INTRODUCTION

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Heparin is a highly sulfated, complex glycosaminoglycan that has been used clinically as an anticoagulant for over 50 yr (1). The biologic actions of heparin result from its polydispersity, heterogenous molecular organization, and ability to interact with various proteins to cause their activation, deactivation, or stabilization (2, 3). Some of these interactions, such as the binding of heparin to antithrombin III, are known to take place at specific oligosaccharide sequences within the heparin polymer (4), and various other nonanticoagulant interactions are also suspected of being highly specific (5). The nonanticoagulant actions of heparin include interaction with various growth factors (5), modulation of cellular proliferation (8, 9), and regulation of angiogenesis (10). Heparin also modulates various proteases and enzymes (11, 12), has significant antiinflammatory properties (13, 14), and regulates inflammatory-cell functions (15).

Recently, inhaled heparin has also been shown to have antiallergic activity (18), and to prevent antigen-induced airway hyperresponsiveness (AHR) in allergic sheep (23). AHR plays a central role in the pathophysiology of asthma (24), and antiallergic as well as antiinflammatory agents have been shown to attenuate AHR (25). Since the biologic actions of heparin depend on its molecular weight (26), it is possible that low-molecular-weight heparins have greater potency in inhibiting allergic bronchoconstriction and postantigen AHR than do higher-molecular-weight heparins. We have recently shown that antiallergic activity of fractionated heparins is indeed molecular-weight dependent, and observed an inverse relationship between heparin molecular weight and antiallergic activity (27). Although unfractionated heparin (UF-heparin) and fractionated low-molecular-weight heparins prevent postantigen AHR in a molecular-weight-dependent manner when given before antigen-challenge, it is not known whether these agents would be effective after antigen challenge. We therefore studied the effects of UF-heparin and three low-molecular-weight heparin fractions (medium-molecular-weight heparin [MMWH]; low molecular-weight heparin [LMWH]; and ultralow-molecular-weight heparin [ULMWH]) on postantigen AHR when these heparin fractions were administered after the antigen challenge.

	METHODS

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Animal Preparation

Twenty-eight adult sheep (mean weight: 30 kg; range: 26 to 33 kg) were included in the study. All sheep were allergic to Ascaris suum antigen and had previously been shown to develop only acute bronchoconstriction following inhalation challenge with this antigen. Inhaled heparin has previously been shown to prevent antigen-induced AHR, which is observed at 2 h after challenge in acutely responding sheep (23).

Measurement of Airway Mechanics

The method used in the study for measuring airway mechanics has been described previously (18, 23). The unsedated sheep were restrained in a cart in the prone position, with their heads immobilized. After topical anesthesia of the nasal passages with 2% lidocaine solution, a balloon catheter was advanced through one nostril into the lower esophagus. The animals were intubated with a cuffed endotracheal tube through the other nostril, using a flexible fiberoptic bronchoscope as a guide. The cuff of the endotracheal tube was inflated only for the measurement of airway mechanics and during aerosol challenge to prevent undue discomfort. Pleural pressure was estimated with the esophageal balloon catheter (filled with 1 ml of air), which was positioned 5 to 10 cm from the gastroesophageal junction. In this position the end-expiratory pleural pressure ranges from 2 to 5 cm H₂O. Once the balloon was placed, it was secured so that it remained in position for the duration of the experiment. Lateral pressure in the trachea was measured with a sidehole catheter (ID: 2.5 mm) advanced through the positioned distal to the tip of the endotracheal tube. Transpulmonary pressure (the difference between tracheal and pleural pressure) was measured with a differential-pressure-transducer-catheter system that showed no phase shift between pressure and flow up to a frequency of 9 Hz (mp 45; Validyne, Northridge, CA). For the measurement of pulmonary resistance (RL), the proximal end of the endotracheal tube was connected to a pneumotachograph (Fleisch No. 1; Dyna Sciences, Blue Bell, PA). The signals of flow and transpulmonary pressure were recorded with a multichannel recorder, which was linked to an 80.386 DOS personal computer for on-line calculation of RL from transpulmonary pressure and flow at isovolume points (respiratory volume was obtained by digital integration). Analysis of at least seven breaths (free from swallowing artifact) was used for the determination of RL. Data were expressed as specific lung resistance (SRL), defined as RL × thoracic gas volume.

