Prevalence of fentanyl in methamphetamine and cocaine samples collected by community-based drug checking services.

BACKGROUND: Overdose deaths involving stimulants and opioids simultaneously have raised the specter of widespread contamination of the stimulant supply with fentanyl. METHODS: We quantified prevalence of fentanyl in street methamphetamine and cocaine, stratified by crystalline texture, analyzing samples sent voluntarily to a public mail-in drug checking service (May 2021-June 2023). Samples from 77 harm reduction programs and clinics originated in 25 US states. Sample donors reported expected drug and physical descriptions. Substances were identified by gas chromatography-mass spectrometry. Negative binomial models were used to calculate fentanyl prevalence, adjusting for potential confounders related to sample selection. We also examined if xylazine changed donors' accuracy of detecting fentanyl. RESULTS: We analyzed 718 lab-confirmed samples of methamphetamine (64%) and cocaine (36%). The adjusted prevalence of fentanyl was 12.5% (95% CI: 2.2%, 22.9%) in powder methamphetamine and 14.8% (2.3%, 27.2%) in powder cocaine, with notable geographic variation. Crystalline forms of both methamphetamine (Chisq=57, p<0.001) and cocaine (Chisq=18, p<0.001) were less likely to contain fentanyl: less than 1% of crystal methamphetamine (2/276) and no crack cocaine (0/53). Heroin was present in 6.6% of powder cocaine samples. Xylazine reduced donors' ability to detect fentanyl, with correct classification dropping from 92% to 42%. CONCLUSIONS: Fentanyl was detected primarily in powder forms of methamphetamine and cocaine. Recommended interventions include expanding community-based drug checking, naloxone and fentanyl test strip distribution for people who use stimulants , and supervised drug consumption sites. New strategies to dampen variability in street drug composition are needed to reduce inadvertent fentanyl exposure.

Tumor Cell-Specific Retention and Photodynamic Action of Erlotinib-Pyropheophorbide Conjugates.

To enhance uptake of photosensitizers by epithelial tumor cells by targeting these to EGFR, pyropheophorbide derivatives were synthesized that had erlotinib attached to different positions on the macrocycle. Although the addition of erlotinib reduced cellular uptake, several compounds showed prolonged cellular retention and maintained photodynamic efficacy. The aim of this study was to identify whether erlotinib moiety assists in tumor targeting through interaction with EGFR and whether this interaction inhibits EGFR kinase activity. The activity of the conjugates was analyzed in primary cultures of human head and neck tumor cells with high-level expression of EGFR, and in human carcinomas grown as xenografts in mice. Uptake of erlotinib conjugates did not correlate with cellular expression of EGFR and none of the compounds exerted EGFR-inhibitory activity. One derivative with erlotinib at position 3, PS-10, displayed enhanced tumor cell-specific retention in mitochondria/ER and improved PDT efficacy in a subset of tumor cases. Moreover, upon treatment of the conjugates with therapeutic light, EGFR-inhibitory activity was recovered that attenuated EGFR signal-dependent tumor cell proliferation. This finding suggests that tumor cell-specific deposition of erlotinib-pyropheophorbides, followed by light triggered release of EGFR-inhibitory activity, may improve photodynamic therapy by attenuating tumor growth that is dependent on EGFR-derived signals.

Tumor cell-specific retention of photosensitizers determines the outcome of photodynamic therapy for head and neck cancer.

Pheophorbide-based photosensitizers have demonstrated tumor cell-specific retention. The lead compound 3-[1'-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH) in a clinical trial for photodynamic therapy of head and neck cancer lesions indicated a complete response in 80% of patients. The question arises whether the partial response in 20% of patients is due to inefficient retention of photosensitizers by tumor cells and, if so, can the photosensitizer preference of individual cancer cases be identified prior to photodynamic therapy. This study determined the specificity of head and neck cancer cells and tumor tissues for the uptake and retention of diffusible pheophorbides differing in peripheral groups on the macrocycle that contribute to cellular binding. The relationship between photosensitizer level and light-mediated photoreaction was characterized to identify markers for predicting the effectiveness of photodynamic therapy in situ. The experimental models were stromal and epithelial cells isolated from head and neck tumor samples and integrated into monotypic tissue cultures, reconstituted three-dimensional co-cultures, and xenografts. Tumor cell-specific photosensitizer retention patterns were identified, and a procedure was developed to allow the diagnostic evaluation of HPPH binding by tumor cells in individual cancer cases. The findings of this study may assist in designing conditions for photosensitizer application and photodynamic therapy of head and neck cancer lesions optimized for each patient's case.

Charged groups on pyropheophorbide-based photosensitizers dictate uptake by tumor cells and photodynamic therapy efficacy.

