Original article

L.M. LICHTENBERGER, T. PHAN, D. FANG, S. EDLER, J. PHILIP, T. LI-GENG, E.J. DIAL

BIOAVAILABILITY OF ASPIRIN IN RATS COMPARING THE DRUG’S UPTAKE INTO GASTROINTESTINAL TISSUE AND VASCULAR AND LYMPHATIC SYSTEMS: IMPLICATIONS ON ASPIRIN’S CHEMOPREVENTIVE ACTION

Department of Integrative Biology and Pharmacology,
The University of Texas Health Science Center at Houston - McGovern Medical School, Houston, TX, USA
Aspirin is an effective analgesic and antiplatelet drug that in addition to its ability to reduce pain, inflammation and fever, appears to have efficacy in the prevention/treatment of a range of diseases including heart disease, numerous cancers and Alzheimer’s. It is important to understand the bioavailability of aspirin and its major metabolite, salicylic acid, since dosage and route of administration can vary for treating differing diseases, and the major side-effects of aspirin, upper gastrointestinal ulceration and bleeding, are dose-dependent. We examined the time course for gastroduodenal uptake of aspirin and the appearance of its major metabolite salicylic acid in blood and lymph after intragastric (to simulate oral) and intraduodenal (to simulate enteric-coating) dosing in rats. Results show that after intragastric dosing, intact aspirin is absorbed primarily by the gastric mucosa and to a lesser extent by the duodenal mucosa. When aspirin is dosed intragastrically or intraduodenally, a much greater concentration of aspirin enters the lymph than the blood. In contrast, the concentration of salicylic acid was higher in blood than in lymph. Lymph levels of both aspirin and salicylic acid were sufficiently high so as to perform a pharmacologic function there, possibly as a chemopreventive agent against colon cancer and potentially the metastatic spread of non-gastrointestinal cancers.
Key words:
aspirin, salicylic acid, lymph, bioavailability, thoracic duct, portal vein, mesenteric lymphatic

INTRODUCTION

Aspirin (ASA) is an effective anti-inflammatory drug that is used clinically for its ability to reduce pain, inflammation and fever due to its action to inhibit both cyclooxygenase (COX)-1 and (at high concentrations) COX-2 and the resulting inhibition of prostaglandin formation (1). ASA also has found uses in preventing cardiovascular disorders such as stroke due to its antiplatelet (COX-1) effect, and in the chemoprevention of certain cancers such as colon (2-10) and breast cancer (11) where the mechanism may involve COX-2 or other mechanisms yet to be fully explained. As more uses for ASA become known (i.e., Alzheimer’s, diabetes) (12, 13), it is important to understand what dose is needed for a particular effect, as the primary side-effect of ASA is a dose-related gastrointestinal (GI) bleeding that can lead to serious morbidity and mortality in susceptible individuals (14). It is thought that low-dose ASA (75 – 325 mg) is effective as an antiplatelet drug due to its ability to irreversibly inhibit platelet COX-1 activity (1). Larger doses of ASA (650 – 2600 mg) are used for COX-2 inhibition and pain relief. After oral ingestion in both man and animals, ASA is quickly absorbed and then metabolized within 15 – 20 minutes to its major metabolite, salicylic acid (SAL) which has negligible anti-platelet activity and is a non-selective COX inhibitor, primarily targeting COX-2. This action may in part, together with its ability to block the NFκ-β activation pathway – a pathway that may be mediated by AMP-kinase (AMPK) as discussed subsequently, may contribute to SAL’s modest anti-inflammatory and anti-carcinogenic activity (15-17). It is intriguing that a drug that is only present for a few minutes in the circulation can have such a major effect on platelets. The explanation for this action is thought to be that because ASA acts rapidly and irreversibly in inhibiting COX-1, any exposure in the systemic circulation may be sufficient to inhibit enough platelets to have an effect. We were interested in whether the site of GI absorption, either the stomach or duodenum, had a measureable effect on circulatory bioavailability. A direct comparison of these routes of administration has not been reported in rats, even though most over-the-counter (OTC) formulations of ASA are enteric-coated to avoid gastric absorption and possible gastric mucosal irritation. In the past, rat studies of ASA bioavailability were performed on oral (18, 19) or intravenous (20-22) routes of administration.