Thoracic Gas Volume

Thoracic gas volume was measured with a body plethysmographic technique (18, 23). The endotracheal tube was connected to a solenoid valve that could be activated from outside the plethysmograph. The plethysmographic pressure and lateral mouth pressure measured between the proximal end of the endotracheal tube and the solenoid valve were measured with a differential gauge (mp 45; Validyne) and strain gauge (Statham Instruments, Hato Rey, PR), respectively, and the readings were displayed on an X-Y oscilloscope provided with a template. The plethysmographic pressure was calibrated manually with a 30-ml syringe at a rate similar to the sheep's spontaneous breathing frequency. After the animal had been enclosed in the plethysmograph, 1 to 2 min were allowed for stabilization of the plethysmographic pressure. The solenoid valve was activated at end expiration, and the slope of the first respiratory cycle against the closed airway was taken for the determination of thoracic gas volume, because subsequent efforts usually produced unsatisfactory slopes caused by the animal straining against the occluded airway. The mean of three measurements was recorded.

Aerosol Delivery System

Aerosols were generated with a disposable medical nebulizer (Raindrop; Puritan Bennett, Lenexa, KS), which produces an aerosol with a mass median aerodynamic diameter (MMAD) of 3.2 µm (geometric SD = 1.9 µm) as determined with a seven-stage Andersen cascade impactor. The output from the nebulizer was directed into a plastic T-piece, which was interconnected between the Harvard animal respirator (Harvard Apparatus, Dover, MA) and the endotracheal tube. To control the aerosol delivery, a dosimeter system was used, consisting of a solenoid valve and a source of compressed air (20 psi), which was activated for 1 s at the beginning of the inspiratory cycle of the Harvard respirator system. All aerosols were delivered at a tidal volume (VT) of 500 ml and a rate of 20 breaths/min.

Bronchoalveolar Lavage

The distal tip of a specially designed 80-cm fiberoptic bronchoscope was wedged into a randomly selected subsegmental bronchus. Lung lavage was performed by the infusion and gentle aspiration of 30-ml aliquots of phosphate-buffered saline (PBS) (pH, 7.4) at 39° C, using 30-ml syringes attached to the working channel of the bronchoscope. The effluent was filtered through a single layer of gauze and placed immediately on ice. The volume of the effluent collected from the bronchoalveolar lavage (BAL) was measured and centrifuged at 420 g for 15 min at 4° C. The supernatant was decanted and centrifuged again at 1,000 × g at 4° C for 15 min. The supernatant was frozen at 80° C for subsequent histamine analysis.

Histamine Radioimmunoassay

Duplicate aliquots from each BAL fluid (BALF) sample were used for histamine radioimmunoassay (RIA), which was done with a commercial kit from Immunotech International (AMA Inc., Westbrook, ME). The sensitivity of the assay is 0.05 to 2.0 nM, and the coefficient of variation (CV) is < 10%. There is less than 0.01% cross-reactivity with histidine, serotonin, or t-methyl histamine (28).

Agents

Ascaris suum extract (Greer Diagnostics, Lenoir, NC) was diluted with buffered saline to a final concentration of 82,000 protein nitrogen units/ml and delivered as an aerosol over a period of 20 min (400 breaths). The dose of antigen delivered was kept constant for all animals, in all antigen experiments. Carbachol (Sigma Chemical Co., St. Louis, MO) was dissolved in PBS for nebulization. UF-heparin (heparin sodium 20,000 USP U/ml; Lyphomed, Deerfield, IL) and various fractionated heparins were derived from porcine intestinal mucosa. MMWH (KABI-2165, mol wt = 5,030 daltons) was obtained from Pharmacia AB (Stockholm, Sweden), and both LMWH (CY216, mol wt = 4,270 daltons) and ULMWH (CY222, mol wt = 2,355 daltons) were obtained from Sanofi Pharma (Gentilly, Cedex, France). UF-heparin and various fractionated heparins were dissolved in 3 ml of bacteriostatic water for injection and were administered as aerosols over periods of 15 to 20 min. The molecular-weight distribution and pharmacologic characteristics of the heparin fractions used in the study are shown in Table 1.

