This conference supported in part by National Institute on Drug Abuse (NIDA) and sponsored by FASEB Summer Research Conference was held at the Copper Mountain Resort in Copper Mountain, CO, from July 18 through July 23, 1999. As the respiratory and pulmonary manifestations associated with inhaled abused substances is an emerging area in both clinical and basic research, the purpose of this conference was to provide a platform to discuss this area of research and the multidisciplinary approaches being employed to unravel the pathological processes and consequences of smoked substances. The secondary goal was stimulate to interdisciplinary cross-fertilization, free interchange of thoughts and ideas as well as future research directions and collaborations. A strong sense of having achieved the conference goals was evident from the lively and enthusiastic discussions among the participants from different backgrounds, generation of new ideas and the potential for collaborative research at the national and international level. For young scholars, it invigorated their enthusiasm to pursue a biomedical career, provided an opportunity to meet potential mentors and allowed for new research collaborations.

The meeting comprised nine major scientific sessions, a poster session designed for young scholars and two afternoon workshops that addressed issues pertaining to the design of studies and the potential use of marijuana for medicinal purposes. Highlights of the presentations and discussions in these sessions are summarized below.

I. Epidemiology of inhaled substances of abuse: scope of the problem: Dr. Timothy Condon, Associate Director, NIDA, opened the conference by highlighting the uniqueness of the conference goal as well as the importance of interdisciplinary research aimed at unraveling the underlying complex pathophysiology associated with the medical consequences of smoked substances of abuse. In order to develop successful intervention programs, it is essential to know the usage pattern of abused substances in the general population. Dr. O'Malley presented data from the Monitoring the Future Study indicating a gradual rise in the prevalence of use of marijuana and cocaine among young individuals in the mid to late 1990s, underscoring the public health importance of these questions. Dr. Elsohly reported an increase in potency of "street" marijuana since 1992 based on analysis of confiscated marijuana, further emphasizing the importance of improving our knowledge of the biologic effects of inhaled D9-tetrahydrocannabinol (THC).

II. Chemistry and toxicology of smoked substances: Data presented in this session focused on defining the contribution of smoke particulates in lung injury. For example, cytotoxic potential of gas-phase components from marijuana and tobacco cigarettes smoke in vitro was greatly diminished due to their water-soluble property at the level of alveoli and peripheral airways (Huber). On the other hand, these cytotoxins caused an increase in cellular production of reactive oxygen species in an endothelial cell line as well as in necrotic cell death suggesting its contribution to cell injury and dysfunction in the lungs of marijuana smokers (Sarafian). Studies also revealed that smoking of "crack" cocaine under controlled conditions caused bronchoconstriction and lung injury in animals due to its conversion to a toxic pyrolysate, methylecgonidine. Since the production of these toxic pyrolysates appears to be affected by conditions such as temperature and air flow rate, this could be a research area for further investigation. Other areas ripe for exploration could be to investigate the association between smoke-induced oxidative stress and cell injury, including apoptotic cell death, which represents an important defense mechanism against malignant transformation.

III. Smoking topography and smoking methodology: relation to drug bioavailability: Presentations in this session demonstrated that the technique (profile) of smoking marijuana and not the strength of marijuana in cigarettes was important in amplifying the potential respiratory and cardiovascular hazards associated with marijuana smoking (Tashkin). Additionally, smoking fewer marijuana cigarettes down to a shorter butt length to deliver more THC and achieve a greater "high" is potentially more harmful to cardio-respiratory health than consuming a comparable amount of marijuana contained in more cigarettes smoked to a longer butt length. A smoke delivery device and its utility to deliver precise doses of inhaled cocaine as well as it potential usefulness for controlled delivery of other smoked substances was discussed by Dr. Hatsukami.

