -RRB- : Constitutively expressed COX-1 is involved in fundamental mechanisms of homeostasis , whereas the inducible COX-2 mediates inflammation . Therapeutic effects of NSAIDs are primarily related to their ability to inhibit COX-2 , whereas some of their most frequent adverse effects may be caused by COX-1 inhibition -LRB- Fig. 1 -RRB- . In contrast to most `` classic '' NSAIDs which block both isoforms , the so-called coxibs preferentially inhibit COX-2 . This may result in better tolerability , namely reduction of gastrointestinal side effects -LSB- 29 , 85 -RSB- . Fig. 1Cyclooxygenase isoforms . Non-steroidal anti-inflammatory drugs -LRB- NSAIDs -RRB- block prostaglandin synthesis by cyclooxygenases -LRB- COX -RRB- . Two isoforms exist of this enzyme : constitutively expressed COX-1 is involved in fundamental mechanisms of homeostasis , whereas the inducible isoform COX-2 mediates inflammation Respiratory and cutaneous adverse events triggered by NSAIDs are among the most common drug-related intolerance reactions in medicine . Typically , these manifest as asthma attacks or urticaria . Pathogenesis of these symptoms seems to be related to COX-1 inhibition -LSB- 76 -RSB- . Therefore , the hypothesis was put forward that coxibs may safely be used in patients with known NSAID intolerance . However , serious intolerance reactions to coxibs have also been observed , thus cautioning too euphoric expectations -LSB- 71 -RSB- . We therefore reviewed the relevant literature in a systematic and structured approach for evidence of coxib tolerance in patients with NSAID intolerance . Methods Search strategy We searched MEDLINE -LRB- 1966 -- 2006 -RRB- , the COCHRANE LIBRARY -LRB- 4th Issue 2006 -RRB- and EMBASE -LRB- 1966 -- 2006 -RRB- up to December 9 , 2006 . The following search terms were used : rofecoxib OR celecoxib OR valdecoxib OR parecoxib OR etoricoxib , combined with hypersensitivity OR intolerance . Those coxibs used in veterinary medicine -LRB- deracoxib , tiracoxib and cimicoxib -RRB- were not included in the literature search . No language or publication restriction was predefined . All publications reporting individual patients with NSAID intolerance and subsequent exposure to a COX-2 Inhibitor were identified and the reference lists of these articles were hand-searched for further publications . If articles could not be retrieved in full text , a copy was requested from the corresponding author and/or journal editor . Inclusion criteria Each publication was appraised for inclusion in a stepwise approach -LRB- Fig. 2 -RRB- . Only publications describing individual patients and providing a rational medication scheme as well as a sufficiently specific outcome report were included in this review . In a first step , evaluation focused on clinical symptoms and severity of adverse events in patients with NSAID intolerance . Therefore , all articles on this topic were included for this analysis . Subsequently , the probability for adverse events was analysed based exclusively on publications of double-blind prospective trials . Fig. 2Flow chart documenting the handling data -LRB- NSAID non-steroidal anti-inflammatory drugs -RRB- Data extraction and synthesis All articles were analysed using a predefined evaluation sheet . Uncertainties were resolved by consensus decisions among the investigators . Data synthesis was qualitative and descriptive . The Common Terminology Criteria for Adverse Events version 3.0 -LRB- CTCAE -RRB- was used to categorize adverse events . Results We identified 230 publications on coxibs and/or NSAID intolerance . Hundred and forty-six references not focusing on individual patients with NSAID intolerance were excluded . Unclear medication schemes or outcome reports led to exclusion of two articles . Eighty-four publications were evaluated for severity and type of adverse reactions to coxibs . Thirteen publications on double-blind studies were used to determine the probability of adverse reactions to coxibs . Severity and type of adverse events to coxibs A total of 119 adverse events among 3,304 patients exposed to coxibs were identified -LSB- 1 -- 18 , 20 -- 24 , 26 -- 28 , 30 -- 35 , 37 -- 58 , 60 -- 75 , 77 -- 84 , 86 -- 89 -RSB- . Adverse events were described for rofecoxib -LRB- 48/1 ,732 -RRB- , celecoxib -LRB- 59/1 ,148 -RRB- , etoricoxib -LRB- 10/328 -RRB- , and valdecoxib -LRB- 2/69 -RRB- , but not parecoxib -LRB- 0/27 -RRB- -LRB- Table 1 -RRB- . All adverse events , except two , have been allergic/urticarial in nature , and none was lethal . One adverse event after administration of rofecoxib and one after celecoxib were reported as life-threatening -LRB- grade 4 -RRB- . The two non-allergic adverse events were described as a grade 1 upper respiratory tract haemorrhage , namely blood in nasal discharge after provocation with rofecoxib , and a grade 1 gastrointestinal symptom , namely nausea and abdominal pain . Table 1Mode and severity of adverse event -LRB- CTCAE common terminology criteria for adverse events -RRB- PatientsEvents -LRB- total -RRB- Urticaria -LRB- grade -RRB- Allergic reactions -LRB- grade -RRB- Allergic rhinitis -LRB- grade -RRB- Airway obstruction -LRB- grade -RRB- Gastroin-testinal symtpms -LRB- grade -RRB- Pruritus -LRB- grade -RRB- Hemorrhage , upper respiratory -LRB- grade -RRB- Rofecoxib1 ,732489 -LRB- 1 -RRB- 8 -LRB- 2 -RRB- 1 -LRB- 3 -RRB- 25 -LRB- 3 -RRB- 1 -LRB- 4 -RRB- 1 -LRB- 2 -RRB- 2 -LRB- 1 -RRB- 001 -LRB- 1 -RRB- Celecoxib1 ,1485919 -LRB- 1 -RRB- 1 -LRB- 2 -RRB- 1 -LRB- 2 -RRB- 33 -LRB- 3 -RRB- 1 -LRB- 4 -RRB- 01 -LRB- 1 -RRB- 03 -LRB- 2 -RRB- 0Etoricoxib328103 -LRB- 1 -RRB- 5 -LRB- 2 -RRB- 1 -LRB- 3 -RRB- 001 -LRB- 1 -RRB- 00Valdecoxib6921 -LRB- 1 -RRB- 1 -LRB- 2 -RRB- 000000Parecoxib2700000000Total3 ,304119486213131 Numbers and grade of adverse events -LRB- in parentheses -RRB- are indicated Evaluation of double-blind studies We identified 13 double-blind studies evaluating tolerability of coxibs in a total of 591 NSAID intolerant patients -LRB- Table 2 -RRB- . The coxibs tested in the context of these studies were rofecoxib -LRB- n = 286 -RRB- , celecoxib -LRB- n = 208 -RRB- , etoricoxib -LRB- n = 56 -RRB- and valdecoxib -LRB- n = 41 -RRB- . Among the 591 patients described , 13 exhibited adverse reactions upon provocation with a coxib . The triggering coxibs were rofecoxib -LRB- 2/286 -RRB- , celecoxib -LRB- 6/208 -RRB- , etoricoxib -LRB- 4/56 -RRB- , and valdecoxib -LRB- 1/41 -RRB- -LSB- 13 , 28 , 48 , 60 , 69 , 74 , 75 , 77 , 78 , 83 , 84 , 87 , 88 -RSB- . One of these reactions was clearly non-allergic , this one being the above-mentioned individual with grade 1 upper respiratory tract haemorrhage . Table 2Snopsis of double blind studies -LRB- NSAID non-steroidal anti-inflammatory drugs -RRB- ReferencesSubstance -LRB- number of patients -RRB- DoseOral provocation with NSAIDFollow-upAdverse events -LRB- number of patients/grade -RRB- -LSB- 87 -RSB- Celecoxib -LRB- 17 -RRB- Celecoxib 200 mgYesClinical examinations and forced expiratory volume hourly for 8 h. 0 -LSB- 74 -RSB- Celecoxib -LRB- 12 -RRB- / rofecoxib -LRB- 3 -RRB- Celecoxib 200 mg/rofecoxib 12.5 mgYesNo information0 -LSB- 75 -RSB- Rofecoxib -LRB- 60 -RRB- Rofecoxib 37.5 mg total doseYesClinical examinations and flow/volume recording hourly0 -LSB- 77 -RSB- Rofecoxib -LRB- 12 -RRB- Rofecoxib 25 mgYesClinical observation every 30 min for 8 h and finally after 24 h.Traces of blood in nasal discharge on days 3 -- 5 of rofecoxib administration -LRB- 1/1 -RRB- , transient urticaria for 30 min after administration of 5 mg rofecoxib , but not after further administration of higher doses of the drug -LRB- 1/1 -RRB- -LSB- 78 -RSB- Celecoxib -LRB- 14 -RRB- Rofecoxib 10Celecoxib 200 -- 400 mg total dose Rofecoxib 37.5 mg total doseYesForced expiratory volume was recorded each hour for 3 h0 -LSB- 60 -RSB- Rofecoxib -LRB- 104 -RRB- Rofecoxib 25 mg total doseYesClinical control for at least 6 h after each challenge and the next day .0 -LSB- 83 -RSB- Celecoxib -LRB- 60 -RRB- Celecoxib 200 mgYesClinical examinations were conducted hourly0 -LSB- 28 -RSB- Celecoxib -LRB- 33 -RRB- Celecoxib 130 mg total doseYesSpirometry and vital signs at 15 - to 30-min intervals after each dose .0 -LSB- 88 -RSB- Celecoxib -LRB- 18 -RRB- / rofecoxib -LRB- 18 -RRB- Celecoxib 300 mg total dose/rofecoxib 37.5 mg total dose1YesVital signs and forced expiratory volume every 15 min for 6 h. 0 -LSB- 84 -RSB- Rofecoxib -LRB- 60 -RRB- Rofecoxib 50 mgYesClinical examinations and forced expiratory volume hourly .0 -LSB- 13 -RSB- Valdecoxib -LRB- 41 -RRB- Valedcoxib 30 mg total doseYesClinical examination and monitoring of pulse/blood pressare until 16 h past last exposureGeneralised urticaria -LRB- 1/2 -RRB- -LSB- 69 , 70 -RSB- Celecoxib -LRB- 54 -RRB- Etoricoxib -LRB- 56 -RRB- Celecoxib 200 mgEtoricoxib 120 mgYesVital signs and pulmonary function was monitored at baseline and hourly for 3 h and the skin , nose , thorax were examined at the same intervalsCelecoxib : urticaria -LRB- 3/1 -RRB- , lip angioedema -LRB- 1/3 -RRB- , eyelid wheals and angioedema -LRB- 1/3 -RRB- , urticaria , rhinorrhea , and conjunctival injection -LRB- 1/1 -RRB- etoricoxib : urticaria -LRB- 3/1 -RRB- , tongue , eyelid and lip angioedema -LRB- 1/3 -RRB- -LSB- 48 -RSB- Rofecoxib -LRB- 19 -RRB- Rofecoxib 37.5 mg total doseYesSerial measurement of forced expiratory volume after 30 , 60 and 120 min0 Discussion This systematic review documents the low probability of allergic/pseudo-allergic reactions induced by coxibs in patients with NSAID intolerance . To our knowledge , this is the first comprehensive analysis of data published on this topic . We have searched all three major medical databases available , namely MEDLINE , COCHRANE LIBRARY , and EMBASE using very broad and general search terms . Following identification of relevant publications , these were evaluated by means of a pre-defined evaluation form . The data available are described in the form of a structured review -LSB- 19 -RSB- . NSAIDs are among the most commonly prescribed therapeutics in the world . Although generally considered safe , their wide and frequent use results in these drugs being among the most common causes of drug-related intolerance reactions . This may at least in part be due to their non-selective inhibition of both cyclooxygenase isoforms . In line with this hypothesis , NSAIDs characterized by pronounced COX-1 inhibition bear a high risk to trigger asthma attacks in patients with aspirin-sensitive asthma bronchiale , whereas preferentially COX-2 inhibiting NSAIDS are better tolerated by these patients -LSB- 36 -RSB- . Our analysis of published studies on this issue further supports this notion , as only 13 of 591 NSAID-intolerant patients showed adverse reactions upon provocation with coxibs in double-blind clinical studies ; all of these were grade 3 or milder . Still , relatively selective COX-2 inhibitors have been identified as triggers of serious intolerance reactions -LSB- 71 -RSB- . This implies that our current understanding of NSAID-triggered intolerance is still imperfect , and its pathogenesis can not be reduced to cyclooxygenase-mediated effects alone , but needs to take into account clinically relevant additional NSAID-mediated effects such as secretion of leukotrienes from mast cells and other leukocytes . It has been suggested that coxibs may confer an elevated risk for acute myocardial infarction and sudden cardiac death , namely after long-term therapy -LSB- 25 -RSB- . As a reaction , several coxibs are no longer available despite a recommendation by the participants of an expert meeting organized by the Food and Drug Administration to grant further prescription of rofecoxib , celecoxib and valdecoxib in the US -LSB- 59 -RSB- . On the other hand , substantial evidence described here points towards a good tolerability of coxibs in patients with NSAID intolerance . Given the wide use of NSAIDs in the short-term treatment of trivial signs and symptoms , the availability of coxibs for these indications would be advantageous for this relevant subpopulation of patients , since this type of application is unlikely to increase cardiac risk .

O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O

non-steroidal anti-inflammatory drugs
adverse drug reaction


Liver Transplantation for Hepatocellular Carcinoma Background Orthotopic liver transplantation -LRB- OLT -RRB- is the best available option for early hepatocellular carcinoma -LRB- HCC -RRB- , although its application is limited by stringent selection criteria , costs , and deceased donor graft shortage , particularly in Asia , where living donor liver transplant -LRB- LDLT -RRB- has been developed . Hepatocellular carcinoma -LRB- HCC -RRB- is the third most common cause of cancer-related death worldwide .1 Orthotopic liver transplantation -LRB- OLT -RRB- is the best therapeutic option for early , unresectable HCC , although it is limited by graft shortage and the need for appropriate patient selection . In the late 1980s , the results after OLT for HCC were disappointing , with high early recurrence and 5-year survival rates ranging between 18 and 40 % .2 This discouraging experience and shortage of deceased donor grafts compelled the transplant community to establish stringent selection criteria to predict posttransplant survival of HCC patients . Recognizing that patients with small , incidentally found tumors had survival rates after liver transplantation equivalent to those after transplantation for benign disease , Mazzaferro et al. in Milan established criteria for OLT in a landmark study published in 1996.3 They showed that a subgroup of patients with radiologic evidence of a single tumor ≤ 5 cm in diameter , or two to three tumors ≤ 3 cm in diameter had 5-year and recurrence-free survival rates of 75 and 83 % , respectively . The Milan criteria were subsequently adopted by the United Network for Organ Sharing -LRB- UNOS -RRB- staging system for allocating organs for OLT in the United States . OLT is limited by a shortage of deceased donor liver grafts , particularly in Asia , where the rate of deceased donors is negligible . To overcome this shortage , living donor liver transplantation -LRB- LDLT -RRB- has been developed with favorable preliminary results . Other areas of study include expanding the criteria for liver transplantation to include larger tumors , as shown by the group from the University of California , San Francisco -LRB- UCSF -RRB- and molecular profiling of HCC to improve prognostication and patient selection . MILAN CRITERIA FOR LIVER TRANSPLANTATION IN HCC In the past 10 years , results of OLT have improved steadily because of careful patient selection pioneered by the introduction of the Conventional Milan Criteria -LRB- CMC -RRB- in 1996.3 The aim of these criteria was to achieve a good outcome in patients who fulfilled the criteria and avoid a poor prognosis in patients who exceeded them . This aim was achieved by the Milan group , who showed that the 10-year overall survival surpassed 70 % in 300 liver transplants for HCC that fulfilled the CMC . Such good results have been confirmed worldwide .4,5 The elements of the CMC , namely size and number of tumors , have been shown in multivariate analysis to be the only independent variables predicting patient survival and tumor recurrence , with other biological prognostic factors playing a role only within the `` size and number '' limits -LRB- Table 1 -RRB- . TABLE 1 . Prognostic factors affecting survival of 250 patients transplanted for HCC according to Conventional Milan Criteria at the National Cancer Institute of Milan10-year survival : multivariate analysisVariableHRCI -LRB- 95 % -RRB- P valueOverall patient survivalConventional Milan criteria -LRB- in vs out -RRB- 3.11.35 -- 6.93.007Tumor-free survivalConventional Milan criteria -LRB- in vs out -RRB- 5.51.39 -- 21.27.01 Microsatellites -LRB- yes vs no -RRB- 3.61.5 -- 8.71.004 Microvascular invasion -LRB- yes vs no -RRB- 3.41.36 -- 8.76.009 Tumor grading -LRB- G3 vs G1-2 -RRB- 3.41.04 -- 11.14.04 The successful outcome of OLT based on the CMC has led to more patients with HCC being routed to transplantation and an increasing number of proposals for expansion of the CMC .6,7 None of these expanded criteria are , however , supported by a robust statistical sample size or prospective validation . In order to investigate proposals to expand the CMC , an unprecedented multicenter study has been conducted in 24 European centers , collecting data from 466 patients transplanted for HCC whose tumors exceeded the CMC at posttransplant pathologic assessment -LRB- http://www.hcc-olt-metroticket.org -RRB- .5,7 The results have been plotted in a tumor size-and-number Cartesian contour plot showing the 5-year survival probability according to the size and number of HCC nodules detected in the explanted liver . Based on a preliminary analysis , a `` HCC forecast chart '' has been developed , which can predict 5-year posttransplant survival rates on the basis of morphological tumor