Experimental Protocol

For every protocol, each animal was studied on three different experiment days. On experiment Day 1, baseline bronchial reactivity to carbachol was determined, whereas on experiment Days 2 and 3 (at least 2 wk apart from one another) the bronchial reactivity to carbachol was redetermined 2 h after the antigen challenge, without or after treatment with UF-heparin and different doses of fractionated heparins. UF-heparin and fractionated heparins were administered immediately after the postantigen measurements of SRL. Group I (n = 4) was treated with UF-heparin (1,000 U/kg); group II (n = 4) was treated with MMWH (KABI-2165, 5 mg/kg); group III (n = 6) was treated with LMWH (CY216, 1.25 mg/kg); and group IV (n = 6) was treated with ULMWH (CY222, 0.04 mg/kg to 0.62 mg/kg). Pretreatment with inhaled UF-heparin, KABI-2165, CY216, and CY222 has previously been shown to prevent antigen-induced bronchoconstriction and AHR (23, 27).

Bronchial reactivity to carbachol. To assess baseline airway responsiveness, cumulative dose-response curves for inhaled carbachol were generated on experiment Day 1 by measuring SRL before and after inhalation of buffered saline, and after each administration of 10 breaths of increasing concentrations of carbachol (0.25%, 0.5%, 1.0%, 2.0%, 3.0%, and 4.0% wt/vol solutions). The bronchoprovocation was discontinued when SRL increased to 400% above the baseline value. The cumulative provocative dose (PD₄₀₀) of carbachol (in breath units) that increased SRL to 400% above baseline was calculated. One breath unit was defined as 1 breath of a 1% carbachol solution. Baseline dose-response curves for carbachol were generated for all sheep at least 2 wk after their last exposure to antigen.

Modification of postantigen AHR by UF-heparin. For the control antigen experiments after baseline measurements of SRL, the sheep in all four study groups were challenged with aerosolized Ascaris suum antigen, and measurements of SRL were repeated within 5 min after challenge. Two hours after challenge, when SRL had returned to its baseline value, a carbachol dose-response curve was generated to determine the postantigen value of PD₄₀₀ as an index of antigen-induced AHR. In order to evaluate the effect of UF-heparin on antigen-induced AHR, this protocol was repeated 2 wk later in group I (n = 4), after the sheep were treated with aerosolized UF-heparin (1,000 U/kg), which was dissolved in 3 ml of bacteriostatic water for injection and administered as an aerosol immediately after the antigen challenge.

Modification of postantigen AHR by fractionated heparins. The effect of each dose of various fractionated low-molecular-weight heparins was studied on different experiment days. There was at least a 2-wk interval between the treatments.

1. Effect of MMWH KABI-2165 (n = 4). On each experiment day, a standard antigen challenge was performed and the PD₄₀₀ of carbachol was determined 2 h after antigen challenge, either without or after treatment with aerosolized KABI-2165 (5 mg/kg) dissolved in 3 ml of bacteriostatic water for injection and administered as an aerosol immediately after the antigen challenge.
2. Effect of LMWH CY216 (n = 6). On each experiment day, a standard antigen challenge was performed and the PD₄₀₀ of carbachol was determined 2 h after antigen challenge, either without or after treatment with aerosolized CY216. CY216 (1.25 mg/kg) was dissolved in 3 ml of bacteriostatic water for injection and administered as an aerosol immediately after the antigen challenge.
3. Effect of ULMWH CY222 (n = 6). On each experiment day, a standard antigen challenge was performed and the PD₄₀₀ of carbachol was determined 2 h after antigen challenge, either without or after treatment with aerosolized CY222. CY222 (0.62 mg/kg; 0.31 mg/kg; 0.16 mg/kg; 0.08 mg/kg; and 0.04 mg/kg) was dissolved in 3 ml of bacteriostatic water for injection and administered as an aerosol immediately after the antigen challenge. The five doses of CY222 were administered at least 2 wk apart from one another.