VI. Impact of marijuana and "crack" cocaine on pulmonary symptoms and lung function: To address the inconsistent clinical findings on measures of lung function reported with marijuana smoking studies, Dr. Sherrill pointed out the importance of understanding the development of lung function with age, the study design and how different smoking methods can affect lung function. For example, significant lung function impairments observed in a small cross-sectional study of marijuana could not be replicated with a much larger cross-sectional or longitudinal study conducted by Tashkin and his group. Dr. Tashkin reported that tobacco affects primarily the small airways and gas-exchanging portion of the lung, potentially leading to chronic obstructive pulmonary disease (COPD), including chronic obstructive bronchitis, small airways disease and/or pulmonary emphysema. This is in contrast as heavy, regular use of marijuana, in the absence of tobacco, is unlikely to result in the development of clinically significant COPD. Marijuana smoking, on the other hand, is associated with symptoms of acute and chronic bronchitis that is consistent with chronic marijuana-related airway injury that could predispose to other smoking-related adverse pulmonary consequences, including predisposition to respiratory infection and bronchogenic carcinoma. Some of the research areas identified during these discussions included human investigations utilizing other available techniques such as, endoscopic airway visualization, bronchial biopsy and bronchoalveolar lavage and epidemiologic studies. In addition focused cellular and animal studies are also needed to investigate smoking-related adverse pulmonary consequences.

"Crack" cocaine smoking has also been associated with acute pulmonary symptoms and lung damage; however, the type of lung injury and the cause of these abnormalities are as controversial as in marijuana research. Findings presented by Dr. Kleerup from a detailed study revealed no significant reduction in the total diffusion capacity of the lung for carbon monoxide, nor in capillary blood volume when assessed following acute experimental cocaine smoking or in chronic habitual "crack" cocaine users. This led to discussions as to the impact of study design, difference in these measures and the role of other confounders. To further evaluate the possible effect of crack cocaine on gas transfer in the lung and its clinical significance, Dr. Cooper presented his findings on the impact of regular use of crack cocaine on maximal exercise performance in a controlled laboratory setting. Except for reductions in maximum oxygen uptake and heart rate, all other exercise measures (sub-maximal exercise responses, ventilatory efficiency, resting arterial blood gases and alveolar-arterial oxygen partial pressure differences) were within normal range in male and female cocaine smokers when compared with age- and gender-matched sedentary control subjects. An interesting finding was the perceived exertion was inappropriately high in the male cocaine smokers, despite VO2max being only 75% of predicted value. This finding suggests a perceptual problem and is consistent with earlier observations in athletes who perceived that they were performing better under the influence of cocaine while, in actual fact, they were impaired in their exercise capacity.

V. Effect of smoked substances on host immunity: Both marijuana and cocaine have been reported to be potent immune modulators; however, the mechanism of the host immune suppression is unclear. Thus, the focus of this session was to advance our knowledge of the underlying mechanisms involved in marijuana- and cocaine-related immune suppression and how these might lead to acute and long-term lung injury. Both delta-9-THC and cannabinol, a plant derived cannabinoid, have been shown to bind to peripheral CB2 receptors, which are coupled to the negative regulation of the cAMP-signaling cascade. Dr. Kaminski reported that the mechanism responsible for the cannabinol-elicited inhibition of interleukin-2 (IL-2) secretion by activated spleen T-cells was due to modulation of the cAMP signal transduction cascades in activated T-cells, resulting in altered IL-2 and IL-4 gene expression. Data presented by Dr. Roth on the assessment of delta-9-THC effects on antigen-specific immunity demonstrated significant alterations in the function of purified human T cells as well as in antigen-presenting dendritic cells. These findings are significant as THC-elicited suppression of innate immunity could predispose marijuana users to opportunistic infections and cancer. Dr. Cabral's studies revealed that THC differentially modulates the capacity of macrophages to process antigens that are necessary for activation of CD4+ T cells and not the peptide presentation to macrophages. Furthermore, this cannabinoid-mediated inhibition of antigen processing by macrophages appears to involve CB2 receptor.

Using an in vivo murine model, Dr. Klein showed that THC suppressed the normal development of Th1 immunity to Legionella infection and this suppression was not due to an increase in IL-4 production but due to a decrease in the functioning of the IL-12 system. Furthermore, the drug effects on the cytokine responses and T helper cell development appear to involve CB1 and CB2 cannabinoid receptors as well as components of the hypothalamic-pituitary-adrenal axis, such as corticosteroids. These intriguing results suggest that the cannabinoid receptors involved could be those expressed in brain areas, such as the hypothalamus, as well as those expressed peripherally on cells of the immune system, an area ripe for further exploration.