This study investigated the impact of anionic and cationic substituents of the pyropheophorbide-based photosensitizers (PS) on uptake and retention by tumor epithelial cells and photodynamic therapy (PDT). A series of PSs were generated that bear carboxylic acid functionalities, alkyl amines with variable length of carbon units or as a quaternary ammonium salt introduced at position 172 of 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH). The nature of the functionalities in the macrocycle made a significant difference in overall lipophilicity (log D values at pH 7.4), and in binding to and retention by human and murine tumor cells. Depending on the presence of functional groups, the PSs showed a change in cellular uptake from diffusion to endocytosis and in the preference for subcellular localization to mitochondria/ER or lysosomes. Two and more carboxylic groups drastically reduced uptake by all cell types. In contrast, PSs with amine and quaternary amine salt showed higher cellular binding, uptake and in vitro PDT efficacy than HPPH. The enhanced cellular uptake of the cationic PSs was accompanied by a loss of tumor cell specificity and contributed to severe systemic toxicity in tumor-bearing mice intravenously injected with the PS and subjected to investigate their therapeutic potential.

Chiral Alkyl Groups at Position 3(1') of Pyropheophorbide-a Specify Uptake and Retention by Tumor Cells and Are Essential for Effective Photodynamic Therapy.

To investigate the importance of the chirality and precise structure at position 3(1') of pyropheophorbide-a for tumor cell specificity and photodynamic therapy (PDT), a series of photosensitizers (PSs) was synthesized: (a) with and without chirality at position 3(1'), (b) alkyl ether chain with a variable number of chiral centers, (c) hexyl ether versus thioether side chain, and (d) methyl ester versus carboxylic acid group at position 172. The cellular uptake and specificity were defined in human lung and head/neck cancer cells. PSs without a chiral center and with an alkyl chain or thioether functionalities showed limited uptake and PDT efficacy. Replacing the methyl group at the chiral center with a propyl group or introducing an additional chiral center improved cellular retention and tumor cell specificity. Replacing the carboxylic acid with methyl ester at position 172 lowered cellular uptake and PDT efficacy. A direct correlation between the PS uptake in vitro and in vivo was identified.

Synthesis, Tumor Specificity, and Photosensitizing Efficacy of Erlotinib-Conjugated Chlorins and Bacteriochlorins: Identification of a Highly Effective Candidate for Photodynamic Therapy of Cancer.

Erlotinib was covalently linked to 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH) and structurally related chlorins and bacteriochlorins at different positions of the tetrapyrrole ring. The functional consequence of each modification was determined by quantifying the uptake and subcellular deposition of the erlotinib conjugates, cellular response to therapeutic light treatment in tissue cultures, and in eliminating of corresponding tumors grown as a xenograft in SCID mice. The experimental human cancer models the established cell lines UMUC3 (bladder), FaDu (hypopharynx), and primary cultures of head and neck tumor cells. The effectiveness of the compounds was compared to that of HPPH. Furthermore, specific functional contribution of the carboxylic acid side group at position 172 and the chiral methyl group at 3(1') to the overall activity of the chimeric compounds was assessed. Among the conjugates investigated, the PS 10 was identified as the most effective candidate for achieving tumor cell-specific accumulation and yielding improved long-term tumor control.

Cell-specific Retention and Action of Pheophorbide-based Photosensitizers in Human Lung Cancer Cells.

This study determined in primary cultures of human lung cancer cells the cell specificity of chlorin-based photosensitizers. Epithelial cells (ECs) preferentially retained 3-[1-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH) and related structural variants. Tumor-associated fibroblasts (Fb) differ from EC by a higher efflux rate of HPPH. Immunoblot analyses indicated dimerization of STAT3 as a reliable biomarker of the photoreaction. Compared to mitochondria/ER-localized photoreaction by HPPH, the photoreaction by lysosomally targeted HPPH-lactose showed a trend toward lower STAT3 cross-linking. Lethal consequence of the photoreaction differed between EC and Fb with the latter cells being more resistant. A survey of lung tumor cases indicated a large quantitative range by which EC retains HPPH. The specificity of HPPH retention defined in vitro could be confirmed in vivo in selected cases grown as xenografts. HPPH retention as a function of the tetrapyrrole structure was evaluated by altering side groups on the porphyrin macrocycle. The presence or absence of a carboxylic acid at position 172 proved to be critical. A benzyl group at position 20 enhanced retention in a subset of cancer cells with low HPPH binding. This study indicated experimental tools that are potentially effective in defining the photosensitizer preference and application for individual patient's cancer lesions.

Highly Effective Dual-Function Near-Infrared (NIR) Photosensitizer for Fluorescence Imaging and Photodynamic Therapy (PDT) of Cancer.

We report herein the synthesis and biological efficacy of near-infrared (NIR), bacteriochlorin analogues: 3-(1'-butyloxy)ethyl-3-deacetyl-bacteriopurpurin-18-N-butylimide methyl ester (3) and the corresponding carboxylic acid 10. In in vitro assays, compared to its methyl ester analogue 3, the corresponding carboxylic acid derivative 10 showed higher photosensitizing efficacy. However, due to drastically different pharmacokinetics in vivo, the PS 3 (HPLC purity >99%) showed higher tumor uptake and long-term tumor cure than 10 (HPLC purity >96.5%) in BALB/c mice bearing Colon 26 tumors. Isomerically pure R- and S- isomers of 3 (3a and 3b, purity by HPLC > 99%) under similar treatment parameters showed identical efficacy in vitro and in vivo. In addition, photosensitizer (PS) 3 showed limited skin phototoxicity and provides an additional advantage over the clinically approved chemically complex hematoporphyrin derivative as well as other porphyrin-based PDT agents, which makes 3 a promising dual-function agent for fluorescence-guided surgery with an option of phototherapy of cancer.