Histamine release in bronchoalveolar lavage. In eight animals, BAL was performed on different experiment days, at least 2 wk apart, before and after segmental antigen challenge, without and after pretreatment with UF-heparin (1,000 U/kg), LMWH CY216 (1 mg/kg), or ULMWH CY222 (1 mg/kg) for assay of histamine in BALF. UF-heparin, CY216 or CY222 were nebulized 1 h before the segmental antigen challenge. A. suum antigen (2.5 ml A. suum and 2.5 ml buffer) was infused via the wedge bronchoscope and BAL was performed 20 min later. The BAL effluent was centrifuged, and the supernatant was saved and frozen at 80° C for subsequent histamine RIA (28).

Statistical Analysis

The data were expressed as mean ± SE. Baseline values of PD₄₀₀ were compared with the postantigen PD₄₀₀ (without and after drug treatment) through Friedman's two-way analysis of variance (ANOVA) followed by nonparametric multiple comparison. The histamine concentration in different BALF samples was compared through Wilcoxon's signed rank test. Significance was accepted at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Antigen-induced AHR

The antigen-induced bronchoconstrictor responses in different groups were reproducible on the control day and on various treatment days. The antigen-induced increases in SRL peaked immediately after antigen challenge and returned to the baseline by 2 h after antigen challenge. On the control day, all animals manifested postantigen AHR at 2 h after antigen challenge, when SRL had returned to baseline.

Previously, we showed that administration of inhaled UF-heparin and fractionated heparins (KABI-2165, CY216, and CY222) before antigen challenge prevented antigen-induced bronchoconstriction and AHR (23, 27). The minimum effective doses of UF-heparin and fractionated heparins that prevented antigen-induced AHR were: UF-heparin = 1,000 U/kg; KABI-2165 = 5 mg/kg; CY216 = 1.25 mg/kg; and CY222 = 0.62 mg/kg. A summary of these data is shown in Figure 1. The doses of the different heparin fractions used in the present study are based on these findings.

View larger version (19K): [in this window] [in a new window]   Figure 1.   Effect of pretreatment with inhaled UF-heparin and fractionated heparins on antigen-induced AHR. AHR data are shown as mean ± SE PD400 (cumulative provocative dose of carbachol that increased SRL by 400%) in breath units; one breath unit is defined as one breath of 1% carbachol solution. The pretreatment data are shown here for comparison. (A) UF-heparin (1,000 U/kg; n = 8); (B) medium-molecular-weight heparin (KABI-2165, 5 mg/kg; n = 7); (C ) low-molecular-weight heparin (CY216, 1.25 mg/kg/ n = 6); (D) ultralow-molecular-weight heparin (CY222, 0.62 mg/kg; n = 7). *Significantly different from baseline (p < 0.05); Significantly different from postantigen (p < 0.05).

Effect of UF-Heparin

The baseline mean ± SE PD₄₀₀ for carbachol was 19 ± 2 breath units, which decreased to 11 ± 0.2 breath units 2 h after antigen challenge (p < 0.05), demonstrating antigen-induced AHR (Figure 2). Administration of UF-heparin (1,000 U/kg) after antigen challenge failed to modify the postantigen AHR (n = 4); PD₄₀₀ was 9 ± 2 breath units, which was not different from the antigen control (p = NS).