Data from human alveolar macrophages (AMs) studies revealed that habitual smoking of either cocaine or marijuana significantly impairs the antimicrobial and tumoricidal activity of human AMs (Baldwin). Moreover, the pattern of impairment was different for each substance, suggesting that cocaine and marijuana mediate their effects by unique and different pathways. Cocaine primarily affects the ability of AMs to kill bacteria and tumor cells, likely by suppressing their ability to generate reactive intermediate molecules such as nitric oxide (NO). While marijuana use has a broader range of effects on AMs including suppression of phagocytosis, inhibition of bacterial and tumor cell killing and a reduction in their ability to produce inflammatory cytokines. Further studies are needed to investigate the mechanism whereby marijuana and cocaine impair alveolar macrophage function, including studies of the effects of these substances on nitric oxide synthesis at the molecular level.

VI. Impact of smoked substance abuse on lung injury and inflammation: Presentations in this session described the pathological changes of the respiratory system associated with smoking of various abused drugs. Dr. Roth reported that regular smoking of marijuana and/or tobacco has significant injurious effects on the central and peripheral airways, even in young and otherwise asymptomatic adults. In this population, the visual bronchitis index revealed lower airway injury and this was correlated with histopathologic evidence of airway inflammation. Dr. Fliegiel presentation indicated extensive morphological alterations in the bronchial and lung parenchyma, as well as macrophages obtained from smokers of marijuana, cocaine and/or tobacco and from primates experimentally exposed to marijuana or sham-smoking. These findings are indicative of the damaging effects of chronic exposure to marijuana and smoked substances. Follow-up studies will explore the long-term injurious potential of these inhaled substances on DNA adducts, assessment of apoptosis and characterization of the expression of pre-neoplastic/early neoplastic cell surface markers. Dr. Virmani discussed the results of a detailed pathologic examination of lungs obtained at post-mortem in cases identified as having abused drugs. Her findings indicated that the route of administration, as well as the drug used, appears to influence the type of pulmonary pathology observed.

VII. Effects of smoked substances on carcinogenesis: Biologic and Epidemiologic evidence: This session addressed research areas aimed at understanding the relationship between smoking marijuana, genetic and biological markers, and risk to the respiratory tract cancer. Polycyclic aromatic hydrocarbons (PAHs) present in the tar fraction of tobacco smoke activate transcription of the CYP1A1 gene that codes for the key enzyme, cytochrome P4501A1, that is primarily responsible for converting procarcinogenic PAHs into active carcinogens. This activation leads to an increase in DNA mutations in lung tissue and an increased risk for developing lung cancer. Recent reports of molecular and genetic alterations in marijuana users suggested that marijuana smoke might also activate CYP1A1 gene. Dr. Roth investigated this possibility using Hepa-1 cells and found that marijuana tar, and more specifically D9-THC, regulates the induction and function of CYP1A1 gene, an observation that is entirely novel. Transcriptional activation of CYP1A1 by D9-THC may help to explain the relatively high frequency of DNA mutations and mucosal abnormalities that occur in marijuana smokers. The inhalation of marijuana smoke delivers both nanogram concentrations of conventional PAHs and milligram quantities of D9-THC to the lung. Induction of CYP1A1 produced by D9-THC could result in greater activation of smoke-related procarcinogens and higher adduct-related injury. However, it is also possible that inhaled D9-THC competes for the active site of CYP1A1, paradoxically reducing the activation of procarcinogens. Further studies are warranted to clarify the interaction of D9-THC with CYP1A1 and the biological role of marijuana smoke as a cancer risk factor.

Dr. Gordy's studies designed to assess the earliest evidence of pulmonary cellular and genetic damage provoked by marijuana smoking initially revealed no significant alterations in DNA adduct formation; however, using a newer comet assay technique, he reported observing significant comet "tails" indicating DNA damage both with marijuana and tobacco smoke but not with tobacco tar alone. His preliminary findings, which showed more evidence of DNA damage induced by smoke from regular marijuana cigarettes than smoke from marijuana "placebo" cigarettes containing no THC, raised the interesting question of whether THC itself may be a direct DNA-damaging agent. He also presented findings that showed the presence of a mutation (exon 6) in p53 tumor suppressor gene in 3 of the 10 subjects tested so far. Since the observed mutation is not listed in the online p53 mutation database, this finding may represent a novel mutation. These ongoing studies of DNA adduct damage and specific mutation occurrence should contribute to a greater understanding of the relative risks and molecular mechanisms of marijuana-induced lung tissue damage and proliferation.