Effect of chirality on cellular uptake, imaging and photodynamic therapy of photosensitizers derived from chlorophyll-a.

We have previously shown that the (124)I-analog of methyl 3-(1'-m-iodobenzyloxy) ethyl-3-devinyl-pyropheophorbide-a derived as racemic mixture from chlorophyll-a can be used for PET (positron emission tomography)-imaging in animal tumor models. On the other hand, as a non-radioactive analog, it showed excellent fluorescence and photodynamic therapy (PDT) efficacy. Thus, a single agent in a mixture of radioactive ((124)I-) and non-radioactive ((127)I) material can be used for both dual-imaging and PDT of cancer. Before advancing to Phase I human clinical trials, we evaluated the activity of the individual isomers as well as the impact of a chiral center at position-3(1) in directing in vitro/in vivo cellular uptake, intracellular localization, epithelial tumor cell-specific retention, fluorescence/PET imaging, and photosensitizing ability. The results indicate that both isomers (racemates), either as methyl ester or carboxylic acid, were equally effective. However, the methyl ester analogs, due to subcellular deposition into vesicular structures, were preferentially retained. All derivatives containing carboxylic acid at the position-17(2) were noted to be substrate for the ABCG2 (a member of the ATP binding cassette transporters) protein explaining their low retention in lung tumor cells expressing this transporter. The compounds in which the chirality at position-3 has been substituted by a non-chiral functionality showed reduced cellular uptake, retention and lower PDT efficacy in mice bearing murine Colon26 tumors.

Photodynamic therapy with 3-(1'-hexyloxyethyl) pyropheophorbide a for cancer of the oral cavity.

PURPOSE: The primary objective was to evaluate safety of 3-(1'-hexyloxyethyl)pyropheophorbide-a (HPPH) photodynamic therapy (HPPH-PDT) for dysplasia and early squamous cell carcinoma of the head and neck (HNSCC). Secondary objectives were the assessment of treatment response and reporters for an effective PDT reaction. EXPERIMENTAL DESIGN: Patients with histologically proven oral dysplasia, carcinoma in situ, or early-stage HNSCC were enrolled in two sequentially conducted dose escalation studies with an expanded cohort at the highest dose level. These studies used an HPPH dose of 4 mg/m(2) and light doses from 50 to 140 J/cm(2). Pathologic tumor responses were assessed at 3 months. Clinical follow up range was 5 to 40 months. PDT induced cross-linking of STAT3 were assessed as potential indicators of PDT effective reaction. RESULTS: Forty patients received HPPH-PDT. Common adverse events were pain and treatment site edema. Biopsy proven complete response rates were 46% for dysplasia and carcinoma in situ and 82% for squamous cell carcinomas (SCC) lesions at 140 J/cm(2). The responses in the carcinoma in situ/dysplasia cohort are not durable. The PDT-induced STAT3 cross-links is significantly higher (P = 0.0033) in SCC than in carcinoma in situ/dysplasia for all light doses. CONCLUSION: HPPH-PDT is safe for the treatment of carcinoma in situ/dysplasia and early-stage cancer of the oral cavity. Early-stage oral HNSCC seems to respond better to HPPH-PDT in comparison with premalignant lesions. The degree of STAT3 cross-linking is a significant reporter to evaluate HPPH-PDT-mediated photoreaction.

Cell-type selective phototoxicity achieved with chlorophyll-a derived photosensitizers in a co-culture system of primary human tumor and normal lung cells.

The ATP-dependent transporter ABCG2 exports certain photosensitizers (PS) from cells, implying that the enhanced expression of ABCG2 by cancer cells may confer resistance to photodynamic therapy (PDT) mediated by those PS. In 35 patient-derived primary cultures of lung epithelial and stromal cells, PS with different subcellular localization and affinity for ABCG2 displayed cell-type specific retention both independent and dependent on ABCG2. In the majority of cases, the ABCG2 substrate 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) was lost from fibroblastic cells more rapidly than from their epithelial counterparts, even in the absence of detectable ABCG2 expression, facilitating selective eradication by PDT of epithelial over fibroblastic cells in tumor/stroma co-cultures. Pairwise comparison of normal and transformed epithelial cells also identified tumor cells with elevated or reduced retention of HPPH, depending on ABCG2. Enhanced ABCG2 expression led to the selective PDT survival of tumor cells in tumor/stroma co-cultures. This survival pattern was reversible through HPPH derivatives that are not ABCG2 substrates or the ABCG2 inhibitor imatinib mesylate. PS retention, not differences in subcellular distribution or cell signaling responses, was determining cell type selective death by PDT. These data suggest that up-front knowledge of tumor characteristics, specifically ABCG2 status, could be helpful in individualized PDT treatment design.