characteristics -LRB- Fig. 1 -RRB- . It is conceivable that similar chart models obtained from large sample sizes could replace the CMC for OLT in patients with HCC . Unlike the CMC or expanded proposals , which are based on tumor morphology , these chart models can incorporate important variables for OLT in HCC , including priority score for HCC and dropout rate based on donor availability at different centers . Preliminary ad interim analysis shows a significant shift of the isograms of the proposed chart depending on the presence of vascular invasion in explant specimens , confirming the need for reliable biological markers of important tumor characteristics , such as vascular invasion . FIG. 1 . HCC forecast chart showing probability of 5-year survival based on tumor size and number in explanted liver . MILAN VERSUS EXPANDED CRITERIA FOR LIVER TRANSPLANTATION Following the 1996 publication by the group from Milan , using a restrictive set of criteria for OLT in patients with HCC , excellent 5-year posttransplant patient survival of at least 70 % has been reported from many centers .4,5 The growing experience and success of OLT for HCC have fueled controversies related to expansion of the Milan criteria for OLT , since many studies have suggested that tumor stage beyond the Milan criteria does not necessarily predict worse survival after OLT .8,9 Among the proposed expanded criteria , the UCSF criteria -LRB- single tumor nodule up to 6.5 cm ; or three or fewer tumors , the largest of which is ≤ 4.5 cm with the sum of the tumor diameters ≤ 8 cm -RRB- reflect a modest expansion of tumor size limits beyond the Milan criteria .6 At least three transplant experiences from Europe and the United States , however , underlined the limitations of applicability of the UCSF criteria in the pretransplant setting , considering that most of the patients adhering to the UCSF were also within the Milan criteria .10 -- 12 Even though the UCSF criteria have been independently validated in several studies , the overlapping population of patients adhering to the UCSF but not the Milan criteria is often negligible and estimated to be < 10 % of the total transplanted population .12,13 Such a limitation was evidenced by a multicenter study from France , in which 39 of 461 patients -LRB- 8.7 % -RRB- had explanted tumors beyond the Milan but within the UCSF criteria .10 Although the 5-year survival of 67 % for patients within the UCSF criteria was equivalent to that of the 183 patients meeting the Milan criteria -LRB- and significantly better than the 34 % 5-year survival rate among 238 patients exceeding both criteria -RRB- , the 44 patients meeting UCSF but exceeding Milan criteria at pretransplant staging had a 5-year survival rate of only 48 % , compared with 60 % observed in the 272 patients within Milan criteria and with 37 % in the 121 patients beyond both criteria . The limitations of pretransplant imaging studies , exemplified by tumor understaging in 20 % of patients , have been a major concern for liberalizing the existing criteria for OLT .5 The UCSF group applied their proposed criteria according to preoperative imaging in 138 patients over a 5-year period ; the 1 - and 5-year recurrence-free probabilities were 95 and 91 % , and the respective probabilities for recurrence-free survival were 91 and 80 % .14 The 106 patients with HCC meeting the Milan criteria -LRB- T1/2 -RRB- had 1 - and 5-year recurrence-free probabilities of 96 and 90 % , respectively , compared with 93 % at both 1 and 5 years for patients with HCC exceeding Milan but meeting UCSF criteria -LRB- T3A -RRB- . Understaging by preoperative imaging was observed in 21 % of T1/2 and 28 % of T3A tumors . When explant tumor stage exceeded the UCSF criteria , the 1 - and 5-year recurrence-free probabilities were 79 and 61 % , compared with 98 and 97 % within the UCSF criteria . Patients meeting T3A criteria did not have a significantly higher incidence of poorly differentiated tumor grade or vascular invasion . In the current era of increasing demand and unrelenting organ shortage , the foundation of the debate regarding expansion of the Milan criteria for HCC may ultimately rest on what the transplant community would consider an acceptable survival after OLT for HCC . Some groups have proposed a 50 % 5-year patient survival to be the minimum acceptable cutoff .5 This mark may have been surpassed by the UCSF group , who have applied expanded criteria to benefit an additional 10 % of patients with HCC with respect to posttransplant survival and tumor recurrence . LIVING DONOR LIVER TRANSPLANTATION FOR HCC : EASTERN EXPERIENCE Liver transplantation offers the best chance of cure for early unresectable HCC . However , its role has been limited by the shortage of deceased donor liver grafts , which is particularly severe in Asia , where the deceased donor organ rates are fewer than 5 donors per million population , compared with 10 -- 35 donors per million population in Western countries .15 In most Asian countries , HCC is the most common cancer and the most frequent indication for OLT , aggravating the unmet demand for liver grafts . The development of living donor liver transplantation -LRB- LDLT -RRB- , especially adult-to-adult right lobe liver transplantation , has allowed more patients with HCC to benefit from OLT .16 LDLT can theoretically provide an unlimited source of liver grafts and eliminate the uncertainty of prolonged waiting times and the risk of dropout due to tumor progression . However , LDLT is a novel treatment for HCC with unresolved issues regarding indications and results . Recent studies on LDLT for HCC suggest favorable long-term survival results .17,18 However , it remains unclear whether the outcome after LDLT for HCC is equivalent to that of deceased donor liver transplantation -LRB- DDLT -RRB- . A study from a Korean group reported similar 3-year survival rates after LDLT and DDLT -LRB- 91.4 vs 89.9 % -RRB- in patients with tumors fulfilling the Milan criteria , after excluding perioperative mortality .13 However , others have found significantly higher rates of tumor recurrence after LDLT compared with DDLT for HCC .19 This may be related to selection bias , as LDLT eliminates the waiting period for grafts . In DDLT the waiting period provides time for a natural selection process in which patients with biologically more aggressive tumors drop out due to tumor progression . In addition , the ischemic-reperfusion injury associated with small-for-size grafts in LDLT and angiogenesis associated with liver regeneration may theoretically promote growth of tumors in the transplanted liver after LDLT , although the actual clinical impact of such biological processes remains unclear .20,21 Liver transplantation is conventionally offered to Child-Pugh class C patients with unresectable early HCC . Recently , there have been heated debates on whether liver transplantation should be used as first-line therapy for Child-Pugh class A patients with early HCC .22 While some previous studies showed that liver transplantation for early HCC may achieve better survival compared with resection , this may be partly related to selection bias in favor of transplantation because patients with more aggressive tumors drop off the waiting list for DDLT .19 Recent studies suggest that for patients with preserved liver function and early stage HCC , hepatic resection can achieve a 5-year survival rate of 70 % , comparable to that after OLT .13,23 The availability of grafts from dedicated live donors has been considered one of the main arguments favoring LDLT as primary therapy for patients with early HCC and preserved liver function . However , even in Asian countries where LDLT is commonly performed , up to half of the patients with early HCC may not have suitable living donors for various reasons , including ABO-incompatibility , hepatitis serology , and patient refusal to accept living donation .18 Furthermore , the risks of donor hepatectomy , with morbidity and mortality rates of 14 -- 21 % and 0.25 -- 1 % , respectively , should be carefully balanced against the benefit of LDLT .19 Risking the life of a donor for HCC patients who have an alternative option of hepatic resection , which achieves long-term survival equivalent to LDLT , is ethically not acceptable to many Asian surgeons . Most Asian centers still consider resection as first-line treatment for HCC patients with preserved liver function and reserve LDLT as an option for salvage transplantation in patients