In addition to the blood circulatory system, the lymphatic system is also available for transporting body fluids. The lymph fluid is a collection of excess interstitial fluid and can carry lipids and also lipophilic drugs as they are absorbed from the GI tract. Lymph from the GI tract can have a high level of fats (triglycerides) that are absorbed from dietary constituents. We speculate that since protonated ASA is a lipophilic substance at acidic gastric pH (pKa ≤ 3.5) and is absorbed quickly from the stomach, it may, in part, be taken up into the lymph and by-pass the liver, allowing it to be protected from immediate hepatic degradation (first-pass effect) and conversion to salicylic acid. In the current study we investigated whether ASA and/or SAL enter the lymphatic circulation following either intragastric or intraduodenal dosing, and how their levels change over time.

MATERIALS AND METHODS

Animal studies were performed with adult male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) that were maintained in the FDA-approved institutional Clinical Laboratory for Animal Medicine and Care. All animal protocols were approved by the institutional Animal Welfare Committee which complies with AALAC and PHS regulations. Rats were fasted overnight to ensure an empty stomach and intestines.

In the first study, the blood and GI tissue levels of ASA and SAL were determined for the first 40 minutes after oral dosing. Conscious rats were dosed intragastrically with ASA at 100 mg/kg and were euthanized after 5, 10, 20 and 40 minutes. This dose of ASA was chosen to be sufficiently high so as to be able to detect circulating ASA. In accordance with US Food and Drug Administration guidance for industry, this dose is approximately equivalent to about one gram of aspirin in a human, or three 325 mg ASA tablets (animal dose in mg/kg divided by 6.2 for rat multiplied by 60 kg for average human weight = 100/6.2 × 60 = 968 mg). A one gram dose has been used in numerous clinical studies (23, 24). Samples of blood from the inferior vena cava, and tissue from the gastric body, proximal duodenum (first 2.5 cm) and distal duodenum were collected for extraction and analysis of ASA and SAL by high-performance liquid chromatography (HPLC). For tissue, after first rinsing to remove GI contents, one part by weight was homogenized in three parts of acetonitrile (HPLC grade). For blood, one part by volume was mixed with three parts of acetonitrile. Samples were centrifuged to pellet particulate matter, and the supernatant was applied to the HPLC instrument.

To estimate bioavailability of aspirin and salicylic acid in blood, gastric and duodenal tissue, calculations of area under the curve (AUC) were made by the method of linear interpolation using the trapezoidal rule. Values for blood and tissue concentrations of aspirin and salicylic acid at 0 to 40 minutes were used.

In the second study, the blood and lymph levels of ASA and SAL were determined for the first 40 minutes after oral dosing. Initially, conscious rats were dosed intragastrically with 1.5 ml of corn oil at 30 minutes prior to surgery, to promote consistent lymph flow. Then the animals were anesthetized with isoflurane for the duration of the experiment. To collect blood, the femoral vein was cannulated by a standard procedure using polyethylene PE-50 tubing (BD-Intramedic-Clay Adams) which was previously flushed with heparin. To collect lymph from the gastric region, the thoracic duct was cannulated by the following method. After a midline incision the thoracic duct was located underneath the aorta and cannulated by a PE-50 tubing catheter of 12 cm length adapted with a silastic tip of 4.5 cm length to give it flexibility and limit mechanical injury to the vessel wall. The PE tubing was then run outside at the lower dorsal wall and attached to a 1 ml syringe via a 16GA-1 BD-needle. Once cannulated, all the tracking gauzes were gently removed, the organs were returned to normal conditions, and fluid and temperature levels were maintained under anesthesia. Baseline lymph was collected for 30 min after which time, blood was collected and then test drugs (vehicle or aspirin) were administered. The animals were dosed intragastrically with ASA at 100 mg/kg and collections of lymph were made at 0 – 10, 10 – 20 and 20 – 40 minutes, with femoral blood collected at 10, 20 and 40 minutes. Blood was collected through a heparinized cannula, immediately centrifuged, and plasma was collected. Samples of plasma and lymph (1 part) were extracted with 3 parts acetonitrile, followed by centrifugation to remove particulate matter prior to HPLC analysis. A separate sample of blood was collected without heparin, allowed to clot, and the serum was used for thromboxane B2 (TXB2) analysis as a measure of aspirin activity on platelet COX-1. Lymph was also analyzed for TXB2.