View larger version (15K): [in this window] [in a new window]   Figure 2.   Modification of postantigen AHR by inhaled UF-heparin (n = 4), medium-molecular-weight heparin (KABI-2165, n = 4), and low-molecular-weight heparin (CY216, n = 6). UF-heparin and fractionated heparins were administered after the postantigen measurements of SRL. AHR data are shown as mean ± SE PD400 (cumulative provocative dose of carbachol that increased SRL by 400%) in breath units; one breath unit is one breath of 1% carbachol solution. (A) UF-heparin (1,000 U/kg); (B) KABI-2165 (5 mg/ kg); (C ) CY216 (1.25 mg/kg). *Significantly different from baseline (p < 0.05).

Effect of Fractionated Heparins

MMWH (KABI-2165, n = 4) and LMWH (CY216, n = 6) also failed to modify the postantigen AHR when administered after antigen-challenge (p = NS). For the MMWH trial, the mean ± SE baseline PD₄₀₀ for carbachol was 23 ± 3 breath units; following antigen challenge, the PD₄₀₀ decreased to 13 ± 2 and 10 ± 3 breath units, without and after treatment with KABI-2165 (5 mg/kg) (Figure 2). Similarly, for LMWH (CY216, 1.25 mg/kg), the mean ± SE baseline PD₄₀₀ for carbachol was 21 ± 2 breath units, which decreased to 10 ± 2 and 12 ± 2 breath units following antigen challenge, without and after treatment with CY216 (Figure 2).

In contrast to UF-heparin, MMWH, and LMWH, administration of ULMWH (CY222) after antigen challenge selectively inhibited antigen induced AHR (p < 0.05). The mean ± SE baseline PD₄₀₀ was 22 ± 2 breath units, which decreased to 10 ± 2 breath units 2 h after antigen challenge. Administration of CY222 after antigen challenge inhibited postantigen AHR in a dose-dependent manner (n = 6); PD₄₀₀ ranged from 22 ± 3 to 25 ± 2 breath units with CY222 in doses of 0.08 mg/kg to 0.62 mg/kg, whereas the 0.04 mg/kg dose was ineffective (12.7 ± 2 breath units) (Figure 3). Compared with the minimum effective pretreatment dose of 0.62 mg/kg (27), CY222 was eight-fold more potent in attenuating AHR when administered after the antigen challenge, with a minimum effective dose of 0.08 mg/ kg (Figure 3).

View larger version (17K): [in this window] [in a new window]   Figure 3.   Dose-dependent modification of postantigen AHR by inhaled ultralow-molecular-weight heparin (CY222). CY222 was administered after the postantigen measurements of SRL (n = 6). AHR data are shown as mean ± SE PD400 (cumulative provocative dose of carbachol that increased SRL by 400%) in breath unit; one breath unit is one breath of 1% carbachol solution. PD400 is shown for the baseline (BSL) and 2 h postantigen without and after treatment with various doses of CY222; each dose was administered at least 2 wk apart from the others. *Significantly different from baseline (p < 0.05); Significantly different from postantigen (p < 0.05).

BAL Histamine Release

The baseline concentrations of histamine in BALF were comparable on different experiment days. Segmental antigen challenge caused a marked increased in the BALF histamine concentration, which increased from a mean ± SE of 2.1 ± 0.8 nM to 75 ± 21 nM for UF-heparin group (n = 8), and from 0.8 ± 0.1 to 85 ± 19 nM for the fractionated heparins group (n = 7). Inhaled UF-heparin and LMWH (CY216) inhibited antigen-induced histamine release by 81% (the BALF histamine concentration increased from 1.2 ± 0.7 nM to 14 ± 6 nM) and 75% (BALF histamine increased from 0.3 ± 0.3 nM to 21 ± 10 nM), respectively (p < 0.05), whereas ULMWH (CY222) caused only 5% inhibition (p = NS) of histamine release (BALF histamine increased from 0.3 ± 0.03 nM to 81 ± 8 nM) (Figure 4).