with recurrent tumors .13,23,24 Another matter of debate in LDLT involves expanding the indications beyond the CMC or UCSF criteria . LDLT is currently being performed in some Asian centers for patients with HCC beyond the Milan criteria , with results that are expectedly worse than those for patients within the Milan criteria .13,17 Some transplant surgeons argue that despite the poorer results , LDLT for advanced HCC may be justified , since donors voluntarily accept the risks of donor hepatectomy to dedicate a graft for HCC patients , who may otherwise have no effective treatment options . However , others argue that the medical profession should not relegate the issue to individual donor autonomy .25 With the lack of clear data showing benefits of LDLT for advanced HCC , the medical community should take a conservative moral stand and limit the use of LDLT for HCC that meets the same criteria as DDLT . LIVING DONOR LIVER TRANSPLANTATION FOR HCC : WESTERN EXPERIENCE Over the past 10 years , LDLT has developed as an alternative to DDLT because of the scarcity of deceased donor livers . Patient survival after LDLT is similar to that after DDLT , despite data from the UNOS database showing lower post-LDLT graft survival , possibly due to the learning curve for LDLT and lower graft-to-recipient body weight ratio .26 In the United States , enthusiasm for LDLT peaked in 2001 , when more than 500 LDLTs were performed ; the aftermath of a widely-publicized donor death in 2002 led to a nationwide retrenchment , with no more than 325 procedures per year since then . Nevertheless , LDLT has an important role in the treatment of HCC . While previously there was concern over possible tumor stimulation due to regeneration after LDLT , it appears that the type of graft -LRB- living vs deceased donor -RRB- has little , if any , impact on post-OLT tumor progression . Typically , LDLT results in a liver graft that is smaller than the expected liver volume of the recipient . The outcome of a relatively small donor organ depends not only on graft size but also on the recipient 's preoperative degree of liver failure and portal hypertension . Compared with patients awaiting transplant for end-stage cirrhosis , candidates with HCC generally have better-preserved liver function and less portal hypertension , and are thus better able to tolerate a small graft . Ready availability is the most important advantage of LDLT for HCC . Posttransplant tumor recurrence is tied to pretransplant tumor stage ; progression of tumor while awaiting transplant can only worsen the prognosis . This fact is reflected in the United States allocation scheme for deceased donor livers , which accords priority to patients with HCC meeting the Milan criteria . Since adoption of the Model of End-Stage Liver Disease organ allocation policy in 2002 , candidates with T2 HCC -LRB- 1 nodule between 2 and 5 cm ; or 2 -- 3 nodules , all ≤ 3 cm -RRB- have enjoyed priority such that in many regions of the United States , waiting times are short , obviating the need for LDLT .27 In some regions , however , longer waiting times and attendant higher dropout rates support the use of LDLT in cases of HCC within the Milan criteria . The Milan criteria were adopted because they identify a subgroup of candidates with HCC for whom transplant results are similar to those in patients transplanted for end-stage cirrhosis without HCC . It is widely recognized , however , that many patients with HCC beyond the Milan criteria can be cured by OLT .8,9 In the United States , adoption of expanded priority criteria for DDLT , as proposed by the UCSF group , is currently under consideration . LDLT can be undertaken in candidates who do not meet criteria for waiting list priority . Broadening criteria based on tumor size and number will inevitably lead to more recurrence ; nevertheless , Roayaie et al. demonstrated 55 % freedom from recurrence at 5 years in patients transplanted with HCC between 5 and 7 cm and no macroscopic vascular invasion .28 Based on data such as these , many centers offer LDLT to patients with HCC meeting expanded criteria that are estimated to yield a 5-year survival of approximately 50 % .5 SELECTION IN HCC : BIOLOGY OR MORPHOLOGY The distinction between biology and morphology in simple terms can be described as behavior versus appearance . Ultimately , the behavior of HCC is the final deciding factor on patient outcome . The ability to predict the biology of HCC is desperately needed for patient selection in OLT . The current staging criteria for HCC takes into account tumor morphology but not tumor biology , with transplantable stages stratified according to tumor size and number . For treatment options other than liver transplantation -LRB- i.e. , liver resection , chemotherapy , and cytoablative therapies -RRB- , tumor morphology as described by the pathological TNM staging system is reasonably adequate . For liver transplantation , however , gross and microscopic morphology are inadequate for predicting outcomes because of the scarcity of the donor organs that must be judiciously allocated . The issue would be less significant if resources were unlimited . Ideally , the TNM or morphology-based staging system would be able to stratify patients into homogeneous groups with the same outcomes . However , the current staging systems are not precise enough to segregate patients with HCC and cirrhosis into such homogeneous groups . As almost all patients with gross invasion of the hepatic venous system , positive lymph nodes , or metastatic disease experience recurrence and are therefore not candidates for transplantation , the N and M components of the TNM classification can immediately be eliminated from the organ allocation scheme . This leaves only the T component upon which we can base our biological prediction , namely tumor size and number . In our efforts to refine our current staging systems , morphological or chemical factors have been studied , including microscopic vascular invasion , encapsulation , plasma albumin mRNA , and serum alpha-fetoprotein.5 To date , however , none of these have improved upon the current systems . Part of the problem has been radiologic techniques are not yet sufficient to perfectly define tumor number and size and will never be able to determine microscopic vascular invasion . Moreover , many new staging systems require pretreatment biopsy , which poses a small but real risk of tumor seeding . To understand the biological behavior and identify genes associated with survival after OLT , Marsh and colleagues at the University of Pittsburgh performed microdissection on explanted tissue and studied DNA mutations near 9 tumor-associated gene loci to create an index of cumulative mutational damage , termed the fractional allelic imbalance -LRB- FAI -RRB- .29,30 They found that FAI and vascular invasion were the strongest independent predictors of tumor-free survival . Thus , incorporation of gene mutational data allows desegregation of HCC patients from imprecise , morphology-based staging systems and allows improved prognostication . CONCLUSIONS While patients with early resectable HCC and preserved hepatic function should undergo surgical resection , in those with unresectable disease due to underlying liver dysfunction , orthotopic liver transplantation -LRB- OLT -RRB- offers the best chance for cure . In the past 10 years , results of OLT have steadily improved because of careful patient selection pioneered by the introduction of the Milan criteria in 1996 . Supported by studies showing that many patients with tumor stage beyond the Milan criteria can be cured by OLT , a number of expanded criteria have been proposed . While expanding the criteria for OLT allows more patients to be eligible for transplantation , arguments against expanding the criteria include the increased risk of vascular invasion and tumor recurrence at higher stages of HCC . The principal limitation of OLT for HCC is the shortage of deceased donor living grafts , especially in Asia . The development of LDLT has allowed more patients to benefit from OLT with favorable preliminary survival results . Given the shortage of organs available for OLT and lack of predictive power of currently used staging systems , improved prognostic tools are needed to predict outcomes after OLT . Molecular markers of cancer progression may add significant discriminatory power to the current staging systems and may improve organ allocation schemes for patients with HCC .