In the third study, times up to 120 minutes after oral dosing were examined to determine the extended levels of SAL in blood and lymph. The thoracic or mesenteric lymph ducts were cannulated for collection of free flowing lymph from the gastric or intestinal regions, respectively, at 30 minute periods for up to 120 minutes. For intestinal mesenteric lymph collection, the preparation was similar to that described above for thoracic duct cannulation. There are two intestinal mesenteric lymph ducts which run along both sides of the superior mesenteric artery (SMA) toward the thoracic main pool. We chose either the left or right duct, based upon the most favorable position for the catheter and carefully dissected the duct’s covered membranes by using a fine tip forceps to isolate an open spot for cannulation. Then the surgical plate was turned to expose the target to a vertical position so the catheter would be inserted toward the duodenum. After successful cannulation, where the lymph fluid could be observed moving out into the catheter, a drop of super glue was applied on top of the insertion spot to hold the cannula in place which was attached to a 1 ml syringe for collection. The femoral vein was cannulated for limited systemic blood collections at the same 30 minute intervals. After a control (zero) time collection, rats were dosed intragastrically with ASA at 100 mg/kg and collections of blood and lymph commenced. Analysis of blood and lymph were as before.

In the fourth study, the mesenteric lymph duct and femoral vein were cannulated in anesthetized rats as before, but the ASA was inoculated directly into the duodenal lumen by catheter. Samples of blood and lymph were collected at 15 and 30 minute intervals for up to 120 minutes. Analysis of blood and lymph were as before. A separate group of animals, similarly cannulated, was used for analysis of TXB2 in blood and lymph at times of 0, 10, 20, and 40 minutes after ASA dosing, for comparison with study 2.

In the fifth study, the mesenteric lymph duct was again cannulated and this time the portal vein was cannulated to provide a blood sample that was collected prior to hepatic exposure and degradation. The ASA was inoculated into the duodenum. Samples of blood and lymph were collected at 10 minute intervals up to 40 minutes. Analysis of blood and lymph were as before.

The samples of blood, lymph and tissue were analyzed by HPLC (Waters Corp.) for their ASA and SAL content compared to standards. Briefly, the protocol for HPLC consisted of injection of 10 µl samples with a Waters Model 2707 autosampler on an Agilent Zorbax 300 SBC18 column (at 45°C) with a Waters Model 2489 UV detector at 230 nm. The elution of the aspirin and salicylic acid was accomplished using an isocratic mixture of 50 mM phosphoric acid/acetonitrile (80%/20% initially), followed by a 20%/80% flush cycle.

Measurement of thromboxane in the serum and lymph was accomplished with a thromboxane B2 EIA kit purchased from Cayman Chemical.

Aspirin used in these studies for oral or duodenal administration was the store brand purchased from Walgreen’s Pharmacy and was prepared by crushing a tablet with mortar and pestle to a uniform particle size and resuspending in water to produce a rapid-release formulation.

Statistical analysis

Statistical analysis comparing blood versus lymph was performed with the Student t test. ANOVA was used in comparing like tissues over time, along with the Tukey HSD test. A value of P < 0.05 was set for significance. Mean values for each group were graphed ± error bars with significant differences noted with an asterisk or dagger symbol.

RESULTS

Study 1

The blood levels of ASA in Fig. 1A are consistent with prior reports (19, 21) that an intragastrically administered dose of ASA is rapidly absorbed and enters systemic circulation in the rat. The process of ASA uptake into femoral vein blood is effectively over by 30 minutes after dosing. It should be noted that only a small fraction (< 2%) of the drug is detectable in systemic circulation 5 – 10 min after intragastric administration, assuming the blood volume of the rat is equivalent to a ratio of 6.4 ml/100 g (25). Also, the SAL levels were increasing during the 40 minute study period, did not decline throughout the 40 minute study period and were consistently higher than the corresponding ASA levels at all time points. This preponderance of SAL over ASA in the circulation is evident from the calculation of AUC, where SAL AUC = 60.1 mg×h/ml compared to ASA AUC = 2.7 µg×h/ml.