View larger version (16K): [in this window] [in a new window]   Figure 4.   Effect of inhaled UF-heparin, LMWH (CY216), and ULMWH (CY222) on antigen-induced histamine release as measured in BALF. Data are shown as mean ± SE histamine concentration in BALF 30 min after segmental antigen challenge, without and after pretreatment with UF-heparin (1,000 U/kg, n = 8), CY216 (1 mg/kg, n = 7), and CY222 (1 mg/kg, n = 7). The baseline concentrations of histamine in BALF were comparable on different experiment days, and ranged between 0.3 ± 0.3 nM and 2.1 ± 0.8 nM.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

AHR to nonantigenic stimuli plays an important role in the pathophysiology of asthma, and multiple factors contribute to this phenomenon, including release of proinflammatory mediators, airway inflammation, enhanced vagal reflex, and altered receptor function (24). Mast-cell-derived mediators released during allergic bronchoconstriction play of pivotal role in antigen-induced AHR. Thus, AHR can be modulated either by an antiallergic agent (e.g., cromolyn sodium) inhibiting mast-cell mediator release, or by the antiinflammatory actions of glucocorticosteroids (25). We have previously shown that UF-heparin and a low-molecular-weight heparin prevent antigen-induced acute bronchoconstriction and AHR, possibly by inhibiting mast-cell mediator release (23, 29). The results of this study further extend our previous observations and demonstrate that: (1) modification of antigen-induced AHR by fractionated heparins is molecular-weight dependent; (2) only ULMWH attenuates AHR when administered after the antigen challenge; (3) UF-heparin and LMWH inhibit antigen-induced histamine release as measured in BALF whereas ULMWH is ineffective in this respect; and (4) ULMWH attenuates postantigen AHR via a mast-cell-independent action.

We have recently observed that prevention of antigen- induced acute bronchoconstriction and AHR by fractionated heparins is molecular-weight dependent (27). An inverse relationship between the molecular weight and antiallergic activity of fractionated heparins was observed, and ULMWH was found to be the most effective agent. ULMWH was 2.5 to 3.5 fold more potent than LMWH and MMWH in preventing allergic bronchoconstriction, whereas against AHR it showed a five- to nine-fold greater potency (27). Although LMWH and MMWH showed greater efficacy in preventing allergic bronchoconstriction, ULMWH was equally effective against AHR and the allergic bronchoconstrictor response (27). The results of this study, in conjunction with previous data (27), show that whereas pretreatment with UF-heparin and fractionated low-molecular-weight heparins prevented antigen-induced AHR, only ULMWH attenuated AHR when administered after the antigen challenge. The failure of LMWH, MMWH, and UF-heparin to attenuate postantigen AHR was not dose-dependent, because considerably higher doses of these fractions, which effectively prevented allergic bronchoconstriction of AHR, were used (27). Whereas the minimum effective dose of ULMWH that prevented antigen-induced ARH was 0.62 mg/kg (27), the results of the present study demonstrate that ULMWH was more potent in attenuating AHR when administered after antigen challenge, as demonstrated by an eight-fold lower dose (80 µg/kg) needed for this effect than that required for prevention of AHR. The reason for the greater potency of ULMWH in attenuation of AHR when administered after antigen exposure is not clear.

It has been suggested that UF-heparin prevents antigen- induced bronchoconstriction and AHR by inhibiting mast-cell mediator release, rather than by a direct effect on airway smooth muscle (18, 22, 23, 30). This was supported by in vivo and in vitro studies demonstrating inhibition of antigen-induced airway-smooth-muscle contraction by UF-heparin, and by the failure of UF-heparin to attenuate agonist-induced contractile responses (18, 30). Heparin also prevented the bronchoconstrictor response induced by compound 48/80, a nonimmunologic mast-cell-degranulating agent (18), and inhibited antigen- induced histamine release from isolated mast cells (22). The antiallergic action of UF-heparin is further supported by in vivo data in the present study, showing prevention by inhaled heparin of antigen-induced histamine release as measured in BALF. The prevention of antigen-induced AHR by UF-heparin was considered relatively specific and unrelated to any antiinflammatory activity on the basis of the failure of heparin to modify the bronchoconstrictor response and AHR mediated by platelet-activating factor (PAF), a proinflammatory mediator (23). Failure of UF-heparin to modify postantigen AHR when administered after the antigen-challenge further supports this concept.