Dr. Dubinett presented data that showed THC-induced suppression of host immune surveillance against lung cancer, since intermittent administration of THC in two different weakly immunogenic murine lung cancer models led to accelerated growth of tumor implants and augmentation of immune inhibitory cytokines at both the tumor site as well as in the spleens of THC-treated mice. Transfer of lymphocytes from THC-treated mice to normal mice resulted in accelerated tumor growth similar to that seen in the THC-treated hosts. In contrast to the findings in immunocompetent mice, THC did not affect tumor growth in tumor-bearing immunodeficient SCID mice, supporting the view that THC promotes tumor growth by an immune-mediated mechanism. The effect appears to be CB2 receptor-mediated since a specific CB2-receptor antagonist inhibits it. Another interesting observation was that in this mouse lung cancer model, cocaine also caused a similar suppression of Th1 immunity as seen with THC, however the underlying mechanism is not yet known.

Dr. Sidney discussed the results of a large retrospective epidemiologic study that examined the relationship of marijuana use to cancer incidence. Compared with nonuser/experimenters, ever- and current use of marijuana was not associated with increased risk of cancer of all sites in analyses adjusted for sociodemographic factors, cigarette smoking, and alcohol use. Marijuana use also was not associated with tobacco-related cancers or with cancer of the following sites: colorectal, lung, melanoma, prostate, breast, and cervix. Among nonsmokers of tobacco cigarettes, marijuana use was associated with an increased risk of prostate cancer and a nearly significantly increased risk of cervical cancer. Dr. Sidney concluded that, in this relatively young cohort study, marijuana use and cancer were not associated in overall analyses, but that associations in nonsmokers of tobacco cigarettes suggested that marijuana use might affect certain site-specific cancer risks. Limitations of this study included the relatively young age of the cohort at study entry and the short duration of follow-up.

Dr. Zhang investigated the relationship between marijuana use and head and neck cancer in a case-control study and found that marijuana use may increase the risk of these cancers and cancers of the tongue and larynx. The relationship between the occurrence of these cancers and marijuana use was dose-dependent. His analysis indicated that marijuana use might interact with mutagen sensitivity and other risk factors to increase the risk of head and neck cancer. These results need to be interpreted with some caution in drawing causal inferences because of certain methodological limitations.

VIII. Marijuana, cocaine and HIV infection: Marijuana and crack cocaine have been suggested to act as co-factors for progression of HIV infection and AIDS; however, the information on the exact mechanisms by which these drugs facilitate AIDS progression is virtually unknown. The presentations in this session aimed at defining these mechanisms by employing both in vivo and in vitro techniques as well as the utility of epidemiologic studies. For example, Dr. Klein showed that in a human T-cell line that expresses message for both CB1 and CB2 receptors (MT-2 cells), cannabinoids do increase HIV infectivity or replication as measured by the fusion assay and p24 expression; however, these receptors may not be involved in this drug effect.

While in vitro studies can directly assess the impact of drugs of abuse on the biology of HIV, they cannot control for the complex interactions that occur when an individual is repeatedly exposed to a drug over time. Moreover, in vitro studies cannot address the possibility that drugs of abuse such as cocaine and marijuana may affect HIV replication indirectly, for example through an intermediate which could be released following absorption of the parent drug(s) in vivo. In light of these limitations, Dr. Baldwin's group recently defined and established a murine model system to evaluate whether drugs of abuse enhance the infectivity and replication of HIV in human lymphocytes in vivo. This model system utilizes the human lymphocyte/ SCID (huPBL/SCID) mouse model, which was first developed by Mosier and colleagues to provide a relevant animal model for assessing HIV pathogenesis in vivo. Dr. Baldwin reported her preliminary results that showed that cocaine was well tolerated by SCID mice and, in the absence of HIV infection, did not affect the implantation and/or distribution of human cells in these mice. Using this model, she found an increase in the total percentage of HIV-infected human cells and a decrease in the number of CD4+ target cells in cocaine-treated HIV-infected huPBL/SCID mice when compared to untreated HIV-infected control animals. This is the first report indicating that cocaine can directly affect HIV replication in vivo. These results also demonstrated that the huPBL/SCID model has the potential to determine how other drugs of abuse, alone or in combination, affect HIV pathobiology in vivo.