O B O B O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O 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Regulation of intestinal hPepT1 -LRB- SLC15A1 -RRB- activity by phosphodiesterase inhibitors is via inhibition of NHE3 -LRB- SLC9A3 -RRB- The H + - coupled transporter hPepT1 -LRB- SLC15A1 -RRB- mediates the transport of di/tripeptides and many orally-active drugs across the brush-border membrane of the small intestinal epithelium . Incubation of Caco-2 cell monolayers -LRB- 15 min -RRB- with the dietary phosphodiesterase inhibitors caffeine and theophylline inhibited Gly -- Sar uptake across the apical membrane . Pentoxifylline , a phosphodiesterase inhibitor given orally to treat intermittent claudication , also decreased Gly -- Sar uptake through a reduction in capacity -LRB- Vmax -RRB- without any effect on affinity -LRB- Km -RRB- . The reduction in dipeptide transport was dependent upon both extracellular Na + and apical pH but was not observed in the presence of the selective Na + / H + exchanger NHE3 -LRB- SLC9A3 -RRB- inhibitor S1611 . Measurement of intracellular pH confirmed that caffeine was not directly inhibiting hPepT1 but rather having an indirect effect through inhibition of NHE3 activity . NHE3 maintains the H + - electrochemical gradient which , in turn , acts as the driving force for H + - coupled solute transport . Uptake of β-alanine , a substrate for the H + - coupled amino acid transporter hPAT1 -LRB- SLC36A1 -RRB- , was also inhibited by caffeine . The regulation of NHE3 by non-nutrient components of diet or orally-delivered drugs may alter the function of any solute carrier dependent upon the H + - electrochemical gradient and may , therefore , be a site for both nutrient -- drug and drug -- drug interactions in the small intestine . 1 Introduction The human intestinal H + - coupled di/tripeptide transporter -LRB- hPepT1 or SLC15A1 -RRB- is responsible for the transport of a significant proportion of dietary protein across the brush-border membrane of the small intestinal epithelium . hPepT1 is also of pharmacological interest as it can transport orally-active drugs such as β-lactam antibiotics and angiotensin converting enzyme inhibitors -LSB- 1 -- 4 -RSB- . More recently a number of amino acid modified pro-drugs such as val-acyclovir , l-DOPA-l-Phe and the anti-hypotensive midodrine have been designed specifically to target hPepT1 and thus to enhance oral bioavailability -LSB- 3 -- 5 -RSB- . When expressed heterologously , hPepT1 functions as a Na + - independent , pH-dependent , H + - coupled transporter -LSB- 6,7 -RSB- . However , until the mid-1980s , it was considered widely that di/tripeptide transport was via a Na + - coupled transport mechanism . Indeed , dipeptides can stimulate Na + absorption in the human small intestine and Na + uptake in human intestinal Caco-2 cell monolayers -LSB- 8 -- 10 -RSB- . Ganapathy and Leibach proposed a model to account for the interaction of H + - coupled di/tripeptide transport and Na + absorption -LSB- 11 -RSB- whereby the intracellular acidification caused by di/tripeptide associated H + influx activated an apical Na + / H + exchanger and thus Na + absorption . This model also explains the partial Na + - dependence of di/tripeptide uptake observed in mammalian small intestine and Caco-2 cell monolayers -LSB- 11,12 -RSB- . Inhibition of Na + / H + exchange by removal of extracellular Na + , prevents the enterocyte from maintaining intracellular pH -LRB- pHi -RRB- during H + / solute influx and , therefore , abolishes the driving force -LRB- the transmembrane H + electrochemical gradient -RRB- for further transport . The apical Na + / H + exchanger specifically activated by di/tripeptide uptake is NHE3 -LRB- SLC9A3 -RRB- which plays a significant role in Na + absorption in the small intestine -LSB- 7,10,13 -RSB- . As hPepT1 is involved in both protein assimilation and drug delivery it is important to elucidate how altering hPepT1 function may affect nutritional status and oral bioavailability . Progress has been made in understanding how intestinal di/tripeptide transport is regulated by hormonal and neural signals as well as by disease states and surgical intervention -LRB- for review see -LSB- 14 -RSB- -RRB- . However , little is known about how co-administered drugs or dietary factors affect hPepT1 function or the function of other brush-border membrane transport proteins . Alteration of cAMP levels within enterocytes by hormonal , neural and paracrine signals is a key step in the regulation of many brush-border membrane nutrient and electrolyte transporters . NHE3 is inhibited acutely by increasing -LSB- cAMP -RSB- i -LSB- 15 -RSB- . Therefore , the aim of this study was to identify how dietary and orally-delivered compounds , which act as phosphodiesterase inhibitors -LRB- and thus increase -LSB- cAMP -RSB- i by preventing breakdown -RRB- , affect hPepT1 function . The dietary phosphodiesterase inhibitors caffeine and theophylline and the orally-delivered drug pentoxifylline all inhibit dipeptide uptake across the brush-border membrane of human intestinal epithelial Caco-2 cell monolayers . This inhibition is not a direct effect on hPepT1 but rather is indirect through inhibition of NHE3 . 2 Materials and methods 2.1 Materials Glycyl -LSB- 1-14C -RSB- sarcosine -LRB- specific activity 57 mCi mmol − 1 -RRB- -RSB- was obtained from Cambridge Research Biochemicals -LRB- Billingham , UK -RRB- . -LSB- 3-3H -RSB- β-Alanine -LRB- specific activity 50 Ci mmol − 1 -RRB- -RSB- was obtained from American Radiolabeled Chemicals -LRB- St. Louis , MO , USA -RRB- . PACAP -LRB- pituitary adenylate cyclase-activating polypeptide -RRB- was from Bachem -LRB- St. Helens , UK -RRB- . Biotrak cAMP enzyme immunoassay was from GE Healthcare -LRB- Chalfont St. Giles , UK -RRB- . 2 ′ ,7 ′ - Bis -LRB- 2-carboxyethyl-5 -LRB- 6 -RRB- - carboxyfluorescein -RRB- acetoxymethyl ester -LRB- BCECF-AM -RRB- was from Invitrogen -LRB- Paisley , UK -RRB- . Transwell polycarbonate filters were from Corning -LRB- Schiphol-Rijk , The Netherlands -RRB- . S1611 was obtained from H.J. Lang -LRB- Aventis Pharma Deutschland GmbH , Frankfurt/Main , Germany -RRB- . Forskolin , phosphodiesterase inhibitors , cell culture media and supplements were from Sigma-Aldrich -LRB- Poole , UK -RRB- . All other chemicals were from Sigma-Aldrich or VWR -LRB- Lutterworth , UK -RRB- and were of the highest quality available . 2.2 Cell culture Caco-2 cells -LRB- passages 100 -- 119 -RRB- were cultured as confluent monolayers on permeable polycarbonate filters , as described previously -LSB- 16 -RSB- . Monolayers were used at 14 -- 22 days post-seeding and fed approximately 24 h prior to use . 2.3 Measurement of dipeptide uptake -LSB- 14C -RSB- Gly -- Sar -LRB- 0.5 μCi ml − 1 , 0.1 -- 10 mM -RRB- or -LSB- 3H -RSB- β-alanine -LRB- 0.5 μCi ml − 1 , 0.1 mM -RRB- uptake across the apical membrane of Caco-2 cell monolayers was measured , essentially as described previously -LSB- 16 -RSB- . Briefly , Caco-2 cells were washed -LRB- in 4 × 500 ml -RRB- and bathed in modified Krebs ' solution -LRB- composition -LRB- mM -RRB- : NaCl , 137 ; KCl , 5.4 ; MgSO4 , 0.99 ; KH2PO4 , 0.34 ; NaH2PO4 , 0.3 ; CaCl2 , 2.8 ; Glucose , 10 -RRB- or Na + - free solution -LRB- identical except choline chloride replaced NaCl and NaH2PO4 was omitted -RRB- . Uptake was measured at apical pH 5.5 or pH 6.5 -LRB- 10 mM MES , adjusted to the correct pH by Tris -RRB- with the basolateral solution being pH 7.4 -LRB- 10 mM HEPES , adjusted to the correct pH by Tris -RRB- in all experiments . Uptake was measured over 15 min -LRB- 37 °C -RRB- at the end of which the monolayers were rinsed thoroughly -LRB- in 3 × 500 ml , ice-cold , pH 7.4 Krebs ' solution -RRB- . Various compounds were added to the buffers for the duration of uptake -LRB- see figure legends for details -RRB- . Cell monolayer-associated radioactivity was determined by scintillation counting . 