In the same animals we verified that aspirin uptake by the gastric tissue occurs within 10 to 15 minutes. Fig. 1B shows that a considerable amount of intact ASA can be measured in the body of the stomach at 5 – 10 minutes after oral dosing with ASA, and that the concentration is near zero by 40 minutes. Gastric tissue had significantly higher ASA concentrations than SAL at the early time points (5 to 10 minutes), which is also reflected in the AUC values (ASA = 100.8 µg×h/mg; SAL = 63.4 µg×h/mg).

Both proximal and distal duodenal tissue was examined, but only the proximal is reported here because the distal values were very similar to the proximal values. Fig. 1C shows that proximal duodenal tissue had about 1/10 of the amount of ASA (AUC = 9.4 µg×h/mg) compared to gastric tissue (Fig. 1B), consistent with a smaller amount of ASA reaching the duodenum after intragastric dosing. SAL levels in duodenal tissue were significantly greater than ASA levels by about 5-fold (SAL AUC = 52.8 µg×h/mg).

Figure 1 Fig. 1. Time course of the appearance of ASA and SAL in: (A) femoral vein, (B) gastric body, and (C) proximal duodenum after intragastric dosing with ASA (100 mg/kg). N = 6/group. *P < 0.05 versus 0 time.

Study 2

This study is the first in which the thoracic lymph duct was cannulated, preventing lymph ASA and SAL from reaching the vascular circulation. The systemic blood levels of ASA peaked at 10 minutes (Fig. 2A) and were about half of that seen in non-cannulated rats (Fig. 1A). Lymph levels of ASA (Fig. 2A) were greater than in the blood, achieving about twice the concentration, and persisted for somewhat longer.

In contrast to the above pattern, SAL levels (Fig. 2B) were greater in the blood collected from the femoral vein than in the thoracic duct lymph at all time points. SAL in the blood of cannulated rats (Fig. 2B) was lower than in non-cannulated rats (Fig. 1A).

We also measured thromboxane in the serum and thoracic duct lymph (Table 1), and demonstrated a similar pattern in both fluids. Serum levels of TXB2 were reduced by 90% at 40 min following aspirin administration. Lymph levels of TXB2 were about half that in the serum, and were reduced by 75% following aspirin dosing.

Figure 2 Fig. 2. Time course of the appearance of: (A) ASA and (B) SAL in thoracic lymph and femoral vein after intragastric dosing with ASA (100 mg/kg). N = 7/group. *P < 0.05 versus 0 time blood and lymph; P < 0.05 versus blood.

Study 3

For the third study, femoral vein blood and thoracic/mesenteric duct lymph levels of SAL were compared at extended times after oral dosing of ASA. Fig. 3A and 3B show there were increasing levels of SAL in both blood and lymph, reaching a plateau around 60 minutes and persisting for at least 2 hours. Blood levels of SAL were about twice the lymph levels at most time points after oral ASA dosing, and were significantly higher at 30 minutes. The thoracic and mesenteric lymph levels of SAL were comparable, consistent with the metabolite being taken up in the gastroduodenal mucosa as shown in Fig. 1B and 1C.

Figure 3 Fig. 3. Time course of the appearance of SAL in: (A) thoracic lymph and femoral vein (N = 3/group) and (B) mesenteric lymph and femoral vein (N = 4/group) after intragastric dosing with ASA (100 mg/kg). *P < 0.05 versus 0 time blood and lymph; †P < 0.05 versus lymph.

Study 4

In the fourth study, it was decided to utilize an intestinal route of administration to simulate where enteric-coated ASA would be absorbed. Fig. 4A shows the ASA results after NSAID dosing into the duodenum. The systemic blood level of ASA is low, but measurable at 15 minutes. The level of ASA in the mesenteric lymphatic is 10 times greater at this same time point. This result shows that after duodenal exposure, ASA does get into the lymph at early time points and at substantially higher levels than the blood. By 30 minutes both the blood and lymph levels of intact aspirin are much lower, but still measureable.