Dr. Bennett described the variations in the patterns of in-patient diagnostic Pneumocystis Carinii Pneumonia (PCP) care and rate of discharge against medical advice among IDUs/ polysubstance abusers, non-IDUs, and homosexual/bisexual. Results from an epidemiologic study presented by Dr. Caiaffa showed that smoking illicit drugs (marijuana and/or cocaine) was independently associated with bacterial pneumonia, while tobacco cigarette smoking was not, presumably because tobacco smoking was nearly universally present in her cohort. Interestingly, smoking illicit drugs had the strongest effect on risk for bacterial pneumonia among HIV+ IV drug abusers with a previous history of PCP. It was pointed out, however, that a number of limitations precluded drawing firm conclusions from these data. Also discussed were the inherent difficulties in analyzing the in vivo effect of inhaled drugs of abuse on viral replication in HIV-infected individuals due to the potential confounding effects of other infections and medications. Epidemiologic studies thus can provide only indirect evidence of the possible relationship between marijuana/cocaine use and HIV replication in vivo, pointing to the need for complementary animal studies, such as those using the huPBC/SCID mouse model, as described above.

IX. Medicinal marijuana: potential impact of pulmonary and immune complications on the therapeutic use of marijuana: During this session, the discussions and presentations centered around the scientific issues pertaining to use of marijuana as an adjunct therapeutic agent for treating HIV-infection/ AIDS. Dr. Abrams pointed out that HIV-infected individuals continue to seek smoked marijuana to combat the nausea induced by highly active antiretroviral therapy (HAART) and to allow them to take their medications as prescribed. Because of the potential for widespread drug-drug interactions with the cyotchrome P450 metabolized protease inhibitors, his team has designed a study to determine the safety/toxicity profile of cannabinoids in persons with HIV disease receiving protease therapy. Specifically addressed was the question whether a metabolic interaction between cannabinoids and protease inhibitors and/or cannabinoids and the immune system may lead to alterations in HIV RNA levels after 21 days of exposure. The trial design was discussed but no data from the trial are as yet available. Once the safety profile of smoked marijuana has been defined in this group of patients, further trials focusing on the efficacy, as well as safety, of medicinal marijuana in HIV-infected individuals with specific symptoms not adequately relieved by conventional medication (e.g., pain, wasting, medication-induced nausea and emesis) may be warranted. Dr. Tashkin discussed the potential respiratory and infectious complications of medicinal marijuana that include spontaneous pneumothorax or pneumomediastinum, acute and chronic bronchitis and predisposition to lower respiratory tract infection, including pneumonia. Evidence suggests that the latter is likely to be due to marijuana-related impairment of the lung's first and second lines of defense against infection as discussed earlier in this meeting. Several recommendations were also suggested to monitor respiratory and infectious complications of medicinal marijuana, e.g., sterilization of marijuana cigarettes, periodic surveillance bacterial and fungal cultures of the sterilized marijuana cigarettes, patients education to contact their physician in the event of symptoms or signs suggesting infection, frequent follow-up visits and use of specific diagnostic procedures, as clinically indicated.

An alternative approach to exploiting the potential medicinal properties of cannabis is to develop synthetic cannabinomimetic compounds that retain these medicinal characteristics but have minimal unwanted effects. Dr. Makriyannis pointed out that for the past 4 or 5 decades a number of research programs spearheaded by the pharmaceutical industry have sought to develop novel, useful medications using the natural ingredients of cannabis as their initial drug prototypes. However, the hopes of developing a wide spectrum of successful medications using this approach have not been realized to date. On other hand, recent developments in cannabinoid biology have provided new impetus to the search for novel cannabinoid compounds with possible therapeutic efficacy. These developments include the discovery of the CB1 and CB2 receptors, as well as two families of lipid compounds that are believed to be the endogenous ligands for the cannabinoid receptors, anadamide and arachidonyl glycerol (and some of its congeners). These four cannabinomimetic targets have become the center of a concerted effort for the discovery of novel medications, with such properties as analgesia (against central and peripheral pain), appetite enhancement, immunomodulation, anti-inflammatory activity and anti-spasmodic activity.

Dr. Khalsa provided an overview of the health effects of marijuana and NIDA's and clinical research programs in relation to the connection between drugs of abuse, AIDS and other disorders. Dr. Thadani concluded the conference with a further discussion of the programmatic objectives of NIDA in stimulating new research in both basic and clinical science to fill important gaps in our current understanding and in attracting new investigators, especially younger investigators and those from under-represented minorities, to the field of drug abuse research.

**This article was prepared by a federal government official as part of official duties.

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