Our findings also demonstrate that the inhibitory effects of UF-heparin and fractionated heparins on antigen-induced histamine release as measured in BALF are molecular-weight dependent. In prior studies, UF-heparin and LMWH not only prevented allergic bronchoconstriction and AHR (23, 27), but also inhibited antigen-induced histamine release as measured in BALF. In contrast, ULMWH prevented allergic bronchoconstriction and AHR (27), but without inhibiting antigen- induced histamine release, as shown in the present study. These data indicate that whereas UF-heparin and fractionated MMWH and LMWH prevent antigen-induced AHR by inhibiting mast-cell mediator release, the attenuation of postantigen AHR by ULMWH is mast-cell independent.

It has been proposed that the antiallergic activity of UF- heparin may be mediated by inhibition of IP₃-dependent mast cell mediator release (23, 31). Low-molecular-weight heparins have also been shown to inhibit inositol-1,4,5-trisphosphate (IP₃)- induced Ca²⁺ release (32) and to attenuate anti-IgE-induced mast-cell degranulation (29). It has been observed that inhibition of IP₃-binding to its receptors by heparin is molecular-weight dependent, and the inhibitory activity decreased as the size of the heparin chain was reduced below 18 monosaccharide units (32). The ability of 18 to 24-monosaccharide-unit fractions of heparin to compete with the labeled ligand was comparable to that of UF-heparin and Fragmin (KABI-2165), whereas 10- to 14-monosaccharide-unit fractions had substantially lower activity, and 8-monosaccharide-unit fractions (mol wt < 2,500 daltons) had none (32). Our present findings are consistent with these observations, and demonstrate that only higher-molecular-weight heparins (including UF-heparin and LMWH) inhibit antigen-induced histamine release as measured in BALF, whereas ULMWH (< 2,500 daltons) is ineffective.

Low-molecular-weight heparins are a heterogenous group of drugs that are derived from UF-heparin by either chemical or enzymatic depolymerization. These derivatives differ from UF-heparin in their anticoagulant and antithrombotic activity, pharmacokinetics, and interaction with various proteins (33). Low-molecular-weight heparins also show marked heterogeneity in their biochemical and pharmacologic characteristics. The three low-molecular-weight heparins used in the present study not only differ in their molecular weights, but also show considerable differences in their pharmacokinetics. ULMWH (CY222) has the highest percent glycosaminoglycan (GAG) content, the lowest molecular weight, and the highest percent of chain length below 2,500 daltons. Low-molecular-weight heparins have increased bioavailability and prolonged biologic activity after subcutaneous administration, probably because of lower binding affinity to endothelial cells (34). However, the bioavailability and pharmacokinetics of inhaled low-molecular-weight heparins in general, and of ULMWH (CH222) in particular, are not known. Pharmacokinetic studies with aerosolized UF-heparin showed it to have significant antiasthmatic activity lasting for as long as 6 to 12 h in sheep and 3 to 6 h in subjects with asthma (35, 36). It is possible that the increased molecular-weight-dependent bioavailability of inhaled ULMWH is partly responsible for its potent in vivo antiallergic activity.

The mechanism of selective inhibition of postantigen AHR by ULMWH is unknown. The GAG heparins have been shown to have antiallergic and antiinflammatory properties including anticomplement action (14), modulation of T lymphocytes (16), inhibition of neutrophil chemotaxis and free radical generation (17), and inhibition of eosinophil influx (15). It has been reported that in guinea pigs and rabbits, UF-heparin and a low-molecular-weight heparinoid (ORG-10172) can prevent antigen and PAF-induced eosinophil influx (15). Although molecular-weight dependence of the antiinflammatory properties of heparin fractions has not been studied, it is possible that selective inhibition of postantigen AHR by ULMWH is related to some unknown antiinflammatory activities.