2.4 Measurement of pHi Intracellular pH -LRB- pHi -RRB- was measured in Caco-2 cell monolayers by microspectrofluorimetry using the pH-sensitive dye BCECF , essentially as described previously -LSB- 10,17 -RSB- . Cell monolayers were acidified by superfusion with apical Gly -- Sar -LRB- 20 mM , 5 min -RRB- in modified Krebs ' solution -LRB- Na + - free , apical pH 5.5 -RRB- . The basolateral solution was Na + - free , pH 7.4 throughout all experiments . pHi recovery was then measured in Na + - containing pH 7.4 apical solution until pHi returned to baseline . The monolayers were incubated with caffeine -LRB- 5 mM , apical and basolateral -RRB- for 10 min . The cells were then exposed to Gly -- Sar once again but in the continued presence of caffeine -LRB- thus total exposure time to the compound before pHi recovery was 15 min -RRB- . pHi recovery was then measured in the continued presence of caffeine . The initial rate of pHi recovery is expressed as H + efflux calculated from the gradient of the first 30 s of recovery -LSB- 18 -RSB- . 2.5 Measurement of intracellular cAMP levels Caco-2 cell monolayers were washed in 4 × 500 ml Krebs ' solution -LRB- pH 7.4 -RRB- . Monolayers were incubated for 15 min in the presence or absence of caffeine , pentoxifylline , theophylline -LRB- all 5 mM -RRB- , or forskolin -LRB- 10 μM -RRB- at apical pH 6.5 and basolateral pH 7.4 . Monolayers were then washed in 3 × 500 ml , ice-cold Krebs ' -LRB- pH 7.4 -RRB- and lysed in 500 μl lysis buffer -LRB- from the Biotrak enzyme immunoassay kit -RRB- for 10 min . -LSB- cAMP -RSB- i was measured following the kit protocol . 2.6 Statistical analysis Data are expressed as mean ± SEM unless stated otherwise . Statistical comparisons of mean values were made using paired two-tailed Student 's t-test or one-way analysis of variance -LRB- ANOVA -RRB- -LRB- using the Tukey -- Kramer or Bonferroni 's multiple comparisons post-test -RRB- as appropriate . Significance was assumed if p < 0.05 . Curves were fitted using FigP software . 3 Results 3.1 Inhibition of dipeptide uptake by caffeine is via inhibition of NHE3 Uptake of the dipeptide Gly -- Sar -LRB- glycylsarcosine -RRB- across the apical membrane of Caco-2 cell monolayers was measured over 15 min at an apical pH -LRB- pH 6.5 -RRB- representative of that found at the surface of the proximal small intestine . Incubation of Caco-2 cell monolayers with the dietary phosphodiesterase inhibitor caffeine -LRB- 5 mM -RRB- at both the apical and basolateral surfaces -LRB- for the duration of the 15 min uptake period -RRB- reduced Gly -- Sar uptake by around 50 % -LRB- from 683.6 ± 18.9 to 351.1 ± 10.7 pmol cm − 2 -LSB- 15 min -RSB- − 1 , n = 17 -- 18 , p < 0.001 , Fig. 1A -RRB- . Caffeine was equally effective at inhibiting Gly -- Sar uptake when added solely to either the apical or basolateral chamber -LRB- data not shown -RRB- . Removal of extracellular Na + reduces Gly -- Sar uptake -LRB- as described previously -LSB- 12 -RSB- -RRB- -LRB- Fig. 1A -RRB- . However , in the absence of extracellular Na + , caffeine had no effect on Gly -- Sar uptake -LRB- uptake being 244.0 ± 9.1 and 242.0 ± 9.5 pmol cm − 2 -LSB- 15 min -RSB- − 1 in the absence and presence of caffeine respectively , n = 17 -- 18 , p > 0.05 -RRB- . Previously we have shown that the Na + - dependence of Gly -- Sar uptake can be attributed to a lack of NHE3 activity in the absence of extracellular Na + -LSB- 10,12,18 -RSB- . To test if the lack of effect of caffeine in the absence of extracellular Na + was due to a requirement for NHE3 activity , dipeptide uptake was measured under other conditions where NHE3 would be inactive -LRB- Fig. 1B -RRB- . At apical pH 6.5 , the NHE3-selective inhibitor S1611 -LRB- 3 μM -RRB- significantly reduced Gly -- Sar uptake in a manner similar to that caused by the removal of extracellular Na + -LRB- p < 0.001 -RRB- . This concentration of S1611 is sufficient to inhibit NHE3 completely but would not inhibit NHE2 or NHE1 -LSB- 19 -RSB- . Previous work has shown that in the absence of Na + , S1611 has no effect on Gly -- Sar uptake into Caco-2 cells -LSB- 12 -RSB- . In addition , when hPepT1 is expressed in isolation in Xenopus laevis oocytes , dipeptide uptake is not inhibited by S1611 -LSB- 7 -RSB- . This observation confirms that the inhibition of dipeptide uptake by S1611 into Caco-2 cells is an indirect effect via inhibition of NHE3 . In the presence of S1611 , Gly -- Sar uptake was no longer inhibited by caffeine -LRB- 5 mM -RRB- . The level of inhibition caused by caffeine alone , by S1611 alone and by both compounds together were not significantly different -LRB- p > 0.05 -RRB- suggesting that caffeine and S1611 inhibit hPepT1 activity through a common mechanism -LRB- i.e. NHE3 inhibition -RRB- . Gly -- Sar uptake increases with decreasing apical pH , consistent with hPepT1 being a H + / dipeptide symporter . However , NHE3 activity decreases with acidification of the apical solution and is inactive by pH 5.5 due to the unfavourable transmembrane pH gradient -LSB- 12 -RSB- . Previous studies have shown that Gly -- Sar uptake at apical pH 5.5 is not inhibited by S1611 or by the removal of extracellular Na + -LSB- 7,12 -RSB- . Similarly , Fig. 1B shows that caffeine -LRB- 5 mM -RRB- has no effect on Gly -- Sar uptake at apical pH 5.5 -LRB- p > 0.05 -RRB- suggesting again that caffeine is inhibiting Gly -- Sar uptake through inhibition of NHE3 . 3.2 The inhibition of hPepT1 by caffeine and related compounds is consistent with inhibition of phosphodiesterase activity Phosphodiesterase inhibitors structurally-related to caffeine and either found in diet -LRB- theophylline -RRB- , or used as orally-delivered therapeutics -LRB- pentoxifylline -RRB- or used as laboratory tools -LRB- IBMX , 3-isobutyl-1-methylxanthine -RRB- were tested for inhibition of hPepT1 -LRB- Fig. 2 -RRB- . Theophylline -LRB- 5 mM , Fig. 2A -RRB- , pentoxifylline -LRB- 5 mM , Fig. 2B -RRB- and IBMX -LRB- 1 mM , Fig. 2C -RRB- reduced dipeptide uptake in the presence of extracellular Na + -LRB- p < 0.001 -RRB- but not in Na + - free conditions -LRB- p > 0.05 -RRB- . Previously , we have shown that the enteric neuropeptide PACAP -LRB- pituitary adenylate cyclase-activating polypeptide -RRB- and the related neuropeptide VIP -LRB- vasoactive intestinal peptide -RRB- reduce dipeptide uptake across the apical membrane of Caco-2 cell monolayers by inhibition of NHE3 -LSB- 18 -RSB- . PACAP activates the VPAC1 receptor which is coupled to adenylate cyclase and , therefore , increases -LSB- cAMP -RSB- i. Fig. 2C shows that when Caco-2 cell monolayers were incubated with PACAP -LRB- 5 nM -RRB- in the basolateral solution for the duration of uptake , Gly -- Sar uptake was reduced . Incubation of the cells with both PACAP and IBMX resulted in a reduction in uptake which was not significantly different from the uptake in the presence of either PACAP or IBMX alone suggesting IBMX , like PACAP , is inhibiting the NHE3-dependent component of Gly -- Sar uptake . Phosphodiesterase inhibitors increase the levels of cAMP within cells by preventing cAMP breakdown . However , caffeine can also elicit cell signalling effects such as antagonism of adenosine receptors and activation of calcium release from intracellular stores -LSB- 20 -- 22 -RSB- . To confirm that the compounds used in this study inhibit hPepT1 function through increasing cAMP levels , -LSB- cAMP -RSB- i in Caco-2 cell monolayers was measured . Monolayers were incubated with caffeine -LRB- 5 mM -RRB- , theophylline -LRB- 5 mM -RRB- , pentoxifylline -LRB- 5 mM -RRB- or the adenylate cyclase activator forskolin -LRB- 10 μM -RRB- which can also inhibit dipeptide uptake -LSB- 12 -RSB- . Cells were lysed and -LSB- cAMP -RSB- i measured by enzyme immunoassay . Caffeine , theophylline and pentoxifylline caused -LSB- cAMP -RSB- i to more than double from control levels of 950 ± 230 fmol cm − 2 -LRB- mean ± SD , n = 3 -RRB- to 2200 ± 830 , 2780 ± 300 and 2330 ± 150 fmol cm − 2 -LRB- mean ± SD , n = 3 -RRB- , respectively . However , the change in cAMP was modest compared to -LSB- cAMP -RSB- i detected in the presence of forskolin -LRB- 92500 ± 12990 fmol cm − 2 -LSB- mean ± SD , n = 3 -RSB- -RRB- . Inhibition of the Na + - dependent -LRB- NHE3-dependent -RRB- component of Gly -- Sar uptake by phosphodiesterase inhibitors was concentration-dependent -LRB- Fig. 3 -RRB- . The IC50 values were 1.0 ± 0.2 mM for caffeine , 0.6 ± 0.1 mM for theophylline and 0.5 ± 0.1 mM for pentoxifylline . The sensitivity of hPepT1 to these compounds is consistent with the effect being through an inhibition of phosphodiesterase activity -LSB- 20,21,23 -- 25 -RSB- . At a concentration of 5 mM all three phosphodiesterase inhibitors almost completely inhibited Na + - dependent Gly -- Sar uptake . 3.3 Pentoxifylline decreases the maximal capacity for dipeptide uptake Gly -- Sar uptake across the apical membrane was measured at apical pH 6.5 at various concentrations of Gly -- Sar -LRB- Fig. 4 -RRB- . In the presence of pentoxifylline -LRB- 5 mM -RRB- Gly -- Sar uptake was reduced compared to control at all Gly -- Sar concentrations . Analysis of the Michaelis -- Menten kinetics confirmed that the maximal capacity -LRB- Vmax -RRB- for Gly -- Sar uptake was significantly reduced from 8246 ± 913 to 5369 ± 117 pmol cm − 2 -LSB- 15 min -RSB- − 1 -LRB- p < 0.05 , n = 6 -RRB- by pentoxifylline but the affinity -LRB- Km -RRB- was not affected -LRB- 1.0 ± 0.2 mM and 1.6 ± 0.6 mM in the absence and presence of pentoxifylline , respectively , p > 0.05 -RRB- . Reduction in the capacity for dipeptide uptake is consistent with a reduction in the driving force -LRB- the H + - electrochemical gradient -RRB- for uptake through inhibition of NHE3 . Previously , we have shown that S1611 and forskolin similarly decrease the Vmax for dipeptide uptake across the apical membrane of Caco-2 cell monolayers -LSB- 12 -RSB- . 3.4 Caffeine inhibits pHi recovery -LRB- NHE3 function -RRB- after dipeptide-induced acidification Previous work demonstrated that NHE3 -LRB- but not NHE1 or NHE2 -RRB- is selectively activated by the intracellular acidification associated with H + / dipeptide symport in Caco-2 cell monolayers -LSB- 10,12 -RSB- . S1611 , the removal of extracellular Na + , or factors that increase -LSB- cAMP -RSB- i , such as forskolin and VIP , all inhibit the ability of the cell to regulate pHi after H + / solute-induced intracellular acidification -LSB- 10,12,18,26 -RSB- . pHi was measured using the pH-sensitive dye BCECF -LRB- Fig. 5 -RRB- . Caco-2 cell monolayers loaded with BCECF were superfused across the apical membrane with Na + - containing , pH 7.4 modified Krebs ' solution . The basolateral solution was maintained as Na + - free , pH 7.4 solution throughout the experiment . Cells were exposed to Gly -- Sar -LRB- 20 mM -RRB- under conditions where hPepT1 activity would be maximal but where there would be no concurrent NHE3 activity -LRB- pH 5.5 , Na + - free -RRB- . After 5 min , Gly -- Sar had induced a large intracellular acidification . When Gly -- Sar was removed and the apical solution returned to a Na + - containing , pH 7.4 solution , pHi recovered rapidly back to baseline levels . The same monolayers were then incubated with caffeine -LRB- 5 mM -RRB- in both the apical and basolateral solutions for 10 min . Gly -- Sar was then reintroduced for 5 min in the continued presence of caffeine . The degree and rate of acidification caused by Gly -- Sar was not altered by caffeine suggesting that hPepT1 is not directly regulated by phosphodiesterase inhibitors . However , the ability of the cell to recover pHi after Gly -- Sar-induced acidification was attenuated after the 15 min exposure to caffeine -LRB- Fig. 5A -RRB- . The degree of attenuation was quantified by calculating the H + efflux rate over the first 30 s of pHi recovery -LRB- Fig. 5B -RRB- . In the presence of caffeine H + efflux was reduced from 57.6 ± 5.8 to 20.5 ± 3.9 μM s − 1 -LRB- n = 4 , p < 0.01 -RRB- . To confirm that phosphodiesterase inhibitors do not have any direct inhibitory effect on dipeptide transport , hPepT1 was expressed in Xenopus laevis oocytes , as described previously -LSB- 7 -RSB- . Incubation of hPepT1-expressing oocytes with phosphodiesterase inhibitors such as caffeine -LRB- 5 mM -RRB- had no effect on dipeptide uptake -LRB- data not shown -RRB- . 3.5 Inhibition of NHE3 by caffeine also inhibits the H + / amino acid transporter hPAT1 -LRB- SLC36A1 -RRB- The indirect nature of the inhibition of hPepT1 by phosphodiesterase inhibitors suggests that any apical solute transporter dependent upon the transmembrane H + - electrochemical gradient will similarly be regulated by these compounds . The H + - coupled amino acid transporter hPAT1 -LRB- SLC36A1 -RRB- has been isolated from Caco-2 cell monolayers -LSB- 27 -RSB- . As well as mediating the uptake of a wide variety of amino acids , hPAT1 can also transport orally-active drugs such as the anti-epileptic vigabatrin -LSB- 28 -RSB- . Previously we have identified that amino acid uptake into hPAT1-expressing oocytes is Na + - independent but hPAT1-mediated amino acid uptake into Caco-2 cells is partially Na + - dependent -LSB- 26,29,30 -RSB- . Intracellular acidification caused by the hPAT1 substrate β-alanine selectively activated Na + / H + exchange by NHE3 -LSB- 26 -RSB- . Like H + - coupled dipeptide uptake , H + - coupled amino acid uptake into Caco-2 cells is inhibited by forskolin , S1611 and VIP in a Na + and pH-dependent manner via inhibition of NHE3 -LSB- 26,29,30 -RSB- . Uptake of the hPAT1 substrate β-alanine -LSB- 16 -RSB- was measured across the apical membrane of Caco-2 cell monolayers at apical pH 6.5 for 15 min -LRB- Fig. 6 -RRB- . Caffeine -LRB- 5 mM -RRB- reduced β-alanine uptake in the presence -LRB- p < 0.001 -RRB- but not the absence of extracellular Na + -LRB- p > 0.05 -RRB- suggesting that H + - coupled amino acid uptake via hPAT1 is also modulated indirectly through regulation of NHE3 . 