Once again, we measured thromboxane levels in the blood and lymph, this time after intraduodenal aspirin dosing, and demonstrated the presence of TXB2 in both fluids. Similar levels of TXB2 in the fluids were seen, and they were both reduced about 80% by aspirin treatment (Table 1).

Table 1. Thromboxane B2 values in serum and lymph after intragastric or intraduodenal dosing with aspirin. Values are expressed as mean and standard error of the mean (S.E.) at baseline (0 time) and 10, 20 and 40 minutes after dosing. In comparison to Pre-ASA (0 min): *P < 0.05, **P < 0.01.
Table 1

Fig. 4B shows SAL levels following duodenal dosing with ASA. These SAL levels had some similarities to those after intragastric dosing (Fig. 2B), in that blood SAL was significantly greater than lymph SAL, and both were elevated at the earliest time period examined and remained high for at least two hours.

Figure 4 Fig. 4. Time course of the appearance of: (A) ASA and (B) SAL in mesenteric lymph and femoral vein after intraduodenal dosing with ASA (100 mg/kg). N = 3/group. *P < 0.05 versus 0 time blood and lymph; P < 0.05 versus lymph.

Study 5

For the fifth study after intraduodenal dosing, blood was collected from the portal vein because that is the vasculature through which the absorbed ASA would pass before entering the liver and undergoing its metabolic processing, and the ASA should be at its highest concentration there. Fig. 5A shows that portal vein ASA was indeed highest at 10 minutes (31.0 µg/ml), achieving a concentration that was much greater than in the femoral vein at a similar time point (3.15 µg/ml; Fig. 4A). Furthermore, ASA in the mesenteric lymph after duodenal dosing of the drug was significantly higher than the blood level at all time-points (Fig. 5A).

In contrast to the pattern observed for ASA, the levels of SAL in the blood from the portal vein were significantly higher than that in the lymph in response to intraduodenal ASA dosing (Fig. 5B), and were 2 – 3 fold higher than that found in the femoral vein at all the time points studied (Fig. 4B).

Figure 5 Fig. 5. Time course of the appearance of: (A) ASA and (B) SAL in mesenteric lymph and portal vein after intraduodenal dosing with ASA (100 mg/kg). N = 6/group. *P < 0.05 versus 0 time blood and lymph; P < 0.05 versus blood.

DISCUSSION

The bioavailability of ASA and SAL are important for understanding how ASA may influence a diverse range of diseases. ASA is available as an orally administered rapid-release product which we attempted to simulate by the intragastric administration of an ASA suspension. ASA is also available in an enteric-coated formulation which we simulated with direct duodenal dosing of the same ASA suspension. In regard to the site of absorption of intragastrically administered aspirin, the results of tissue analysis show that freely suspended ASA is preferentially absorbed in the stomach and to a lesser extent in the duodenum after an oral dose. The gastric body contained higher levels of ASA than the proximal duodenum, consistent with the greater amount of intact ASA there, indicating that there was significant metabolism of ASA in gastric tissue and/or fluid as ASA was being absorbed during gastro-duodenal aboral propagation. SAL levels in the duodenum were consistent with the lower levels of intact ASA in the duodenal lumen and tissue. The combination of gastric and duodenal absorption helped achieve an early ASA blood level of 25 µg/ml (Fig. 1A), which represents 1 – 2% of the administered product (calculated based on a blood volume of 16 ml for a 250 g rat: 25 µg/ml × 16 ml/25,000 µg = 0.016 or 1.6%). These novel tissue results support the use of rapid-release ASA where immediate effects of aspirin are needed.

Of potentially more interest, however, this report is the first to directly compare blood and lymph levels of ASA and SAL following ASA dosing into the gastric or duodenal lumen. After intragastric dosing, we determined that the concentration of intact ASA in thoracic duct lymph exceeded the levels detectable in systemic circulation (Fig. 2A). The findings with intraduodenally-administered ASA are similar (Fig. 4A), and relevant to the clinical situation because many of the over-the-counter (OTC) ASA drugs are formulated with an enteric coating to by-pass the stomach and promote intestinal (duodenal) absorption to protect the stomach from the mucosal erosive property of ASA. Indeed, clinical studies have shown that enteric-coated ASA is absorbed slower than rapid-release ASA (23, 26), and is associated with fewer minor gastrointestinal lesions (27), although there is evidence that the dyspeptic symptoms of the drug are not significantly reduced with enteric-coating (28). This may be due to lower gut injury caused by EC-aspirin as indicated in a recent capsule-endoscopy study (29).

Our unique finding that lymph levels of ASA were consistently greater than those in the systemic blood, when examined at early time points, is consistent for both oral and duodenal dosing. It appears that ASA administered into the stomach or duodenum is taken into the lymphatics draining the specific area of the upper GI tract (thoracic duct-stomach; mesenteric lymphatics-duodenum) at higher levels than the blood, achieving at least a 4- to 10-fold greater concentration than the systemic circulation and could contribute a pharmacologic function there (in the lymphatics). As well, ASA by entering the lymphatic system can circumvent first-pass hepatic metabolism of the drug which in turn will drain into the vascular system and help maintain a greater blood level of ASA. This effect of circumvention is evident in the difference between the blood levels of ASA in the cannulated and non-cannulated animals (Fig. 2A versus Fig. 1A), where the cannulated lymph animals had about 70% lower blood levels of ASA. The possible lymphatic shunting of aspirin to circumvent first-pass hepatic metabolism is schematically depicted in Fig. 6.

Figure 6 Fig. 6. Schematic depiction of aspirin and salicylic acid transport in the lymphatic vasculature system from the stomach after intragastric dosing (partitioning into the gastric lymphatics which drain into the thoracic duct) and duodenum after intraduodenal dosing (partitioning into the mesenteric lymphatics). Subsequently, lymphatic contents drain into the blood. Aspirin and salicylic acid are also taken up into the venous vascular system and portal vein leading to the liver, followed by release of metabolic products through the hepatic vein into the general circulation.

The question of whether intact ASA is present in lymph and at what concentration may have practical clinical implications. Many cancer cells are known to enter the lymphatic system and use that circulatory pathway as a means to metastasize. ASA has been recommended for prevention of certain solid tumor cancers, including colorectal cancer (30), although its mechanism is not entirely clear. We speculate that one way ASA may promote chemoprevention is by an action in lymph to suppress metastatic spread of cancer cells. The finding here that mesenteric lymph contains significant ASA, added to the fact that colon cancer cells may metastasize through the mesenteric lymph, support the concept that ASA may be exerting a chemopreventive effect by virtue of the drug being taken up by the thoracic duct/mesenteric lymphatics. The exact nature of the chemopreventive effect is not known, but could be related to direct effects on cancer cells such as induction of apoptosis and inhibition of proliferation. ASA could also be working on the lymphatic endothelium to control its dilation which would affect passage of cancer cells (31, 32). Lastly, it is possible that aspirin may target and inhibit the activity of platelets present in the systemic blood and lymphatic vasculature. This is a particularly intriguing possibility, as platelets have been demonstrated to promote cancer cell growth and metastatic activity, by directly stimulating epithelial-mesenchymal-transition (EMT) of pre-malignant cells (33). Following up on this possibility, we have evidence that aspirin dosing will inhibit the thromboxane levels in both thoracic duct and mesenteric lymph (Table 1). Until recently, the source of thromboxane was thought to be from platelets. However, the source of this lymph thromboxane is not known, and could originate from platelets outside of the lymphatics or inside the lymphatics such as from lymphovenous hemostasis (34), or from nonplatelet (endothelial) cells (35) that may be disturbed by surgery and/or cannulation. Regardless of the source of thromboxane in the lymphatic vasculature, it is clearly sensitive to aspirin inhibition in a time-dependent manner. This finding is consistent with the systemic effect of aspirin on COX-1 inhibition and apparently applies to both the vascular and lymphatic circulations.