4 Discussion The di/tripeptide transporter hPepT1 acts as a high-capacity route for solutes across the first barrier to oral-bioavailability , the brush-border membrane of the small intestine . Many , orally-active peptidomimetics and amino acid-conjugated pro-drugs have been identified as hPepT1 substrates -LSB- 3,4 -RSB- . There is an increasing number of examples of physiological regulation -LRB- hormonal , neural , paracrine -RRB- of hPepT1 and of regulation of hPepT1 in certain disease states and after surgery -LRB- reviewed by -LSB- 14 -RSB- -RRB- . Another , less studied , factor which may affect the degree to which drugs are absorbed across the small intestinal epithelium is interaction with co-administered drugs or components of diet . Exposure of Caco-2 cell monolayers to the hPepT1 substrate Gly -- Gln for 4 days resulted in a subsequent increase in capacity for dipeptide uptake and in hPepT1 expression -LSB- 31 -RSB- . Another study found that an array of flavonoids , which are found ubiquitously in foods of plant origin , either inhibit , have no effect or increase the hPepT1-mediated uptake of the antibiotic cefixime into Caco-2 cell monolayers -LSB- 32 -RSB- . In this study we identify that incubation of human intestinal epithelial cells with either dietary or orally-active therapeutic phosphodiesterase inhibitors reduces Gly -- Sar uptake through a reduction in hPepT1 capacity . The data presented here show that the inhibition of Gly -- Sar uptake by phosphodiesterase inhibitors is both Na + - and pH-dependent -LRB- Figs. 1 and 2 -RRB- suggesting that inhibition is not a direct effect on hPepT1 but rather through NHE3 . When NHE3 is inhibited -LRB- e.g. by the removal of extracellular Na + or by addition of S1611 -RRB- the cells are no longer able to maintain pHi during solute-induced acidification and , therefore , the driving force -LRB- the transmembrane H + electrochemical gradient -RRB- for further dipeptide uptake is reduced . Previously , we have shown that hPepT1 can be inhibited by other factors which are known to increase cAMP in intestinal epithelial cells such as the enteric neuropeptides VIP and PACAP -LSB- 18 -RSB- . Although caffeine , theophylline and pentoxifylline can elicit effects through pathways other than increasing cAMP , a number of factors suggest that they are acting here as phosphodiesterase inhibitors . Firstly , incubating Caco-2 cell monolayers with all three compounds produced an increase in -LSB- cAMP -RSB- i . The increase is relatively small compared to that produced by forskolin . However , this could be due to the cAMP signal being compartmentalised -LRB- as demonstrated in cardiomyocytes -LSB- 33 -RSB- -RRB- and thus the local change may be much greater than the measured , global change . Secondly , phosphodiesterases can also regulate the levels of cGMP but none of the compounds tested here produced a significant change in -LSB- cGMP -RSB- i -LRB- data not shown -RRB- . Lastly , the concentrations of caffeine , theophylline and pentoxifylline required to produce inhibition of hPepT1 -LRB- IC50 = 1.0 , 0.6 and 0.5 mM , respectively , Fig. 3 -RRB- are much closer to those for inhibition of phosphodiesterases than for other effects such as antagonism of adenosine receptors -LRB- IC50 ≈ 2 -- 80 μM -RRB- and release of intracellular calcium -LRB- EC50 ≈ 5 -- 20 mM -RRB- -LSB- 20 -- 22,34 -- 36 -RSB- . Theophylline -LRB- 100 μM -RRB- has been shown to abolish increased glucose absorption resulting from activation of A2 receptors by luminal adenosine -LRB- 5 mM -RRB- in mouse small intestine in vivo -LSB- 37 -RSB- . However , in addition to the significantly higher affinity for theophylline , A2 receptors are positively coupled to adenylate cyclase so antagonism of the receptor should cause a decrease in -LSB- cAMP -RSB- i and not the increase noted here -LSB- 22,37 -RSB- . Caffeine -LRB- and to a lesser extent theophylline -RRB- is found in high levels in many beverages either naturally or by addition . For example , the average cup of coffee contains 85 mg of caffeine -LSB- 38 -RSB- . Pentoxifylline is given orally as 400 mg tablets to treat intermittent claudication as part of peripheral arterial disease -LSB- 39 -RSB- . Although it is difficult to predict the actual concentration at the surface of the small intestinal epithelium it is conceivable that caffeine and pentoxifylline may reach concentrations high enough to elicit an effect on hPepT1 similar to that measured here using Caco-2 cell monolayers . There is evidence that caffeine given orally in doses equivalent to diet can elicit changes in small intestinal function . A study using human volunteers found that ingestion or intraluminal jejunal perfusion of caffeine -LRB- 75 -- 300 mg -RRB- caused a rapid reversal of net fluid absorption to net secretion -LSB- 40 -RSB- . Although the cell signalling pathway by which caffeine stimulated fluid secretion was not investigated , this study does indicate that the concentrations of caffeine used here in vitro may be sufficient to cause changes in the small intestinal epithelium in vivo . Since the initial reports of phosphodiesterase activity it has become evident that there are many types of phosphodiesterase . Currently there are 11 different PDE families each of which can contain several isoforms and splice variants -LSB- 24,41 -RSB- . Many of these isoforms are tissue or cell-specific and recent studies have shown that they can be expressed in distinct sub-cellular locations where they act to limit site , amplitude and duration of cyclic nucleotide signals -LSB- 24,33 -RSB- . Little is known about which PDE isoforms are found in the small intestinal epithelium . Studies reporting tissue distribution of PDE mRNA expression often include a small intestinal sample but generally it is not clear whether the PDE is expressed specifically in enterocytes . A single study of PDE3 isoforms identified that PDE3B but not PDE3A mRNA was highly abundant in the epithelium and underlying smooth muscle layers of developing small intestine of rat -LSB- 42 -RSB- . Caffeine , theophylline , IBMX and pentoxifylline generally inhibit all PDE isoforms with roughly equal efficacy -LSB- 25,43,44 -RSB- . However , since many PDE isoforms and variants are located in distinctive cell and sub-cellular locations they are now considered to be good targets for specific therapeutic agents . Often the resulting drugs are given orally , for example , the PDE5 inhibitor sildenafil used to treat erectile dysfunction -LSB- 45 -RSB- . PDE5 is expressed at the mRNA level in the small intestine -LSB- 46,47 -RSB- . Further work is required to identify which PDE isoforms are expressed in small intestinal epithelial cells both along the proximal -- distal and crypt -- villus axes and which isoforms play a functional role in regulating solute transport at the brush-border membrane . In conclusion , we have shown that non-nutrient components of diet and orally-delivered therapeutic agents both regulate activity of the nutrient and drug transporter hPepT1 . This regulation is an indirect effect through inhibition of apical NHE3 function , which acts as a pH homeostatic mechanism during H + - coupled dipeptide transport . Functional cooperativity between hPepT1 and NHE3 results not only in the absorption of di/tripeptides but also stimulation of Na + absorption -LSB- 8 -- 10 -RSB- . The H + - coupled amino acid transporter hPAT1 -LRB- SLC36A1 -RRB- is regulated by caffeine in a similar , indirect manner to hPepT1 -LRB- Fig. 6 -RRB- . Thus , through modulation of a single homeostatic mechanism -LRB- NHE3 -RRB- , a luminal compound may influence several different absorptive mechanisms . The results presented here suggest that any regulation of NHE3 activity by luminal contents , co-administered drugs or by neurohormonal signals may have significant impact on the general absorptive state of the cell . Any other nutrient transporter dependent on the maintenance of the H + electrochemical gradient for function may be regulated in a similar manner .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

pept1
na+/h+ exchange
intestinal absorption
drug transport