In support of the possible role for ASA on lymphatics, in preclinical studies ASA was reported to inhibit metastasis to regional lymph nodes in a mouse model of lung cancer (36). In addition, it is well established that drugs that can be transported through the GI lymphatic system can avoid first-pass hepatic metabolism (37). Thus, considerable efforts during the past decade have been focused on targeting of lymphatic transport for uptake of engineered liposomes and lipidic nanoparticles as a means of delivery of drugs for chemotherapeutic and other purposes (38, 39). Studies clearly show that duodenal dosing of lipid-encapsulated drugs can promote lymphatic uptake of those drugs (40-42). Because ASA is a lipophilic compound, it naturally possesses the appropriate characteristics for uptake into the lymph.

The concentration at which ASA is active in vitro is reported to be in the range of 4 to 20 µg/ml for inhibition of stimulus-induced platelet aggregation (43, 44), and 180 µg/ml and higher for direct anti-cancer effects (45). This level is similar to the range of the 3 µg/ml found in our rat study after duodenal drug dosing and 9 µg/ml after oral administration of a comparable dose. In addition, our finding that portal vein levels of ASA after duodenal dosing are greater (ranging between 25 – 30 µg/ml) than femoral vein levels, supports even greater exposure of blood to sufficient levels of intact ASA to allow for platelet inhibition. Our findings in the rat are consistent with results of clinical studies, where enteric coating has been shown to delay absorption of intact ASA compared to rapid-release ASA (23, 26, 46), and is associated with a reduced antiplatelet effect (28).

Lymph levels of ASA are of particular interest as they may be sufficient to have an anti-cancer action. Our studies showed both thoracic duct and mesenteric lymph levels of 18 – 46 µg/ml after intragastric and intraduodenal dosing, respectively (Figs. 2A, 4A and 5A). These concentrations are at the low end of that required for direct anti-cancer effects, as mentioned above, but we only tested a single dose of ASA. It should be noted that a higher dose of ASA at 100 mg/kg, bid, has been used successfully to block cancer metastatic spread in at least one lymphatic-related cancer model (36).

It is also of interest to consider the levels of SAL in blood and lymph, as it may be contributing to some of the same effects as ASA, particularly in the anti-cancer area. The SAL concentration was consistently greater in the blood (femoral and portal) than in the lymph at all time points after dosing with ASA, whether by oral or duodenal administration. However, the levels of SAL in the lymph were substantial, and both blood and lymph levels remained elevated for at least two hours (the longest time point examined). The concentrations of SAL achieved in either fluid were within the range that is reported to possess in vitro activity, such as recent reports showing that SAL possesses the same or better activity as ASA to decrease cancer cell viability (47), reduce the oncometabolite 2-hydroxyglutarate (48), downregulate cyclin A2/CDK2 proteins (49), and decrease c-Myc protein levels in multiple cancer cell lines (50). It also should be noted that SAL’s chemopreventive action, may in part be attributable to the aspirin metabolite activating cAMP-activated protein kinase (AMP-kinase) a known tumor suppressor and inhibitor of inflammation - whose expression and/or activity is reduced in a number of cancers (51).

In summary, our studies show that ASA can be absorbed from the stomach or duodenum, depending on its formulation, and result in significant concentrations of active ASA and SAL in the blood and lymph. These combined salicylate levels in the lymph may help explain, at least in part, the remarkable chemopreventive actions of ASA.

Authorship contributions: Lichtenberger, Phan, Edler, Philip, Li-Geng and Dial participated in research design; Phan, Edler, Philip, Fang and Li-Geng conducted experiments; Phan, Edler, Philip, Fang, Li-Geng and Dial performed data analysis; Lichtenberger, Li-Geng, Edler and Dial wrote or contributed to the writing of the manuscript.

Acknowledgments: This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grants T35 007676, P30 056338].

Conflict of interests: Dr. Lenard Lichtenberger is a shareholder in PLx Pharma Inc.

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R e c e i v e d : August 2, 2016
A c c e p t e d : October 31, 2016
Author’s address: Prof. Lenard Lichtenberger, Department of Integrative Biology and Pharmacology, McGovern Medical School, 6431 Fannin, Houston TX 77030, USA. e-mail: lenard.m.lichtenberger@uth.tmc.edu